biodiesel production from microalgae

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BIODIESEL PRODUCTION FROM MICROALGAE Mostafa El-Sheekh and Abd El-Fatah Abomohra Botany Department, Faculty of Science, Tanta University, Tanta, Egypt

ABSTRACT Microalgae have long been considered as a promising feedstock for biomass production. The worldwide annual production of algal biomass in 2004 was estimated to be 5 Gg. One-fifth, approximately, of this biomass is used to nourish fish and shellfish that are cultivated in aquaculture hatcheries. Recently, microalgae were discussed to be used as a renewable feedstock for biodiesel production as they have some advantages when compared to traditional biofuel feedstocks. The selection of the most suitable species is based on several key parameters such as lipid and fatty acid productivities. With predictions that crude oil prices will reach record breaking increases, algal based biofuels are gaining widespread attention. One of renewable and carbon-neutral fuel applications exploiting algal components is transesterification of lipids to biodiesel. Microalgae cultivation can be done in open-culture systems called “ponds” or in highly controlled closed-culture systems called “photobioreactors, PBRs”. After algal growth, there are many methods for harvesting of microalgae such as centrifugation, filtration and gravity sedimentation which may be preceded by a flocculation step. Choosing of the suitable harvesting method depends on algal species, growth medium, algae production, end product and production cost. The present article discusses the steps of conversion of microalgae for biodiesel production including selection of the suitable microalgal strain for large scale outdoor cultivation, different systems used for algal biomass production, methods of harvest of algae and lipid extraction, conversion of the extracted lipids into biodiesel and advantages of microalgae for biodiesel production.

Keywords: algae, biodiesel, biofuel, microalgae, transesterification, renewable energy

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Corresponding author: [email protected]

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Mostafa El-Sheekh and Abd El-Fatah Abomohra

1. INTRODUCTION Fossil fuel is important for the global economy for energy production required for lighting, transportation and heating. The need of communities for fossil fuel increased as a result of increasing population and expanding economy. In addition, the expanded use of fossil fuels increased the atmospheric CO2 concentration causing the climate changes, which now affect all parts of the world. Furthermore, fossil fuel is a limited resource and it will run out [1]. These factors motivated the development of renewable energy sources that can replace fossil fuels. Nowadays, algal-based biofuels are gaining widespread attention. Fuel applications exploiting algal components include transesterification of lipids to biodiesel [2, 3], saccharification of carbohydrates to ethanol [4], gasification of biomass to syngas [5], cracking of hydrocarbons to gasoline [6], and biosynthesis of hydrogen gas [7]. The main advantages of biodiesel, other than being a renewable energy source, is that its burning is much cleaner than that of fossil fuel, environmentally friendlier, and it can be used in the present diesel engines without modifications. Algae are one of the most exciting future solutions for our energy crisis, especially that of transportation fuel. Algae need very low requirements to grow including carbon dioxide, sun light, and water. They can grow in nonarable land or in waste water. Algae grow fast and have lipid content higher than that of seed plants. They have short generation time, i.e., they can double their mass every few hours and produce at least 30 times more oil per acre than seed plants. Moreover, because algae can grow under severe conditions, they can grow on arid coastal lands, unsuitable for conventional agriculture, using wastewater or sea water. The bottleneck in biodiesel production from microalgae is to identify the strains with high oil productivity and to develop cost-effective growing and harvesting systems. Figure 1 shows the pathways of biodiesel production from microalgae and byproducts of the production process.

Figure 1. Microalgal biodiesel refinery producing multiple products from algal biomass.

Biodiesel Production from Microalgae

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2. SELECTION AND GROWTH OF MICROALGAE FOR BIODIESEL There are different metabolic behaviors in microalgae including; (1) autotrophic, i.e., algae using light as a sole energy source, they convert light energy to chemical energy using CO2 through photosynthesis; (2) mixotrophic, i.e., algae performing photosynthesis as the main energy source, but they need both organic compounds and CO 2; (3) heterotrophic, i.e., algae utilizing only organic compounds as energy and carbon source and (4) photoheterotrophic, i.e. algae utilizing light to use organic compounds as carbon source [8]. Algae can change the metabolic pathway according to the changes in the environmental conditions. The ability of algae to fix CO2 is a method of removing CO2 from flue gases of power plants and, thus, can be used to reduce emissions of greenhouse gases [9]. Algae used for biodiesel production should be selected to grow in photoautotrophic mode to reduce the cultivation cost and to utilize CO2, as much as possible, for CO2 sequestration. The chemical composition of algae differs in different growth phases, in the exponential growth phase algae contain more protein, while in the stationary phase they have more lipid, carbohydrates and glycogen [10, 11]. Lipid content of algae varies noticeably among individual species or strains within and between taxonomic groups (Table 1). Table 1. Screening for Biomass productivity, lipid content and lipid productivity of 30

microalgal strains [12, 13].

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Many screening studies reported that green algae represent the largest taxonomic groups from which oleaginous candidates have been identified [11, 12, 13]. This may not be because of the higher lipid content of green algae than other algal taxa, but rather because many of green algae can easily be isolated from diverse habitats and grow faster than species from other taxonomic groups [11]. In contrast to higher plants, algae show greater variation in fatty acid composition. Some algae have the ability to synthesize medium-chain fatty acids (e.g., C8, C10, C12 and C14), whereas others produce long-chain fatty acids (> C20). For instance, a C10 fatty acid in the filamentous cyanobacterium Trichodesmium erythraeum comprising 27–50% of the total fatty acids [14]. On the other hand, the long chain fatty acid docosahexaenoic acid (C22:6n-3) comprising ~ 24% of total fatty acids was found in the dinophyte Gymnodinium sanguineum [15].

3. MICROALGAL BIOMASS PRODUCTION SYSTEMS Cultivation of microalgae can be done in open-culture systems (Figure 2) such as lakes or ponds or in highly controlled closed-culture systems called photobioreactors (PBRs, Figure 3).

Figure 2. Open cultivation system for growing algae a) diagram shows the constituents of the open pond, b) Picture of race-track style open-pond.


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Figure 3. Photobioreactor system for growing algae. a) diagram shows the constituents of a tubular photobioreactor, b) picture of tubular vertical photobioreactor, c) picture of flat plate photobioreactor.

Open-culture systems are normally cheaper, more durable than photobioreactors. However according to Richmond [16], ponds use more energy to homogenize nutrients and the water level cannot be kept much lower than 15 cm (or 150 L m-2) for the microalgae to receive enough solar energy to grow. Open ponds are more susceptive to weather conditions, not allowing control of water temperature, evaporation and lighting. Also, they may produce large quantities of microalgae, but occupy more extensive land area and are more susceptible to contaminations from other microalgae or bacteria [10]. On the other hand, PBRs are more controlled systems and can be optimized according to the biological and physiological characteristics of the cultivated algal species, allowing cultivation of algal species that cannot be grown in open ponds. PBRs are considered to have several advantages over open ponds; offer better control over culture conditions and growth parameters (pH, temperature, mixing, CO2 and O2), prevent evaporation, reduce CO2 losses, allow to attain higher microalgae densities or cell concentrations, higher volumetric productivities, offer a more safe and protected environment, preventing contamination or minimizing invasion by competing microorganisms [10]. Despite their advantages, PBRs suffer from several drawbacks that need to be considered and solved. Their main limitations include: overheating, bio-fouling, oxygen accumulation, difficulty in scaling up, the high cost of building and operating of algal biomass cultivation, cell damage by shear stress and deterioration of material used for the photo-stage [16].

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4. HARVEST AND EXTRACTION Algal biomass must be harvested and processed to release the products such as TAGs, which can then be transesterified to produce biodiesel. This step may contribute to 20–30% of the total biomass production cost [17]. There is no one universal harvesting method for harvesting microalgae. In order to remove large volumes of water and obtain large biomass, harvest method may involve one or more steps. The common harvesting methods include sedimentation, flocculation, centrifugation, filtration and ultra-filtration. The choice of suitable harvesting method depends on algal species, growth medium, desired end product, and production cost benefit [18]. Flocculation may precede the above mentioned harvesting methods [17] to increase the effective “particle” size and hence ease sedimentation, centrifugal recovery, and filtration [19]. Filtration under pressure or vacuum can be used to harvest large volumes of biomass, but for large scale production filtration can be relatively slow and consequently unsatisfactory [10]. In addition, filtration is better suited for large and filamentous microalgae such as Spirulina platensis but cannot recover smaller organisms such as Dunaliella and Chlorella [17]. Membrane microfiltration and ultra-filtration are other possible alternatives to conventional filtration for recovering algal biomass. These filtration processes are more suitable for fragile cells but can’t be used in large scale production as well and are more expensive. Richmond [16] reported that the desired product quality is one of the main criterions for selecting a proper harvesting procedure. Gravity sedimentation may be used for low value products which possibly enhanced by flocculation. On the other hand, centrifugation is recommended to be used for high value products such as recovery of high quality algae for food or aquaculture. Albeit the high cost of centrifugation, it is suitable to rapidly concentrate any type of microorganisms. Additionally, centrifuges can be easily cleaned or sterilized to effectively avoid bacterial contamination or fouling of raw product [10]. Another criterion for selecting a proper harvesting procedure is the potential of the harvesting method to adjust the density or the acceptable level of moisture in the resulting biomass [16]. Biomass recovered by gravity sedimentation contains water content much higher than centrifugally recovered biomass. Mata et al. [10] reported that the cost of thermal drying is much higher than those of mechanical dewatering such as filtration or centrifugation. In order to reduce the production cost of dewatering, combination of methods can be used, e.g. mechanical dewatering as a first step for pre-concentration and then post-concentration by centrifugation or thermal drying. However, dewatering must be quickly processed after separation of algal biomass to prevent the biomass to get spoiled. A key requirement during extraction is that the oil should be released and extracted without significant contamination by other cellular components, such as DNA or chlorophyll [20]. In addition, the lipid extraction process should be more selective towards biodiesel precursor acylglycerols than polar lipids and non-acylglycerol neutral lipids including free fatty acids, hydrocarbons, sterols, ketones, carotenes, and chlorophylls that are not convertible to biodiesel [21]. Moreover, the selected technology should be efficient (both in terms of time and energy), non-reactive with the lipids, relatively cheap (both in terms of capital cost and operating cost), and safe [22]. Extraction can be broadly categorized into two methods, mechanical methods which include expeller press and ultrasonic-assisted extraction, and chemical methods which include organic solvent extraction and supercritical fluid extraction.


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However, each of these methods has drawbacks; the mechanical press generally requires drying the algae, which is energy consuming, using of ultrasonic is an efficient method for small scale extraction, supercritical extraction requires high pressure equipment which is energy consuming and expensive. Although the use of chemical solvents present safety and health issues, many manufacturers of algae oil use chemical solvents in extracting oil. The principles underlying organic solvent extraction of microalgal lipids are anchored on the basic chemistry concept of ”like dissolving like”. According to Halim et al. [23], the proposed mechanism for organic solvent extraction is shown in Figure 4 and it takes place by the following steps: The first step takes place by penetration of non-polar organic solvent (such as hexane or chloroform) through the cell membrane to the cytoplasm of the algal cell. Then the second step comes by interaction of the organic solvent with the neutral lipids using similar van der Waals forces forming an organic solvent-lipids complex. This organic solvent–lipids complex, diffuses (according to the concentration gradient) across the cell membrane and the static organic solvent film surrounding the cell into the bulk organic solvent. Consequently, the neutral lipids are extracted out of the cells and remain dissolved in the non-polar organic solvent. The interaction between organic solvent and cell wall results in the static organic solvent film (Figure 4). This film surrounds the microalgal cell and remains undisturbed by any solvent flow or agitation. Some neutral lipids are, however, found in the cytoplasm as a complex with polar lipids linked via hydrogen bonds to proteins in the cell membrane. On the other hand, polar organic solvent (such as methanol or isopropanol) is able to disrupt the lipid–protein associations by forming hydrogen bonds with the polar lipids in the complex [21, 22]. The mechanism in which the non-polar/polar organic solvent mixture extracts membrane-associated lipid complexes (Figure 4) takes place by the following steps [23]:

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Figure 4. The proposed mechanism for organic solvent extraction. Pathway at the left part shows the mechanism for non-polar organic solvent. Pathway at the right part shows the mechanism for nonpolar/polar organic solvent mixture. neutral lipids, polar lipids, non-polar organic solvent and polar organic solvent [23].

The organic solvent (both non-polar and polar) penetrates through the cell membrane into the cytoplasm and interacts with the lipid complex. During this interaction, the non-polar organic solvent surrounds the lipid complex and forms van der Waals associations with the neutral lipids in the complex, while the polar organic solvent also surrounds the lipid complex and forms hydrogen bonds with the polar lipids in the complex. The hydrogen bonds are strong enough to displace the lipid–protein associations binding the lipid complex to the cell membrane. An organic solvent–lipids complex is formed and diffuses across the cell membrane and the static organic solvent film surrounding the cell into the bulk organic solvent.

5. CONVERSION OF ALGAE OIL TO BIODIESEL Biodiesel is a mixture of fatty acid methyl esters obtained by transesterification of vegetable oils or animal fats [10]. These lipid feedstocks are composed by 90–98% per weight of triglycerides and small amounts of monoglycerides, diglycerides, free fatty acids, and residual amounts of phospholipids, tocopherols, sulphur compounds, and traces of water [24]. Transesterification is a multiple step reaction, including three reversible steps in series, where triglycerides are converted to diglycerides, then diglycerides are converted to


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monoglycerides, and monoglycerides are then converted to esters (biodiesel) and glycerol (by-product). The overall transesterification reaction is described in Figure 5. For the transesterification reaction, oil or fat and a short chain alcohol (usually methanol) are used as reagents in the presence of a catalyst (like NaOH). Although the alcohol:oil theoretical molar ratio is 3:1, the molar ratio of 6:1 is generally used to complete the reaction accurately [10].

Figure 5. Transesterification of triacylglycerides extracted from algal oil for fatty acid methyl ester (biodiesel).

6. FOOD VERSUS FUEL Using of edible plants (mainly sugarcane, maize and oilseeds) for biofuel production has a domino effect, since it resulted in all grain prices to double. This leads to trickle through food chain and the price of all food should double soon and little grain will be available for food emergency aid [25]. Also, burning of grain for fuel to run luxury cars has bioethical issue when people are undernourished. Gressel [25] mentioned that more arable land than is available in the USA would be required for a 15% blend in fuels to use soybean or maize as a source of biofuel for US (Table 2). The use of food for fuel can replace a small proportion of the fossil fuel used, and thus cannot have any major effect on fuel prices, with a major effect on food and feed prices. Table 2. Cropping area needed to replace 15% of transport fuels in the USA [25].

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Stephenson et al. [26] studied the improvement of the production of biodiesel from rape seed and stated oil productivity of 1.4 t ha-1 y-1. Hill et al. [27] estimated oil productivity of soya by 0.48 t ha-1 y-1 and Uphama et al. [28] stated oil productivity of Jatropha by 2.4 t ha-1 y-1. In a recent study, we cultivated Scenedesmus obliquus in large scale to compare biodiesel production from crop seeds with S. obliquus. In our study, the annual productivity of oil from S. obliquus was calculated as 22 t ha-1 y-1 which is more than 800% higher than Jatropha. This makes microalgae to be the main source of biodiesel that has the potential to displace fossil diesel.

7. ADVANTAGES OF ALGAE FOR BIODIESEL PRODUCTION Many research reports mentioned the different advantages of using microalgae for biodiesel production in comparison with other available feedstocks [3, 10, 11, 29, 30, 31], we can conclude it in the following points: 1. Algae are easy to cultivate since they can grow with a little attention using waste or marine water which is unsuitable for human use [10]. 2. They have much higher growth rates and productivity when compared to other feedstocks, requiring much less land area [3]. 3. Different microalgae species can be adapted to live in harsh conditions. Thus, it is possible to find species best suited to local environments or specific growth conditions, which is not available with other current biodiesel feedstocks, e.g. rapeseed, sunflower and palm oil [10]. 4. Microalgae can produce different types of other renewable fuels such as methane, hydrogen and ethanol [32]. 5. Microalgae convert sun energy into chemical energy in a process of photosynthesis, completing an entire growth cycle every few days [29]. Moreover they can grow almost anywhere, although the growth rates and lipid content can be accelerated by the addition or removing of specific nutrients [33]. 6. The utilization of microalgae for biofuels production can also serve other purposes. Some possibilities currently being considered were described by Wang et al. [34] as follow  Removal of CO2 from industrial flue gases by algae bio-fixation, reducing the green house gases (GHG) emissions.  Wastewater treatment by removal of NH4+, NO3-, PO43-, making algae to grow using these water contaminants as nutrients.  After oil extraction the resulting algae biomass can be used as organic fertilizer due to its high N:P ratio, or simply burned for electricity and heat generation.  Depending on the microalgal species other valuable compounds may also be extracted, such as fats, polyunsaturated fatty acids, natural dyes, sugars, pigments, carotenoids, antioxidants, high-value bioactive compounds and other fine chemicals.


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For user acceptance, microalgal biodiesel should comply with existing standards. In the European Union, the standard EN 14214 is one of standards used for testing biodiesel intended for vehicle use [35]. One of those parameters used to test the quality of oil for biodiesel production is the degree of unsaturation [36]. In a recent study, we estimated the potential of S. obliquus oil as raw material for diesel fuel with regard to the fatty acid composition. We recorded that the portion of polyunsaturated fatty acids (PUFAs) decreased in favor of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) as the growth advanced. These results agreed with Mansour et al. [37] who reported an increase in the relative proportions of both SFA and MUFA 16:0 and 18:1, respectively, in the dinoflagellate Gymnodinium sp. at the cost of PUFAs. This increase was associated with growth phase transition from the exponential to the stationary phase. On the other hand, total unsaturation of oil is indicated by its iodine value. Iodine number measures the total unsaturation (double bonds) within the fatty acid methyl ester (FAME) product. It means the grams of iodine required to react with 100 g of FAME sample. Polymerization of fuels results in high iodine value, leading to injector fouling. It also leads to poor storage stability. Standard EN 14214 [35] requires the iodine value of biodiesel not exceeding 120 g iodine/100 g biodiesel. In our study, we recorded the iodine value of S. obliquus by 78 g iodine/100 g oil which meets the standard specifications [38]. Additionally, measurement of other parameters including density, viscosity, flash point, cold filter plugging point, ester content, cetane number and saponification value are important to detect the suitability of microalgae oil as a feedstock for biodiesel.

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