Microalgae as Feedstocks for Biodiesel Production

Fossil-based fuels including oil, coal and gas play a pivotal role in modern world energy market. These fossil fuels, according to world energy outlook 2007, will remain the major sources of energy and are expected to meet about 84% of energy demand in 2030. However, fossil fuels are non-renewable and will be finally diminished. It has been recently estimated that the global oil, coal and gas last only approximately for 35, 100 and 37 years respectively, based on a modified Klass model (Shafiee & Topal, 2009). In order to sustain a stable energy supply in the future, it is necessary to develop other sources of energy, e.g., renewable energy. Renewable energy is derived from natural processes that are replenished constantly, including hydropower, wind power, solar energy, geothermal energy, biodiesel, etc. An estimated $150 billion was invested in renewable energy worldwide in 2009, around 2.5 times of the 2006 investment (Figure 1). It is well known that transport is almost totally dependent on petroleum-based fuels, which will be depleted within 40 years. An alternative fuel to petrodiesel must be technically feasible, easily available, economically competitive, and environmentally acceptable (Demirbas, 2008). Biodiesel is such a candidate fuel for powering the transport vehicles. Biodiesel refers to a biomass-based diesel fuel consisting of long-chain alkyl (methyl, propyl or ethyl) esters. In addition to being comparable to petrodiesel in most technical aspects, biodiesel has the following distinct advantages over petrodiesel (Knothe, 2005a): 1. derived from renewable domestic resources, thus reducing dependence on and preserving petroleum; 2. biodegradable and reduced exhaust emissions, being environment-friendly; 3. higher flash point, being safer for handling and storage; and 4. excellent lubricity. Like petrodiesel, biodiesel operates in compression ignition engines. Biodiesel is miscible with petrodiesel in all ratios. Currently, the blends of biodiesel and petrodiesel instead of net biodiesel have been widely used in many countries and no engine modification is


Introduction
Fossil-based fuels including oil, coal and gas play a pivotal role in modern world energy market.These fossil fuels, according to world energy outlook 2007, will remain the major sources of energy and are expected to meet about 84% of energy demand in 2030.However, fossil fuels are non-renewable and will be finally diminished.It has been recently estimated that the global oil, coal and gas last only approximately for 35, 100 and 37 years respectively, based on a modified Klass model (Shafiee & Topal, 2009).In order to sustain a stable energy supply in the future, it is necessary to develop other sources of energy, e.g., renewable energy.Renewable energy is derived from natural processes that are replenished constantly, including hydropower, wind power, solar energy, geothermal energy, biodiesel, etc.An estimated $150 billion was invested in renewable energy worldwide in 2009, around 2.5 times of the 2006 investment (Figure 1).It is well known that transport is almost totally dependent on petroleum-based fuels, which will be depleted within 40 years.An alternative fuel to petrodiesel must be technically feasible, easily available, economically competitive, and environmentally acceptable (Demirbas, 2008).Biodiesel is such a candidate fuel for powering the transport vehicles.Biodiesel refers to a biomass-based diesel fuel consisting of long-chain alkyl (methyl, propyl or ethyl) esters.In addition to being comparable to petrodiesel in most technical aspects, biodiesel has the following distinct advantages over petrodiesel (Knothe, 2005a): 1. derived from renewable domestic resources, thus reducing dependence on and preserving petroleum; 2. biodegradable and reduced exhaust emissions, being environment-friendly; 3. higher flash point, being safer for handling and storage; and 4. excellent lubricity.Like petrodiesel, biodiesel operates in compression ignition engines.Biodiesel is miscible with petrodiesel in all ratios.Currently, the blends of biodiesel and petrodiesel instead of net biodiesel have been widely used in many countries and no engine modification is 134 required (Singhania et al., 2008).These blends of biodiesel with petrodiesel are usually denoted by acronyms, for example B20 which indicates a blend of 20% biodiesel with petrodiesel (Knothe, 2005a).The global markets for biodiesel are entering a period of rapid and transitional growth.In the year 2007, there were only 20 nations producing biodiesel for the needs of over 200 nations; by the year 2010, more than 200 nations become biodiesel producing nations and suppliers (Thurmond, 2008).Global biodiesel production has massively increased to 16.6 billion liters per year over the last nine years (Figure 2).Much of the growth is happening in just three countries: the United States, Brazil and Germany, which together account for over half of biodiesel (Checkbiotech, 2009).The International Energy Agency's report suggests that world production of biodiesel could top 25 million tons per year by 2012 if the recent trends continue.Biodiesel can be produced from a variety of feedstocks, including plant oils, animal fats and waste oils as well as microalgae (Demiras, 2008).Each feedstock has its advantages and disadvantages in terms of oil content, fatty acid composition, biomass yield and geographic distribution.Depending on the origin and quality of feedstocks, changes may be required for the production process of biodiesel.The use of plant oils as biodiesel feedstocks has been long recognized and well documented in numerous studies (Abdullah et al., 2009;de Oliveira et al., 2005;Graef et al., 2009;Hawash et al., 2009;Hill et al., 2006;Jain & Sharma, 2010;Nakpong & Wootthikanokkhan, 2010;Patil & Deng, 2009;Rashid & Anwar, 2008;Sahoo & Das, 2009;Saka & Kusdiana, 2001).These feedstocks include the oils from soybean, rapeseed, palm, canola, peanut, cottonseed, sunflower and safflower.Based on the geographic distribution, soybean is the primary source for biodiesel in USA, palm oil is used as a significant biodiesel feedstock in Malaysia and Indonesia, and rapeseed is the most common base oil used in Europe for biodiesel production (Demiras, 2008).The vast majority of these plants are also used for food and feed production, which means that possible food versus fuel conflicts are present.Thus, the use of these plant oils as feedstocks for biodiesel seems insignificant for the developing countries which are importers of edible oils (Meher et al., 2008).In addition to these edible oils, various non-edible, tree-borne oils from jatropha, karanja, jojoba and neem are the potential biodiesel feedstocks (Jain & Sharma, 2009;Meher et al., 2008;Sahoo & Das, 2009).Jatropha and karanja are two oilseed plants that are not widely exploited due to the presence of toxic components in the oils.In India, they are popularly used as biodiesel feedstocks.In addition to the plant oils, animal fats and waste oils are the potential sources for commercial biodiesel production (Thompson et al., 2010).Among these feedstocks, tallow, lard, yellow grease and waste cooking oils have received most interest (Banerjee et al., 2009;Canakci, 2007;da Cunha et al., 2009;Dias et al., 2009;Diaz-Felix et al., 2009;Oner & Altun, 2009;Phan & Phan, 2008).However, animal fats and waste oils usually contain large amounts of free fatty acids, which can be as high as 41.8% (Canakci, 2007).Free fatty acids cannot be directly converted to biodiesel in alkali-catalyzed transesterificatoin but react with alkali to form soaps that inhibit the separation of biodiesel from glycerin and wash water fraction (Huang et al., 2010).A two-step process was developed for these high fatty acid feedstocks: acid-catalyzed pretreatment and alkali-catalyzed transesterificaton.Because animal fats and waste oils have relatively high level of saturation (Canakci, 2007), the biodiesel from these sources exhibits poor cold flow properties.Microalgae represent a wide variety of aquatic photosynthetic organisms with the potential of producing high biomass and accumulating high level of oil.The production of biodiesel from microalgal oil has long been recognized and been evaluated in response to the United States Department of Energy for research in alternative renewable energy (Sheehan et al., 1998).Currently, the commercialization of algae-derived biodiesel is still in its infancy stage.Using microalgae as biodiesel feedstocks has received unprecedentedly increasing interest, including but not restricted to microalgal strain selection and genetic engineering, mass cultivation for biomass production, lipid extraction and analysis, transesterification technologies, fuel properties and engine tests (Abou-Shanab et al., 2011;Brennan & Owende, 2010;Demirbas, 2009;Greenwell et al., 2010;Miao & Wu, 2006;Pruvost et al., 2011;Radakovits et al., 2010;Rodolfi et al., 2009;Ross et al., 2008;Sydney et al., 2011).Considering their unique characteristics, microalgae have been considered as the most promising feedstock of biodiesel that has the potential to displace fossil diesel (Chisti, 2007).This review mainly focuses on the potential of using microalgae as biodiesel feedstocks, biodiesel production pipeline, and possibility of employing genetic engineering for improving microalgal productivity.

Potential of using microalgae as biodiesel feedstocks
Microalgae represent a large and diverse group of prokaryotic or eukaryotic photosynthetic microorganisms that are in unicellular or multicellular form.Examples of prokaryotic microorganisms are cyanobacteria (commonly referred to as blue-green algae) that are closely related to Gram-negative bacteria and eukaryotic ones are for example green microalgae and diatoms (Graham et al., 2009).Microalgae can be found in a wide range of environmental conditions, including water, land, and even unusual environments such as snow and desert soils (Lee, 2008).It is estimated that there are more than 50,000 species around the world, among which only about 30,000 have been studied and analyzed (Mata et al., 2010).Extensive collections of microalgae have been established by researchers in different countries, including the Freshwater Microalgae Collection of University of Coimbra (Portugal), the Collection of the Goettingen University (Germany), the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP, USA), the University of Texas Algal Culture Collection (USA), the CSIRO collection of Living Microalgae (CCLM, Australia), the National Institute for Environmental Studies Collection (NIES, Japan), the American Type Culture Collection (ATCC, USA), and the Freshwater Algae Culture Collection of Institute of Hydrobiology (China).Together more than 10,000 microalgal strains are available to be selected for use in a broad range of applications, for example, as biodiesel feedstocks.The use of microalgae for biodiesel production has long been recognized and its potential has been widely reported by many research studies recently (Abou-Shanab et al., 2011;Afify et al., 2010;Ahmad et al., 2011;Cheng et al., 2009;Damiani et al., 2010;Gouveia et al., 2009;Liu et al., 2010;Rodolfi et al., 2009;Yoo et al., 2010).Microalgae reproduce themselves autotrophically using CO 2 from air and light through photosynthesis.Compared with higher plants, microalgae exhibit higher photosynthetic efficiency and grow much faster, finishing an entire growth cycle within a few days (Christi, 2007).Typical growth rates are presented in Figure 3 as the doubling time for each microalgal species.A low doubling time corresponds to a high specific growth rate.Microalgae double themselves with an average time of 26 h, and some can even reproduce within 8 h.Moreover, they can be adapted to grow in a broad range of environmental conditions, suggesting the possibility of finding species best suited to local environments which is not suitable for cultivating oil plants (e.g.palm, soybean and rapeseed).In addition to growth rate, lipid content is another important factor to assess the potential of microalgae for biodiesel production.Over the past few decades, thousands of algae and cyanobacterial species have been screened for high lipid production, and numerous oleaginous species have been isolated and characterized.The lipid contents of these oleaginous algae are species-and/or strains-dependent, vary greatly, and may reach as high as 68% of dry weight, as shown in Table 1.Generally, microalgae synthesize a low content of lipids under nutrient replete conditions (Figure 4), with membrane lipids (e.g., phospholipids and glycolidips) being the main components; whereas under stress conditions such as nitrogen deficiency, a great increase in total lipids was observed (Figure 4) with neutral lipids in particular triacylglycerols (TAGs) being the dominant components (Hu, 2004).TAGs are considered to be superior to phospholipids or glycolipids for biodiesel feedstocks because of their higher percentage of fatty acids and lack of phosphate (Pruvost et al., 2009).Unlike higher plants in which individual classes of lipids may be synthesized and localized in a specific cell, tissue or organ, algae produce these different lipids in a single cell (Hu et al., 2008b).The synthesized TAGs are deposited in lipid bodies located in cytoplasm of algal cells (Damiani et al., 2010;Rabbani et al., 1998).The important properties of biodiesel such as cetane number, viscosity, cold flow, oxidative stability, are largely determined by the composition and structure of fatty acid esters which in turn are determined by the characteristics of fatty acids of biodiesel feedstocks, for exmaple carbon chain length and unsaturation degree (Knothe, 2005b).Fatty acids are either in saturated or unsaturated form, and the unsaturated fatty acids may vary in the number and position of double bones on the acyl chain.Based on the number of double bones, unsaturated fatty acids are clarified into monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs).The fatty acid profile of a great many algal species has been investigated and is shown in Table 2.The synthesized fatty acids in algae are commonly in medium length, ranging from 16 to 18 carbons, despite the great variation in fatty acid composition.Specifically, the major fatty acids are C16:0, C18:1 and C18:2 or C18:3 in green algae, C16:0 and C16:1 in diatoms and C16:0, C16:1, C18:1 and C18:2 in cyanobacteria.It is worthy to note that these data are obtained from algal species under specific conditions and vary greatly when algal cells are exposed to different environmental or nutritional conditions such as temperature, pH, light intensity, or nitrogen concentration (Guedes et al 2010;James et al., 2011;Sobczuk & Chisti, 2010;Tatsuzawa et al., 1996).Generally, saturated fatty esters possess high cetane number and superior oxidative stability; whereas unsaturated, especially  (Knothe, 2008).
In this regard, it is suggested that the modification of fatty esters, for example the enhanced proportion of oleic acid (C18:1) ester, can provide a compromise solution between oxidative stability and low-temperature properties and therefore promote the quality of biodiesel (Knothe, 2009).Thus, microalgae with high oleic acid are suitable for biodiesel production.
Currently the commercial production of biodiesel is mainly from plant oils and animal fats.However, the plant oil derived biodiesel cannot realistically meet the demand of transport fuels because large arable lands are required for cultivation of oil plants, as demonstrated in Table 3.Based on the oil yield of different plants, the cropping area needed is calculated and expressed as a percentage of the total U.S. cropping area.If soybean, the popular oil crop in United States is used for biodiesel production to meet the existing transport fuel need, 5.2 times of U.S. cropland will need to be employed.Even the high-yielding oil plant palm is planted as the biodiesel feedstock, more than 50% of current U.S. arable lands have to be occupied.The requirement of huge arable lands and the resulted conflicts between food and oil make the biodiesel from plant oils unrealistic to completely replace the petroleum derived diesel in the foreseeable future.It is another case, however, if microalgae are used to produce biodiesel.As compared with the conventional oil plants, microalgae possess significant advantages in biomass production and oil yield and therefore the biodiesel productivity.In terms of land use, microalgae need much less than oil plants, thus eliminating the competition with food for arable lands (Table 3).
In addition to biodiesel, microalgae can serve as sources of other renewable fuels such as biogas, bioethanol, bio-oil and syngas (Chisti, 2008;Demirbas, 2010;Mussgnug et al., 2010).Moreover, microalgal biomass contains significant amounts of proteins, carbohydrates and other high-value compounds that can be potentially used as feeds, foods and pharmaceuticals (Chisti, 2007).Thus, integrating the production of such co-products with biofuels will provide new insight into improving the production economics of microalgal biodiesel.Microalgae can also be used for sequestration of carbon dioxide from industrial flue gases and wastewater treatment by removal of nutrients (Chinnasamy et al 2010;Fulke et al., 2010;Levine et al., 2011;Yang et al., 2011).Coupled with these environment-beneficial approaches, the production potential of microalgae derived biodiesel is desirable.

Biodiesel production from microalgae
The biodiesel production from microalgal oil shares the same processes and technologies as those used for other feedstocks derived oils.However, microalgae are microorganisms living essentially in liquid environments and thus have particular cultivation, harvesting, and downstream processing techniques for efficient biodiesel production.The microalgal biodiesel production pipeline is schematically presented in Figure 5, including strain selection, mass culture, biomass harvesting and processing, oil extraction and transesterification.

Microalgae selection
There are more than 50,000 microalgal species around the world.Selection of an ideal species is of fundamental importance to the success of algal biodiesel production.
Theoretically, an ideal species should own the following desirable characteristics: rapid growth rate, high oil content, wide tolerance of environmental conditions, CO2 tolerance and uptake, large cell size, easy of disruption, etc.However, it is unlikely for a single species to excel in all above mentioned characteristics.Thus, prioritization is required.Commonly, fast-growing strains with high oil content are placed on the priority list for biodiesel production.Fast growth makes sure the high biomass productivity and reduces the contamination risk owing to out-competition of slower growers.High oil content helps increase the process yield coefficient and reduce the cost of downstream extraction and purification.The selected species should be suitable for mass cultivation under local geographic and climatic conditions, for example, the inland prefers freshwater algae while the coastal place desires marine algal species.Ease of harvesting is an often-overlooked criterion and should be taken into account.Algal biomass harvest requires significant capital and accounts for up to 30% of total biomass production cost (Molina Grima et al., 2003).Therefore, it is desirable to choose algal species with properties that simplify harvesting, including large cell size, high specific gravity and autofloculation potential (Griffiths & Harrison, 2009).These properties can greatly influence the process economics for biodiesel production from algae.An additional algal characteristic is the suitability of lipids for biodiesel production; for example, neutral lipids in particular TAG are superior to polar lipids (phospholipids and glycolipids) for biodiesel and C18:1 has advantages over other fatty acids for improving biodiesel quality (Knothe, 2009).

Microalgae cultivation 3.2.1 Factors affecting algal lipids and fatty acids
Microalgae require several things to grow, including a light source, carbon dioxide, water, and inorganic salts.The lipid content and fatty acid composition are species/strainspecific and can be greatly affected by a variety of medium nutrients and environmental factors.Carbon is the main component of algal biomass and accounts for ca 50% of dry weight.CO 2 is the common carbon source for algal growth.But some algal species are also able to utilize organic carbon sources, for example sugars and glycerol (Easterling et al., 2009;Liu et al., 2010).Sugars particularly glucose are preferred and can be used to boost production of both algal biomass and lipids (Liu et al., 2010).Nitrogen is an important nutrient affecting lipid metabolism in algae.The influence of nitrogen concentration on lipid and fatty acid production has been investigated in numerous algal species.Nitrate was suggested to be superior to other nitrogen sources such as urea and ammonium for algal lipid production (Li et al., 2008).Generally, low concentration of nitrogen in the medium favors the accumulation of lipids particularly TAGs and total fatty acids.But in some cases, nitrogen starvation caused decreased synthesis of lipids and fatty acids (Saha et al., 2003).Nitrogen concentration also affects algal fatty acid composition.For example, in cyanobacteria, increased levels of C16:0 and C18:1 and decreased C18:2 levels were observed in response to nitrogen deprivation (Piorreck & Pohl, 1984).In the marine alga Pavlova viridis, nitrogen depletion resulted in an increase in saturated, monounsaturated fatty acids and C22:6 (n-3) contents (Li et al., 2005).Nitrogen starvation brought about a strong increase in the proportion of C20:4 (n-6) in the green algal Parietochloris incisa (Solovchenko et al., 2008).Similar to nitrogen, silicon is a key nutrient that affects lipid metabolism of diatoms, and can promote the accumulation of neutral lipids as well as of saturated and monounsaturated fatty acids when depleted from culture medium (Roessler, 1988).Other types of nutrient deficiency include phosphorus and sulfur limitations are also able to enhance lipid accumulation in algae (Khozin-Goldberg & Cohen, 2006;Li et al., 2010b;Sato et al., 2000).These types of nutrient deficiency, however, do not always lead to elevated overall lipid production, because they at the same time exert negative effect on algal growth and contribute to the reduced algal biomass production that compromises the enhanced lipid yield resulting from increased lipid content.Therefore, the manipulation of these nutrients needs to be optimized to induce lipid accumulation while maintaining algal growth for maximal production of lipids.Iron is a micro-nutrient required in a tiny amount for ensuring algal growth.Within a certain range of concentrations, high concentrations of iron benefit algal growth as well as cellular lipid accumulation and thus the overall lipid yield in the green alga Chlorella vulgaris (Liu et al., 2008).Among the environmental factors, light is an important one that has a marked effect on the lipid production and fatty acid composition in algae (Brown et al., 1996;Damiani et al., 2010;Khotimchenko & Yakovleva, 2005;Napolitano, 1994;Sukenik et al., 1989;Zhekisheva et al., 2002Zhekisheva et al., , 2005)).Generally, low light intensity favors the formation of polar lipids such as the membrane lipids associated with the chloroplast; whereas high light intensity benefits the accumulation of neutral storage lipids in particular TAGs.In H. pluvialis, for example, high light intensity resulted in a great increase of both neutral and polar lipids, but the increase extent of neutral lipids was much greater than that of polar lipids, leading to the dominant proportion of neutral lipids in the total lipids (Zhekisheva et al., 2002(Zhekisheva et al., , 2005)).Although the effect of light intensity on fatty acid composition differs among the algal species and/or strains, there is a general trend that the increase of light intensity contributes to the enhanced proportions of saturated and monounsaturated fatty acids and the concurrently the reduced proportion of polyunsaturated fatty acids (Damiani et al., 2010;Sukenik et al., 1989;Zhekisheva et al., 2002Zhekisheva et al., , 2005)).Temperature is another important environmental factor that affects profiles of algal lipids and fatty acids.In response to temperature shift, algae commonly alter the physical properties and thermal responses of membrane lipids to maintain fluidity and function of membranes (Somerville, 1995).In general, increased temperature causes increased fatty acid saturation and at the same time decreased fatty acid unsaturation.For example, C14:0, C16:0, C18:0 and C18:2 increased and C18:3 (n-3), C18:4, C20:5 and C22:6 decreased in Rhodomonas sp., and C16:0 increased and C18:4 decreased in Cryptomonas sp. when temperature increased (Renaud et al., 2002).As for the effect of temperature on cellular lipid content, it differs in a species-dependent manner.In response to increased temperature, algae may show an increase (Boussiba et al., 1987), no significant change or even a decrease (Renaud et al., 2002) in lipid contents.Other environmental factors such as salinity, pH and dissolved O 2 are also important and able to affect algal lipid metabolism.
In addition to the nutritional and environmental factors, growth phase and aging of the culture affect algal lipids and fatty acids.Commonly, algae accumulate more lipids at stationary phase than at logarithmic phase (Bigogno et al., 2002;Mansour et al., 2003).Associated with the growth phase transition from logarithmic to stationary phase, increased proportions of C16:0 and C18:1 and decreased proportions of PUFAs are often observed.Besides, it is suggested that algal lipids and fatty acids can be greatly affected by cultivation modes.Algae growing under heterotrophic mode usually produce more lipids in particular TAG and higher proportion of C18:1 than under photoautotrophic mode (Liu et al., 2011).

Raceway ponds and photobioreactors
Currently, the commonly used culture systems for large-scale production of algal biomass are open ponds and enclosed photobioreactors.An open pond culture system usually consists of a series of raceways-type of ponds placed outdoors.In this system, the shallow pond is usually about one foot deep; algae are cultured under conditions identical to their natural environment.The pond is designed in a raceway configuration, in which a paddle wheel provides circulation and mixing of the algal cells and nutrients (Chisti, 2007).The raceways are typically made from poured concrete, or they are simply dug into the earth and lined with a plastic liner to prevent the ground from soaking up the liquid.Compared with photobioreactors, open ponds cost less to build and operate, and are more durable with a large production capacity.However, the open pond system has its intrinsic disadvantages including rapid water loss due to evaporation, contamination with unwanted algal species as well as organisms that feed on algae, and low biomass productivity.In addition, optimal culture conditions are difficult to maintain in open ponds and recovering the biomass from such a dilute culture is expensive.Unlike open ponds, enclosed photobioreactors are flexible systems that can be employed to overcome the problems of evaporation, contamination and low biomass productivity encountered in open ponds (Mata et al., 2010).These systems are made of transparent materials with a large surface area-to-volume ratio, and generally placed outdoors using natural light for illumination.The tubular photobioreactor is the most widely used one, which consists of an array of straight transparent tubes aligned with the sun's rays (Chisti, 2007).The tubes are generally no more than 10 cm in diameter to maximize sunlight penetration.The medium broth is circulated through a pump to the tubes, where it is exposed to light for photosynthesis, and then back to a reservoir.In some photobioreactors, the tubes are coiled to form what is known as a helical tubular photobioreactor.Artificial illumination can be used for photobioreactor.But it adds to the production cost and thus is used for the production of high value products instead of biodiesel feedstock.The algal biomass is prevented from settling by maintaining a highly turbulent flow within the reactor using either a mechanical pump or an airlift pump (Chisti, 2007).The result of photosynthesis will generate oxygen.The oxygen levels will accumulate in the closed photobioreactor and inhibit the growth of algae.Therefore, the culture must periodically be returned to a degassing zone, an area where the algal broth is bubbled with air to remove the excess oxygen.In addition, carbon dioxide must be fed into the system to provide carbon source and maintain culture pH for algal growth.Photobioreactors require cooling during daylight hours and temperature regulation in night hours.This may be done through heat exchangers located either in the tubes themselves or in the degassing column.
Table 4 shows the comparison between open ponds and photobioreactors for microalgae cultivation.
Photobioreactors have obvious advantages over open ponds: offer better control, prevent contamination and evaporation, reduce carbon dioxide losses and allow to achieve higher biomass productivities.However, enclosed photobioreactors cost high to build and operate and the scale-up is difficult, limiting the number of large-scale commercial systems operating globally to high-value production runs (Greenwell et al., 2010).In this context, a hybrid photobioreactor-open pond system is proposed: using photobioreactors to produce contaminant-free inoculants for large open ponds.

Biomass harvesting and concentration
Algal harvesting is the concentration of diluted algal suspension into a thick algal paste, with the aim of obtaining slurry with at least 2-7% algal suspension on dry matter basis.Biomass harvest is a very challenging process and may contribute to 20-30% of the total biomass production cost (Molina Grima et al., 2003).The most common harvesting methods include sedimentation, filtration, centrifugation, sometimes with a pre-step of flocculation or flocculation-flotation.Flocculation is employed to aggregate the microalgal cells into larger clumps to enhance the harvest efficiency by gravity sedimentation, filtration, or centrifugation (Molina Grima et al., 2003).The selection of a harvesting process for a particular strain depends on size and properties of the algal strain.The selected harvest method must be able to handle a large volume of algal culture broth.Filtration is the most commonly used method for harvesting algal biomass.The process can range from micro-strainers to pressure filtration and ultra-filtration systems.Vacuum filtration is feasible for harvesting large microalgae such as Coelastrum proboscideum and Spirulina platensis but unsuitable for recovering small size algal cells such as Scenedesmus, Dunaliella, or Chlorella (Molina Grima et al., 2003).Membrane-based microfiltration and ultrafiltration have also been used for harvesting algal cells for some specific application purposes, but overall, they are more expensive.Centrifugation is an accelerated sedimentation process for algae harvesting.Generally, centrifugation has high capital and operation costs, but its efficiency is much higher than natural sedimentation.Because of its high cost, centrifugation as an algae harvesting method is usually considered only feasible for high value products rather than biofuels.

Biomass processing for oil extraction
After harvesting, chemicals in the biomass may be subject to degradation induced by the process itself and also by internal enzyme in the algal cells.For example, lipase contained in the cells can rapidly hydrolyze cellular lipids into free fatty acids that are not suitable for biodiesel production.Therefore, the harvested biomass need be processed rapidly.Drying is a major step to keep the quality of the oil.In addition, the solvent-based oil extraction can be difficult when wet biomass is used.Various drying methods such as sun drying, spray drying, freeze drying, and drum drying can be used for drying algal biomass (Mata et al., 2010).Due to the high water content of algal biomass, sun-drying is not a very effective method for algal powder production.Spray drying and freeze drying are rapid and effective, but also expensive and not economically feasible for biofuel production.Because of the high energy required, drying is considered as one of the main economical bottlenecks in the entire process.
There are several approaches for extracting oil from the dry algal biomass, including solvent extraction, osmotic shock, ultrasonic extraction and supercritical CO 2 extraction.
Oil extraction from dried biomass can be performed in two steps, mechanical crushing followed by solvent extraction in which hexane is the main solvent used.For example, after the oil extraction using an expeller, the leftover pulp can be mixed with cyclohexane to extract the remaining oil.The oil dissolves in the cyclohexane and the pulp is filtered out from the solution.These two stages are able to extract more than 95% of the total oil present in the algae.Oil extraction from algal cells can also be facilitated by osmotic shock or ultrasonic treatment to break the cells.Osmotic shock is a sudden reduction in osmotic pressure causing cells to rupture and release cellular components including oil.The algae lacking the cell wall are suitable for this process.In the ultrasonic treatment, the collapsing cavitation bubbles near to the cell walls cause cell walls to break and release the oil into the solvent.Supercritical CO 2 is another way for efficient extraction of algal oil, but the high energy demand is a limitation for commercialization of this technology (Herrero et al., 2010).

Oil transesterification
Algal oil contained in algal cells can be converted into biodiesel through transesterification.
Transesterification is a chemical conversion process involving reacting triglycerides of vegetable oils or animal fats catalytically with a short-chain alcohol (typically methanol or ethanol) to form fatty acid esters and glycerol (Figure 6).This reaction occurs stepwise with the first conversion of triglycerides to diglycerides and then to monoglycerides and finally to glycerol.The complete transesterification of 1 mol of triglycerides requires 3 mol of alcohol, producing 1 mol of glycerol and 3 mol of fatty esters.Considering that the reaction is reversible, large excess of alcohol is used in industrial processes to ensure the direction of fatty acid esters.Methanol is the preferred alcohol for industrial use because of its low cost, although other alcohols like ethanol, propanol and butanol are also commonly used.In addition to heat, a catalyst is needed to facilitate the transesterification.The transesterification of triglycerides can be catalyzed by acids, alkalis or enzymes.Acid transesterification is considered suitable for the conversion of feedstocks with high free fatty acids but its reaction rate is low (Gerpen, 2005).In contrast, alkali-catalyzed transesterification has a much higher reaction rate, approximately 4000 times faster than the acid-catalyzed one (Fukuda et al., 2001).In this context, alkalis (sodium hydroxide and potassium hydroxide) are preferred as catalysts for industrial production of biodiesel.The use of lipases as transesterification catalysts has also attracted much attention as it produces high purity product and enables easy separation from the byproduct glycerol (Ranganathan et al., 2008).However, the cost of enzyme is still relatively high and remains a barrier for its industrial implementation.In addition, it has been proposed that biodiesel can be prepared from oil via transesterification with supercritical methanol (Demirbas, 2002).

Genetic engineering of microalgae 4.1 Microalgal lipid biosynthesis
Although lipid metabolism, in particular the biosynthesis of fatty acids and TAG, is poorly understood in algae, it is generally recognized that the basic pathways for fatty acid and TAG biosynthesis are similar to those demonstrated in higher plants.
Algae synthesize the de novo fatty acids in the chloroplast using a single set of enzymes.A simplified schedule for saturated fatty acid biosynthesis is shown in Figure 7. Acetyl-CoA is the basic building block of the acyl chain and serves as a substrate for acetyl CoA The fatty acid elongation is terminated when the acyl group is released from ACP by an acyl-ACP thioesterase that hydrolyzes the acyl ACP and produces free fatty acids or by acyltransferases that transfer the fatty acid from ACP to glycerol-3-phosphate or monoacylglycerol-3-phosphate.These released fatty acids serve as precursors for the synthesis of cellular membranes and neutral storage lipids like TAG.
It has been proposed that the biosynthesis of TAG occurs in cytosol via the direct glycerol pathway (Figure 8).Generally, acyl-CoAs sequentially react with the hydroxyl groups in glycerol-3-phosphate to form phosphatidic acid via lysophosphatidic acid.These two reactions are catalyzed by glycerol-3-phospate acyl transferase and lysophosphatidic acid acyl transferase respectively.Dephosphorylation of phosphatidic acid results in the release of DAG which accepts a third acyl from CoA to form TAG. This final step is catalyzed by diacylglycerol acyltransferase, an enzymatic reaction that is unique to TAG synthesis.In addition, an alternative pathway that is independent of acyl-CoA may also be present in algae for TAG biosynthesis (Dahlqvist et al., 2000).This pathway employs phospholipids as acyl donors and diacylglycerols as the acceptors and might be activated when algal cells are exposed to stress conditions, under which algae usually undergo rapid degradation of the photosynthetic membranes and concurrent accumulation of cytosolic TAG-enriched lipid bodies (Hu et al., 2008b).the quality of biodiesel (Knothe, 2008(Knothe, , 2009)).Oleic acid is converted to linoleic acid (C18:2) in a single desaturation step, catalyzed by a Δ12 desaturase enzyme encoded by the FAD2 gene.Inactivation of this desaturation step can greatly increase the proportion of oleic acid in soybean and may represent a possible strategy for elevated accumulation of oleic acid in algae.
Genetic engineering can also be used potentially to improve tolerance of algae to stress factors such as temperature, salinity and pH.These improved attributes will allow for the cost reduction in algal biomass production and be beneficial for growing selected algae under extreme conditions that limit the proliferation of invasive species.Photoinhibition is another technical challenge to be addressed by genetic engineering.When the light intensities exceed the value for maximum photosynthetic efficiency, algae show photoinhibition, a common phenomenon for phototrophy under which the growth rate slows down.Engineered algae with a higher threshold of light inhibition will significantly improve the economics of biodiesel production.
Engineering algae for biodiesel production is currently still in its infancy.Significant advances have only been achieved in the genetic manipulation of some model algae.It is likely that many of these advances can be extended to industrially important algal species in the future, making it possible to use modified algae as cell factories for commercial biodiesel production.Nevertheless, many challenges yet remain open and should be addressed before profitable algal biodiesel become possible.

Conclusion and perspectives
Microalgae have the potential for the production of profitable biodiesel that can eventually replace petroleum based fuel.Algal-biodiesel production, however, is still too expensive to be commercialized as no algal strains are available possessing all the advantages for achieving high yields of oil via the economical open pond culturing system.Current studies are still limited to the selection of ideal microalgal species, optimization of mass cultivation, biomass harvest and oil extraction processes, which contribute to high costs of biodiesel production from microalgae.Future cost-saving efforts for algal-biofuel production should focus on the production technology of oil-rich algae via enhancing algal biology (in terms of biomass yield and oil content) and culture-system engineering coupled with advanced genetic engineering strategies and utilization of wastes.In addition to oils, microalgae also contain large amounts of proteins, carbohydrates, and other nutrients or bioactive compounds that can be potentially used as feeds, foods and pharmaceuticals.Integrating the production of such co-products with biodiesel is an appealing way to lowering the cost of algal-biofuel production.

Acknowledgment
This work was supported by a grant from Seed-Funding Programme for Basic Research of the University of Hong Kong.

Table 1 .
Lipid content and productivity of various microalgal species. www.intechopen.com

Table 2 .
Fatty acid composition of various algal species (% of total fatty acids) polyunsaturated, fatty esters have improved low-temperature properties

Table 3 .
Comparison of microalgae with other biodiesel feedstocks.