Today, the need to ensure energy security is more urgent than ever. The manner in which energy is produced and consumed is of crucial importance to sustainable development, as energy has deep relationships with each of its three dimensions - the economy, the environment and social welfare (IEA 2009). The scale and breath of the energy challenge is enormous, far greater than many people realize, but it can and must be met. World energy consumption increases from 472 quadrillion Btu in 2006 to 552 quadrillion Btu in 2015 and 678 quadrillion Btu in 2030-a total increase of 44 percent over the projection period (EIA 2009).
Total proven oil reserves worldwide were estimated to be worth the equivalent of 40 years of consumable oil, based on a 1988 worldwide oil production rate of 64.2 million barrels per day. Proven oil reserves include residual oil in oil fields where production drilling has already begun. Projected oil reserves are slightly greater than proven oil reserves, but are not however infinite. The lack of stability of future energy supplies has motivated the development of alternative energy sources in order to eliminate the possibility of a future energy shortage (FAO 1997).
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Renewable energy increases diversity of energy supplies and can replace diminishing fossil fuel resources over the long run. Their use in place of fossil fuels can substantially reduce greenhouse gases and other pollutants (IEA 2009). To find clean and renewable energy sources ranks as one of the most challenging problems facing mankind in the medium to long term. The associated issues are intimately connected with economic development and prosperity, quality of life, global stability, and require from all stakeholders tough decisions and long-term strategies (Teresa et al. 2010).
Solar energy is renewable, whereas all other fuels including those of fossil and nuclear origin are limited in amount and are exhaustible. One efficient method of capturing solar energy is through the use of the photosynthetic process to produce biomass (a renewable raw material resource for the production of food, fuel and chemicals) through appropriate conversions (FAO 1997).
The Best Solution Of the Problem ----- ALGAE!
According to Khosla 2008, a good fuel should meet the CLAW requirements:
C - CARBON emissions
L - Low to no additional LAND use; benefits for using degraded land to restore biodiversity and organic material
A - AIR quality improvements- i.e., low carbon emissions
W - Limited WATER use.
Biofuels ---the renewable fuels made from biomass that can be used to supplement or replace the fossil fuels, including petroleum and diesel, used in transport, are made from renewable resources such as cellulose, corn, plant oils etc. But the obstacles in the production of biofuels are Competition with food and fiber production for use of arable land; cost; regional market structure; biomass transport; lack of well managed agricultural practices in emerging economies; water and fertilizer use; conservation of bio-diversity; logistics and distribution networks (IEA 2007).
It is believed that the majority of oil and natural gas originated from algae in ancient oceans. Oil (petroleum) consists of liquid hydrocarbons, which arc compounds composed of carbon and hydrogen. At least 80% w/w of oil is carbon. The remainder is principally hydrogen, but sulfur and oxygen may each account for up to 5% of the weight of oil. The burning heating volume of oil is relatively high owing to its liquid state, and is comparable to that of coal (FAO 1997). Thus, scientists are now trying to follow the nature's way again.
Microalgae -- the untapped resource with more than 25,000 species of which only 15 are in use. In recent years, microalgal culture technology is a business-oriented line owing to their different practical applications. In near future, algal biomass will serve as a renewable energy source (Raja et al., 2008). Microalgae can provide feedstock for several different types of renewable fuels such as biodiesel, methane, hydrogen, ethanol, among others. Algae biodiesel contains no sulfur and performs as well as petroleum diesel, while reducing emissions of particulate matter, CO, hydrocarbons, and SOx (Teresa et al. 2010). They contain lipids and fatty acids as membrane components, storage products, metabolites and sources of energy. Algal fatty acids and oils have a range of potential applications. Algal oils posses characteristics similar to those of fish and vegetable oils, and can thus be considered as potential substitutes for the products of fossil oil (FAO 1997).
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Algae thrive in the presence of sunlight, CO2, and water. They multiply quickly and can be harvested year-round ( Schenk et al, 2008). Unlike conventional biofuel feed stocks algae do not require vast and often valuable tracts of land and ample freshwater to grow, advocates say. Instead, algae can be grown on nonagricultural land in a fraction of the space and with brackish water or wastewater. In addition, algae are potentially far more productive than other leading oil crops such as palm, canola, and soy. Some companies are reporting that they can produce up to 6000 gallons of fuel per acre per year (gal/ac/yr) from algae, even though they're not yet operating on a large scale. In comparison, palm yields 650 gal/ac/yr; canola, 150 gal; and soy, 50 gal. And because algae consume CO2, algae companies plan to link up with power plants, cement factories, and other industrial plants to capture heat-trapping CO2 that would otherwise waft into the atmosphere. All these things conjointly made Amanda Mascarelli to believe that algae are the fuel of the future (Mascarelli, 2009).
Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensified focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous high value compounds (Rosenberg et al. 2008).
Some major advantages of using microalgae-derived biofuels are:
Micro-algae are capable of all year round production, therefore, oil productivity of microalgae cultures exceeds the yield of the best oilseed crops, e.g. biodiesel yield of 12,000 l ha_1 for microalgae (open pond production) compared with 1190 l ha_1 for rapeseed (Schenk et al. 2008).
They grow in aqueous media, but need less water than terrestrial crops therefore reducing the load on freshwater sources (Dismukes et al.2008).
Micro-algae can be cultivated in brackish water on non-arable land, and therefore may not incur land-use change, minimizing associated environmental impacts (Searchinger et al. 2008), with no compromise on the production of food, fodder and other products derived from crops (Chisti Y. 2007).
Micro-algae have a rapid growth potential and many species have oil content in the range of 20-50% dry weight of biomass, the exponential growth rates can double their biomass in periods as short as 3.5 h (Chisti 2007, Metting 1996, Spolaore et al. 2006 ).
With respect to air quality maintenance and improvement, microalgae biomass production can effect biofixation of waste CO2 (1 kg of dry algal biomass utilize about 1.83 kg of CO2) (Chisti 2007).
Nutrients for microalgae cultivation (especially nitrogen and phosphorus) can be obtained from wastewater, therefore, apart from providing growth medium, there is dual potential for treatment of organic effluent from the agri-food industry (Cantrell et al. 2008).
algae cultivation does not require herbicides or pesticides application (Rodolfi et al. 2008)
They can also produce valuable co-products such as proteins and residual biomass after oil extraction, which may be used as feed or fertilizer. (Spolaore et al.2006) And can also be fermented to produce ethanol or methane (Hirano et al. 1997).
The biochemical composition of the algal biomass can be modulated by varying growth conditions, therefore, the oil yield may be significantly be enhanced (Qin 2005).
Micro-algae are capable of photobiological roduction of 'biohydrogen' (Ghirardi et al.2000).
The attribute for potential biofuel production, CO2 fixation, biohydrogen production, and bio-treatment of wastewater accentuate the potential applications of microalgae.
Because of the variety of high-value biological derivatives, with many possible commercial applications, microalgae can potentially revolutionize a large number of biotechnology areas including biofuels, cosmetics, pharmaceuticals, nutrition and food additives, aquaculture, and pollution prevention (Rosenberg et al., 2008, Raja et al., 2008)
Despite the inherent potential of algae as a biofuel resource, many challenges have impeded the development of algal biofuel technology to commercial viability that could allow for sustainable production and utilization.
Species selection must balance requirements for biofuel production and extraction of valuable co-products (Ono and Cuello 2006).
Attaining higher photosynthetic efficiencies through the continued development of production systems (Pulz and Scheinbenbogan 1998).
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Development of techniques for single species cultivation, evaporation reduction, and CO2 diffusion losses (Ugwu et al,, 2008).
Potential for negative energy balance after accounting for requirements in water pumping, CO2 transfer, harvesting and extraction (Hirano et al. 1998).
Few commercial plants are in operation, therefore, there is a lack of data for large scale plants (Pulz 2001).
Incorporating flue gases which are unsuitable in high concentration owing to the presence of poisonous compounds such as NOx and SOx (Brown 1996).
Though it is not cost effective yet to compete with fossil diesel without additional support (for example government subsidies) research is being done to turn it economically viable, both in academia and in industry (Kanel and Guelcher 1999, Bijl et al. 2004, Yokochi et al. 2003). In a long term, as crude oil reserves diminish and price per barrel increases in a daily basis, other alternatives must become available, and thus, it is now the time to search, develop and implement those (Teresa et al. 2010).
Although the microalgae oil yield is strain-dependent it is generally much greater than other vegetable oil crops, as shown in Table 1 that compares the biodiesel production efficiencies and land use of microalgae and other vegetable oil crops, including the amount of oil content in a dry weight basis and the oil yield per hectare, per year (Teresa et al. 2010).
Table 1. Comparison of Microalgae with other biodiesel feed stocks:
Seed oil content
(% oil by weight in biomass)
(L oil/ha year)
(m2 year/kg biodiesel)
(kg biodiesel /ha year)
Corn / Maize (Zea maysL.)
Hemp (Cannabis sativa L.)
Soybean (Glycine max L.)
Jatropha (Jatropha curcasL.)
Camelina (Camelina sativa L.)
Canola/ Rapeseed (Brassica napus L.)
Sunflower (Helianthus annus L.)
Castor (Ricinus communis)
Palm oil(Elais guineensis)
Microalgae (low oil content)
Microalgae (medium oil content)
Microalgae (high oil content)
(Chisti 2007, Teixeira and Morales 2007, Kheira and Atta 2008, Callaway 2004, Hili and Feinberg 1984, Kulay and Silva 2005, Mobius Biofuels, LLC 2008, Nielsen 2008, Peterson and Hustrulid 1998, Rathbauer et al. 2002, Reijnders and Huijbregts 2008, Vollmann et al. 2007, Zappi et al. 2003).
The large-scale production of microalgae is a promising method of producing a renewable feedstock for a wide variety of fuel products currently refined from crude petroleum. These microalgae-derived products include lipid extraction products (triglycerides, fatty acids, and hydrocarbons) and catalytic conversion products (paraffins and olefins) (Hili and Feinberg 1984).
The opnion of Yousaf Chisti about algal fuel is that "microalgal biodiesel is the only renewable biodiesel that has the potential to completely displace liquid transport fuels derived from petroleum. Existing demand for liquid transport fuels could be met sustainably with biodiesel from microalgae, but not with bioethanol from sugarcane. Algal biomass needed for production of large quantities of biodiesel could be grown in photobioreactors, but a rigorous assessment of the economics of production is necessary to establish competitiveness with petroleum-derived fuels. Achieving the capacity to inexpensively produce biodiesel from microalgae is of strategic significance to an environmentally sustainable society. Extensive efforts are already underway to achieve commercial- scale production of microalgal oil, but for the moment barely any biodiesel is being made from microalgae (Chisti 2008).
Co-processes in microalgae production:
The combined production of renewable energy and material resources with unique environmental applications for GHG emission mitigation and wastewater treatment is one of the hallmarks of microalgal research (Reith et al. 2004).
The utilization of microalgae for biofuels production can also serve other purposes. Some possibilities currently being considered are listed below.
CO2 Biomitigation: The use of algae to capture carbon dioxide as a method for green house gas mitigation has been reported. A small fraction of the sunlight energy that bathes Earth is captured by photosynthesis and drives most living systems. Life on Earth is carbon based and the energy is used to fix atmospheric carbon dioxide into biological material (biomass), indeed fossil fuels that we consume today are a legacy of mostly algal photosynthesis. Algae can be thought of as marine and fresh water plants that have higher Photosynthetic efficiencies than terrestrial plants and are more efficient capturing carbon. They have other favorable characteristics for this purpose. (Mike Packer 2009). Mass cultures of microalgae have potential utilization in the production of biofuels and chemicals, food and feed, and for CO2 fixation and water purification (Wang et al. 2008, Kadam. 1997, Benemann et al. 2003).
Wastewater treatment by removal of NH4 +, NO3, PO4, making algae to grow using these water contaminants as nutrients (Wang et al. 2008) and also provide a pathway for the removal of chemical and organic contaminants, heavy metals and pathogens from wastewater while producing biomass for biofuel production. Photosynthetic oxygen from microalgae production reduces or eliminates the need for external mechanical aeration (Munoz and Guieysse 2006).
However, algal wastewater treatment plants have high land requirements for open pond systems and high capital costs for photobioreactor systems (Brennan and Owende 2009).
It was found that Spirulina sp. acted as a biosorbent, thus was able to absorb heavy metal ions (Cr3+, Cd2+, and Cu2+). Biosorption properties of microalgae depended strongly on cultivation conditions with photoautrophic species showing greater biosorption characteristics (Chojnacka et al. 2005).Chlorella vulgaris was grown in wastewater from a steel making plant with the aim of developing an economically feasible system to remove ammonia from wastewater and CO2 from flue gas (Yun et al. 1997).
After oil extraction the resulting algae biomass can be processed into ethanol, methane, livestock feed, used as organic fertilizer due to its high N:P ratio, or simply burned for energy cogeneration (electricity and heat) (Wang et al. 2008).
Depending on the microalgae species other compounds may also be extracted, with valuable applications in different industrial sectors, including a large range of fine chemicals and bulk products, such as fats, polyunsaturated fatty acids, oil, natural dyes, sugars, pigments, antioxidants, high-value bioactive compounds, and other fine chemicals and biomass (Li et al. 2008, Li et al 2008b, Raja et al 2008).
Growth requirements of Algae:
Algae are recognized as one of the oldest life forms ( Falkowski and Raven.1997). They are primitive plants (thallophytes), i.e. lacking roots, stems and leaves, have no sterile covering of cells around the reproductive cells and have chlorophyll a as their primary photosynthetic pigment ( Lee 2008). Algae structures are primarily for energy conversion without any development beyond cells, and their simple development allows them to adapt to prevailing environmental conditions and prosper in the long term (Falkowski and Raven 1997).
There are four distinct groups within the algae.
Prokaryotes. The cyanobacteria are the only prokaryotic algae.
Eukaryotic algae with chloroplasts surrounded by the two membranes of the chloroplast envelope.
Eukaryotic algae with the chloroplast surrounded by one membrane of chloroplast endoplasmic reticulum.
Eukaryotic algae with the chloroplast surrounded by two membranes of chloroplast endoplasmic reticulum.
Algae can either be autotrophic or heterotrophic; the former require only inorganic compounds such as CO2, salts and a light energy source for growth; while the latter are non photosynthetic therefore require an external source of organic compounds as well as nutrients as an energy source. Some photosynthetic algae are mixotrophic, i.e. they have the ability to both perform photosynthesis and acquire exogenous organic nutrients (Lee 2008).
The basic requirements for microalgal phototrophic growth include carbon dioxide, light, as well as other macro- and micro-nutrients. Carbon source can be obtained from power plants, which release large 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. (see the Figure below) (Huang et al. 2010)
Figure: Open Pond Photosynthetic system OPPS (Huang et al. 2010)
Following are the important growth requirements for algae:
Under natural growth conditions phototrophic algae absorb sunlight, and assimilate carbon dioxide from the air and nutrients from the aquatic habitats. Therefore, as far as possible, artificial production should attempt to replicate and enhance the optimum natural growth conditions (Brennan and Owende 2009). The use of natural conditions for commercial algae production has the advantage of using sunlight as a free natural resource ( anssen et al. 2003)
However, this may be limited by available sunlight due to diurnal cycles and the seasonal variations; thereby limiting the viability of commercial production to areas with high solar radiation. For outdoor algae production systems, light is generally the limiting factor (Pulz et al. 1998)
2. Carbon dioxide:
Microalgae can fix CO2 efficiently from different sources, including the atmosphere, industrial exhaust gases, and soluble carbonate salts. Combination of CO2 fixation, biofuel production, and wastewater treatment may provide a very promising alternative to current CO2 mitigation strategies.
Microalgae can fix carbon dioxide from different sources, which can be categorized as (1) CO2 from the atmosphere,
CO2 from industrial exhaust gases (e.g., flue gas and Microalga Cultivation N/P-Rich Wastewater flaring gas), and
Fixed CO2 in the form of soluble carbonates (e.g., NaHCO3 and Na2CO3).
The merit of CO2 bio-mitigation locates primarily in the fact that biomass produced in the process of CO2 fixation can be converted efficiently into biofuels for energy production (Wang et al. 2008).
Under natural growth conditions, microalgae assimilate CO2 from the air (contains 360 ppmv CO2). Most microalgae can tolerate and utilize substantially higher levels of CO2, typically up to 150,000 ppm. (Bilanovic et al. 2009, Chiu et al. 2009). Therefore, in common production units, CO2 is fed into the algae growth media either from external sources such as power plants. (Brown.1996, Hsueh et al. 2007, Vunjak-Novakovic et al. 2005, Doucha et al. 2005, Kadam 2002). Carbon can also be supplied in the form of soluble carbonates such as Na2CO3 and NaHCO3. (Emma Huertas et al. 2000, Colman and Rotatore 1995)
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. (Chisti 2007)
Other inorganic nutrients required for algae production include nitrogen, phosphorus and silicon. (Suh and Lee 2003). While some algae species can fix nitrogen from the air in the form of NOx (David et al. 2000, Moreno et al. 2003). Most microalgae require it in a soluble form with urea being the best source .(Hsieh and WuW-T. 2009).
Phosphorus is of lesser importance and is required in very small amounts during algal growth cycle.(Celekli et al. 2009) but, Phosphorous must be supplied in excess of basic requirement because phosphates ions bond with metals ions, therefore, not all the added P is bioavailable ( Chisti 2007).
Importance of silicon is confined to productive growth of certain groups of algae such as diatoms (Martin-JeÂ´zeÂ´quel et al. 2000).
Microalgae like other plant-based biofuel resources provide the mechanism for collection, conversion and storage of solar energy into chemical form. For biofuel production, the major factors cited as determining economically viable production include: productivity (viz., strain selection, photosynthetic efficiency, and productivity of lipids), production and harvesting costs (Borowitzka 1992).
Selection, Screening, and Culturing of Algae:
The selection of appropriate algae strains is an important factor in the overall success of biofuel production from microalgae
The algal strain should be selected on the basis of the following criteria:
Have high lipid productivity.
Be robust and able to survive the shear stresses common in photo-bioreactors.
Be able to dominate wild strains in open pond production systems.
Have high CO2 sinking capacity.
Have limited nutrient requirements.
Be tolerant to a wide range in temperatures resulting from the diurnal cycle and seasonal variations.
Provide valuable co-products.
Have a fast productivity cycle.
Have a high PE (Photosynthetic efficiency).
Display self flocculation characteristics.
(Rosenberg, et al. 2008, Sheehan et al. 1998, Bruton et al. 2009).
At the moment, no known algal strain is capable of meeting all these requirements concurrently (Teresa et al. 2010). Actually, site specific adaptation is the key to commercial microalgae production. This allows the algae to be exposed to the prevailing environmental conditions, which is a distinct advantage over imported strains (Sheehan et al. 1998).
While many microalgae strains naturally have high lipid content (ca. 20-50% dry weight), it is possible to increase the concentration by optimizing the growth determining factors (Hu, et al. 2008)..
Lipid content can be increases by the control of nitrogen level (Widjaja et al. 2009, .Weldy and Huesemann 2007, Wu W-T and Hsieh. 2008). It also varies with light intensity (Qin. 2005, Weldy and Huesemann 2007). Temperature and salinity are also known for effecting the lipids concentration in the cells (Qin 2005, Wu W-T and Hsieh 2008).
CO2 concentration an important factor as well that affects the amount of lipids (Chiu et al. 2009, de Morais and Costa 2007). The harvesting procedure is also reported to effect final lipid content (Chiu et al. 2009, .Widjaja et al. 2009). However, increasing lipid accumulation will not result in increased lipid productivity as biomass productivity and lipid accumulation are not necessarily correlated (Rodolfi et al. 2008, Sheehan et al. 1998).
Initial research focused on the isolation of high lipid content microalgae that could be cultivated in large-scale open pond cultivation for biodiesel production (Weissman and Tillett 1992, Laws et al. 1986, Benemann et al. 1977, Weissman et al. 1988, Weissman et al. 1988b).
And also can be combined with capturing CO2 from coal-fired power plants as biological emission control process
The primary findings of the above research were:
Increment in oil accumulation in algal cells due to nitrogen-deficiency is inversely proportional to oil productivity of entire cultures due to lower total productivity resulting from lower nutrient levels.
Open pond production is most appropriate for large-scale microalgae production due to low costs.
Maintenance of uncontaminated mono-specific microalgae cultures in open ponds for sustainable high production is exceedingly difficult (Brown and, Zeiler 1993, Kadam and. Power 1997, Chelf et al. 1991).
The most effective method of improving microalgae lipid accumulation is nitrogen limitation, which not only results in the accumulation of lipids, but also results in a gradual change of lipid composition from free fatty acids to triacylglycerol (TAG) (Widjaja et al. 2009). TAGs are more useful for conversion to biodiesel (Meng et al. 2009). Because of this variety of high-value biological derivatives, with many possible commercial applications, microalgae can potentially revolutionize a large number of biotechnology areas including biofuels, cosmetics, pharmaceuticals, nutrition and food additives, aquaculture, and pollution prevention (Rosenberg et al. 2008, Raja et al. 2008).
Recovery of microalgal biomass
The recovery of microalgal biomass generally requires one or more solid-liquid separation steps is a challenging phase of the algal biomass production process (Wang et al. 2008), and accounts for 20-30% of the total costs of production according to one source (Gudin and Therpenier 1986).
The processes involved include flocculation, filtration, flotation, and centrifugal sedimentation; some of which are highly energy intensive. Low cell densities (typically in the range of 0.3-5 g l_1) when there is limited light penetration, and the small size of some algal cells (typically in the range of 2-40 mm), make the recovery of biomass difficul (Li et al. 2008).The selection of harvesting technology is crucial to economic production of microalgal biomass (Schenk 2008).
A factor such as strain selection is an important consideration since certain species are\ much easier to harvest. For example, the cyanobacterium Spirulina's long spiral shape (20-100 mm long) naturally lends itself to the relatively cost-efficient and energy-efficient microscreen harvesting method (Benemann and Oswald 1996).
Lipid classes in Microalgae:
Polyunsaturated fatty acids content of algae depend not only on the species, but also on factors related to culture conditions: the composition of the medium, the aeration rate, light intensity, duration of the photoperiod, temperature and the age of culture.The algae produce a great variety of fatty acids and lipids. The main fatty acids are saturates and the cis isomers of unsaturates, with 12 to 22 carbon and up to six double bonds. Extraction of lipids from algae requires attention to their polarity. Polarity is related to distribution of lipids within the algal cell and association of lipid to non-lipid components (Medina et al 1998). Thus, lipids present in algae can be classified into neutral and polar that are subdivided as noted in Table 2. PUFAs may be present as triglycerides, phospho- and glycolipids. Triglycerides are usually regarded as energy storage products, whereas phospholipids and glycolipids are structural lipids contained in the cell walls.
Table 2. Classification of Lipids present in Algae:
Free fatty acids
(Pohl and Zurheide 1982).
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:
the formation of acetyl coenzyme A (acetyl-coA) in the cytoplasm;
the elongation and desaturation of carbon chain of fatty acids; and
the biosynthesis of triglycerides in microalgae.
(Huang et al. 2010)
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 was described by Yang et al.in 2000
It was concluded that glyceraldehydes 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 (Huang et al. 2010).
It was 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 (Akazawa and Okamoto 1980).
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. 2), 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 thioester 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 (Stumpf 1984).
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. 3). Long-chain fatty acids (C20-C22) often exist in microalgae and the content varies from species to species (Meireles et al. 2003).
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 (Huang et al. 2010).
Figure 2. Reaction process of the FFA biological synthesis system (Shen and Wang 1989).
Figure 3. The elongation and desaturation of carbon chain of fatty acids (Guschina and Harwood 2006)
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. 4. One of the hydroxyl in L-a-phosphoglycerol reacts with 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 (Huang et al. 2010).
Figure 4: The biosynthesis of triglycerides in microalgae (Huang et al. 2010).
EXTRACTION OF OIL FROM ALGAE:
Pretreatment of the samples
Lipids from microorganisms may generally be extracted in the wet state directly after harvesting. The cells do not need to be homogenized since they are readily broken by suspending in the extracting solvent. In some cases, cell breakage may be necessary to allow better penetration of the solvent into the cell and increase the lipid yield. This may be accomplished using one of the established cell-disruption techniques such as sonication, homogenization, freezing and grinding (Medina et al 1998).
a. mechanical shearing in a bead mill: For large-scale cell disruption of algae, mechanical shearing in a bead mill appears most suitable. Bead mills have been used to disintegrate the algae Scenedesmmus obliquus and Spirulina platens'is (Hedenskog and Ebbinghaus 1972).
b. Ultrasonication is another potentially useful disruption method. Ultrasound at high acoustic intensities is known to disrupt microbial cells in suspension (Chisti and Moo-Young 1986). Dunstan et al. extracted lipids from green algae (Chlorophyceae and Prasinophyceae) with chloroform-methanol-water (1:2:0.8 by vol) mixture and between each extraction the samples were sonicated at 20Â°C (Dunstan et al. 1992).
c. Extraction from freeze-dried samples has also been used. Freeze-drying breaks up the cells and turns the algal material into a loose, fine powder; making homogenization unnecessary (Ahlgren, and Merino 1991).
However, direct extraction of fatty acids from wet P. tricornuturn biomass (after harvesting by centrifugation) with 96% ethanol produced only slightly lower yields (90%) than those obtained from lyophilized biomass (96%); therefore cost of extraction may be reduced by omitting lyophilization (Molina Grima et al 1996).
Extraction of lipids and/or fatty acids
Extraction methods should be fast, efficient and gentle in order to reduce degradation of the lipids or fatty acids. The extraction solvents should be inexpensive, volatile (for ready removal later), flee from toxic or reactive impurities (to avoid reaction with the tipids), able to form a two-phase system with water (to remove non-lipids), and be poor extractors of unwanted components (e.g., proteolipids, small molecules). Effectiveness of solvents for the different classes of lipids should also be considered. For complete extraction, all the linkages between the lipids and other non-lipid cell components must be broken and, at the same time, the disruption agents used must not degrade the lipids (Medina et al 1998).
There are three main types of associations in which lipids participate:
Hydrophobic or van der Waals interactions, in which neutral or non-polar lipids, such as glycefides, are bound by relatively weak forces through their hydrocarbon chains to other lipids and to hydrophobic regions of proteins.
Hydrogen bonding and electrostatic association by which polar lipids are bound to proteins and
Covalent association, although this type of interaction is less frequent. The energy of the weak hydrophobic interactions which link the stored lipids never exceeds 2 kcal/mole, so they may be disrupted by non-polar organic solvents, such as chloroform, hexane or ether (Chuecas and Riley 1969).
Hydrogen bonds of membrane-associated polar lipids have energy of 0.2-12 kcal/mole and may be disrupted only by polar organic solvents, such as methanol, ethanol and other alcohols, and also by water (a solvent having a high dielectric constant). To extract lipids linked by stronger electrostatic forces such as ionic bonds, it is necessary to shift the pH value somewhat toward the acidic or alkaline region (Kates1988, Zhukov and Vereshchagin 1981).
Lipids are retained in living matter also by mechanical confinement. For example, the poor permeability of the cell walls to solvents hinders extraction. In such cases adding a small amount of water to the extractant may increase the lipid yield. Water causes swelling of the cellular structures rich in polysaccharides, thereby facilitating access of extracting solvents to the lipids. Thus the presence of water in the extractant is absolutely necessary for quantitative extraction of polar lipids (Zhukov and Vereshchagin 1981)
Some biological materials contain enzymes that degrade lipids during extraction. Typically, alcohol-containing solvent mixtures inactivate many of the lipid-degrading phosphatidases and lipases (Kates. 1988, Zhukov and Vereshchagin 1981).
Alcohol also aids disruption of lipid-protein complexes and dissolution of the lipids. However, the alcoholic solvents also extract some cellular contaminants such as sugars, amino acids, salts, hydrophobic proteins and pigments. Therefore, crude alcoholic extract must be treated to remove these water-soluble contaminants (Kates 1988).
Separation of lipid mixtures, for example, the crude lipid extract from the microalga Spirulina was reported to contain 4.34% of the original dry weight while the purified lipid extract contained only 3.62%. (Ahlgren and Merino 1991).
Methods of purifying crude lipid extracts are usually based on differences in affinity of the polar lipids and the contaminants for a certain solvent. The crude extract may be treated with polar solvents such as chloroform (Folch et al 1957, Bligh and Dyer1959, Kates 1988).
Hexane is also effective for the purification of lipids (Molina Grima et al. 1994)., or diethyl ether, in which the non-lipid contaminants are less soluble. These procedures do not completely extract most polar lipids (e.g., proteolipids) because of their low solubility in these solvents (Medina et al. 1998).
Ibhfiez Gonzalez et al. carded out the purification of the fatty acid extract from Phaeodactylum tricornumm biomass in a three steps operation:
extraction of unsaponifiable lipids from the hydroalcoholic extract of fatty acid (as sodium salts) with hexane;
acidification to pH 1 to form the flee fatty acid; and
extraction of fatty acids from the hydroalcoholic solution with hexane (Ibhfiez Gonz,ilez et al. 1997).
Other less used purification methods are molecular adsorption on silicic acid or alumina, and the use of ion-exchange and gelfiltration (Zhukov and Vereshchagin 1981). None of these methods guarantee degradation-free recovery of completely pure lipids.The chemical nature of the lipid must also be taken into consideration in designing a recovery scheme to prevent oxidation (Medina et al 1998).
Following are some methods which are considered effective:
a. An efficient extraction system for microorganisms is 2:1 (v/v) mixture of chloroform and methanol (Folch et al.1957, Kochert 1978).
b. In a simplified and improved procedure, Bligh and Dyer used a single-phase extractant, chloroform-methanol-water (1:2:0.8 by volume, including the water present in the sample) for extraction of lipids from fresh tissues. The single phase extraction mixture was diluted with chloroform and water to yield a biphasic system (2:2:1.8 ratio of chloroform-methanol-water) chosen such that the lower layer was almost pure chloroform and contained the purified lipids; the upper layer was nearly all methanolwater and contained the non-lipids. The extraction was simple, fast and gentle (room temperature). Lipid extraction was complete and the separation of lipids and non-lipids was nearly quantitative (Bligh and Dyer 1959). The method is applicable to a variety of materials (animal or plant tissue, and microorganisms), but with microorganisms (including microalgae), homogenization is not necessary and the extraction takes at most two hours at room temperature (Kates 1988). This procedure is routinely used for lipid extraction from microalgae. Among disadvantages of the procedure are the toxicity and flammability of the solvents (chloroform, methanol) (Hara and Radin 1978)
c. Hexane-isopropanol (3:2 v/v) is a low-toxicity solvent. Lipid extraction with this system followed by a wash of the extract with aqueous sodium sulfate to remove the non-lipid contaminants has some advantages over the Bligh and Dyer system. The extract contains less non-lipid material; interference in processing by proteolipid protein contamination is avoided; the two phases separate rapidly during the washing step; the solvent density is low enough to permit centrifugation of the homogenate as an alternative to filtration; the solvents are cheaper; the solvents can be eliminated by vacuum evaporation to dryness; and the washed extract can be directly applied to a chromatographic column with UV detection. However, the hexane-isopropanol system gave substantially lower lipid yields when used with microalgae (Ahlgren and Merino1991, Molina Grima et al. 1994).
d. Butanol and ethanol are also low-toxicity solvents that are relatively cheap, sufficiently volatile and only slightly carcinogenic, but lipid extracts needs to be further purified, for example, by a chloroform-methanol-water phase separation. Nagle and Lemke compared the extraction efficiency of these solvents, the hexane-isopropanol (2:3 v/v), and the Bligh and Dyer system (as control). Test were performed on the diatom Chaetoceros muelleri and the alga Monoraphidium minutum. With the former, the most effective system was butanol (90% of the yield obtained with the chloroform-methanolwater 1:2:0.8 by vol); the yield with hexane-isopropanol (2:3 v/v) was 78%, and with ethanol the yield was 73%. Recovery of lipids from lyophilized/, galbana biomass using the various solvent systems is detailed in Table below (Molina Grima et al. 1994).
e. The hexane: ethanol (96%) (1:2.5 v/v) single-phase extractant was used, first, to extract lipids from I. galbcma lyophilized biomass and then to purify them by adding hexane and water to form a biphasic system in a proportion chosen in such a manner that the lower layer was practically 100% hexane and contained the purified lipids and the upper layer, nearly all ethanol-water, contained the non-lipids. Although a high lipid yield (79.6%) was obtained with this system, high proportion of lipids (27.4%) remained in the ethanol-water phase together with the non-lipid contaminants (Table 3), implying that a greater consumption of hexane is required to extract them. As shown in Table 7 the lipid yield increased with increasing alcohol content or alcohol polarity in the extraction solvent mixture (Nagle and Lemke 1990).
Table 3. Yields (%) of extracts and raffinates (in brackets) obtained by lipid extraction from Isochrysis galhana
(1:2:0.8 by volume)
Ethanol 96 %
Ethanol 96 %- water
Î£ n3 PUFAs
Î£ Fatty acids
76 ml of solvent system per gram of lyophilized biomass, 1h, room temperature (Molina Grima et al. 1994).