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Rajkumar, R., Yaakob, Z., and Takriff, M. S. (2014). "Potential of the micro and macro algae for biofuel production: A brief review," BioRes. 9(1), 1606-1633.

Abstract

The world seems to be raising its energy needs owing to an expanding population and people’s desire for higher living standards. Diversification biofuel sources have become an important energy issue in recent times. Among the various resources, algal biomass has received much attention in the recent years due to its relatively high growth rate, its vast potential to reduce greenhouse gas (GHG) emissions and climate change, and their ability to store high amounts of lipids and carbohydrates. These versatile organisms can also be used for the production of biofuel. In this review, sustainability and the viability of algae as an up-coming biofuel feedstock have been discussed. Additionally, this review offers an overview of the status of biofuel production through algal biomass and progress made so far in this area.


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Potential of the Micro and Macro Algae for Biofuel Production: A Brief Review

Renganathan Rajkumar,* Zahira Yaakob, and Mohd Sobri Takriff

The world seems to be raising its energy needs owing to an expanding population and people’s desire for higher living standards. Diversification biofuel sources have become an important energy issue in recent times. Among the various resources, algal biomass has received much attention in the recent years due to its relatively high growth rate, its vast potential to reduce greenhouse gas (GHG) emissions and climate change, and their ability to store high amounts of lipids and carbohydrates. These versatile organisms can also be used for the production of biofuel. In this review, sustainability and the viability of algae as an up-coming biofuel feedstock have been discussed. Additionally, this review offers an overview of the status of biofuel production through algal biomass and progress made so far in this area.

KeywordsMicroalgae; Macroalgae; Biomass; Lipid; Biofuel; Oil production; Bioconversion; AlgacultureWastewater treatment; Malaysia

Contact information: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, University Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia;

* Corresponding author: micro_rajkumar@yahoo.co.in

INTRODUCTION

The energy requirements of the global community are rising year by year. Currently, fossil fuels are a prominent source of transportation fuels and energy. The world’s demand for oil is expected to rise 60% from the current level by 2025 (Khan et al. 2009). In view of the increasing oil demand and the depleting oil reserves, development of innovative techniques for the production of biofuels from novel renewable biomass feedstock sources are gaining importance all over the world. Production of biofuels from traditional agricultural crops such as corn, oil palms, and soybeans using arable lands and fresh water will greatly impact food production.

Biomass, whether terrestrial or aquatic, is considered a renewable energy source. Relative to alternative energy sources, the aquatic biomass represents the strategy that is most ready to be executed on a large scale without any economic or environmental penalty (Aresta etal. 2005). Among these, algae are endowed with a unique adaptability to grow in diverse habitats, either in marine or fresh waters (IEA Report 1994). In the past, research mainly focused on their usage as food, animal feed, bio-fertilizer, and in aquaculture.

Algae have received a great deal of attention as a novel biomass source for the generation of renewable energy. Apart from other biomass sources, algae contains a high biomass yield per unit of light and area, can have a lot of starch or oil content, does not require fresh water or agricultural land, and the requirements for nutrients can be fulfilled by either wastewater or seawater. Algae produce an array of organic molecules, particularly carbohydrates and lipids. These biomolecules can be used to extract a fuel known as biofuel. Algae are both unicellular and multicellular autotrophic aquatic life forms.

Microalgae can provide several different kinds of renewable biofuel. These include methane produced by the anaerobic digestion of the algal biomass (Spolaore et al. 2006), biodiesel synthesized from the micro algal oil (Thomas 2006), and biohydrogen produced by a photobiological mechanism (Gavrilescu and Chisti 2005). The idea of producing microalgal biofuel is not a new one (Kapdan and Kargi 2006), but it is now being viewed seriously in view of the increasing price of petroleum. Serious interest is also motivated by concern about global warming that is associated with the use of fossil fuels (Sawayama et al. 1995).

Macroalgae are generally fast growing and are able to reach sizes up to 60 m in length (McHugh 2003). Growth rates of macroalgae far exceed those of terrestrial plants. For example, brown algae biomass of the average productivity was approximately 3.3 to 11.3 kg dry weight m−2 yr−1 for non-cultured algae and up to 13.1 kg dry weight m−2 over 7 month for cultured algae compared with 6.1 to 9.5 kg fresh weight m−2 yr−1 for sugar cane, a most productive land plant (Kraan 2010). They are seasonally available in the natural water basins. Cultivation of macroalgae at sea, which does not require arable land and fertilizer, offers a possible solution to the energy crisis. Macroalgae are mainly utilized for the production of food and the extraction of hydrocolloids, and it is possible to produce ethanol from algae (Goh and Lee 2010). Macroalgal biomass contains high amounts of sugars (at least 50%), which can be used in ethanol fuel production (Wi et al. 2009).

This review explores the opportunities for energy products, encompassing both fresh and marine habitat macro- and microalgae. This paper also discusses the variety of algal resources and their environment, along with the manufacture systems that have been demonstrated for use, as well as algal mass cultivation.

BACKGROUND

History and the Prospects of Research for Algal Biofuel Production

Biofuel production and the environment have been crucial issues in today’s world. Several researchers have described the need for biofuels and the kinds of materials that can serve this purpose (Naika et al. 2010; Antoni et al. 2007). Based on productivity per unit area, algae constitute one of the most effective raw materials that could be exploited for the biofuel production. Algal biomass is capable of producing a host of end products including energy, chemicals, food, cosmetics, fertilizer, and agents for wastewater treatment and/or CO2sequestration. This could reduce production costs, since there would be a variety of products to serve as sources of revenue, as the cost-effectiveness of these is crucial for the economic and commercial viability of these algal products.

Algal biomass can be used as raw material for biofuel production viapyrolysis (bio-oil), or for bio-gas and bio-ethanol generation through fermentation. Macro and micro algae for bioenergy production should satisfy several criteria as listed below (Carlsson et al. 2007): i) they should be highly productive; ii) they should be easily harvestable; iii) they should be able to withstand water currents in the open ocean; and iv) they should be produced at a cost that is equal or lower than the other available sources. Scientific research has been started on the utilization of the various species of algae in waste water/seawater treatment in order to transform them into biofuels by means of various technological processes ranging from the esterification to anaerobic digestion (Kraan 2010).

Macroalgae

Macroalgae constitute the most important component in the marine ecosystems that serve for the marine bioresources preservation by preventing eutrophication and pollution (Notoya 2010). Macroalgae belong to the lower plants, in that they do not have roots, stems, and leaves. Instead, they are composed of a thallus (leaf-like) and sometimes a stem and a foot. Some species enclose gas-filled structures to help in buoyancy. They can grow very fast and in sizes of up to tens of meters in length (Luning and Pang 2003). Macroalgae differ in various aspects, such as morphology, longevity, and ecophysiology. Based on their pigmentation, they are classified into Phaeophyta (brown), Rhodophyta (red), and Chlorophyta (green) algae (Chan et al. 2006). In their natural environment, macro-algae grow on rocky substrates and form stable, multi-layered, perennial vegetation, capturing almost all available photons. Approximately 200 species of macroalgae are used worldwide, about ten of which are intensively cultivated, such as the Phaeophyta, Laminaria japonica and Undaria pinnatifida, the Rhodophyta, EucheumaGracilaria, Porphyra and Kappaphycus, and the Chlorophyta, Enteromorpha and Monostroma (Luning and Pang 2003). Figure 1 shows examples of some commercially exploited macroalgae.

Fig. 1. Some commercially exploited macroalgae A) Gracilaria dura; B) Acanthophora spicifera; C) Hypnea esperi; D) Padina pavonica

The world production of macroalgae reached 8 million tons in 2003 (McHugh 2003). Many countries have now embarked on establishing large scale macroalgae cultivation in their territories. Recent research (www.unbsj.ca/sase/biology/chopinlab) has shown the potential of macroalgae for large-scale culture in the Atlantic waters of Canada, France (Kaas 2006), Germany (Buck and Buchholz 2004), Ireland (Kraan et al. 2000), Isle of Man, UK (Kain et al. 1990), and Spain (Peteiro and Freire 2009). In Asian countries such as China, India, Philippines, South and North Korea, Indonesia, and Japan, macroalgae is being cultivated for various needs such as food, feed, chemicals, cosmetics, and pharmaceutical products (Carlsson et al.2007).

Importance of Macroalgal Biomass

With substantial processing required for fossil fuels and the higher cost of vegetable oils, there has been a great deal of interest in the algal culture. Apart from that, algal biofuel production presents the following advantages:

1. Production of biofuel from the macroalgae cultivation in seawater is a new approach, since 70% of the earth’s surface is covered by water. Macroalgae possess a unique life cycle. They are more productive in view of the fact that more than five harvests can be made in a year.

2. In addition, macroalgae can succeed in salty water with only sunlight and available nutrients from the seawater. They do not need any chemical fertilizer. Thus, large amounts of energy and money could be saved. These characteristic features favor the sustainability of the production of macroalgae-based bioethanol.

3. Production of bioethanol from terrestrial plants leaves a large impact on the environment in general and on human beings in particular due to eutrophication, acidification, and ecotoxicity. This is mostly caused by agricultural practices by the generation of waste water (Luo et al. 2009).

4. In general, macroalgae can live in a variety of environmental conditions. There is a wide range of organisms that grow along the coastal areas. With the advancement of genetic engineering, it is now possible to develop a suitable species of macroalgae for bioethanol production (Goh and Lee 2010). Genetically engineered macroalgae would need to be cultivated in enclosed bioreactors. These characters bring about high confidence for future improvement of macroalgae in renewable energy area such as bioethanol.

5. Converting the macroalgal biomass to ethanol rather than using terrestrial plant biomass have some important benefits, i.e., no negative impact on the food security. The relatively high sugar content and lower lignin content than lignocelluloses facilitates high mass production (Adams et al. 2009; Wi et al. 2009)

6. Algal biomass can be cultivated in the unused vast ocean of the coastal area within the limited economic zone. In fact, utilization of sea water for the algal biomass production has great potential to relieve the water crisis. As for the ecology, macroalgae supplies oxygen to the sea and helps reduce the accumulation of carbon dioxide in the atmosphere (Goh and Lee 2010).

7. Several algal species are known for their ability to remove heavy metals from water, which can be useful to the environment (Aderhold et al. 1996). Certain algal species have the ability to produce high amounts of carbohydrates instead of lipids as preserved polymers. These are the ideal candidates for bioethanol production, as carbohydrates from algae can be extracted and then converted to fermentable sugars.

8. Apart from bioethanol production, algal biomass can be used for the production of an enormous variety of supplementary products i.e., protein, pigments, plastics, etc. (Reith et al. 2005; Wijffels 2009). In addition to replacing fossil fuels, thereby mitigating climate alteration, algal biomass can also serve in the recycling of heavy nutrients in the near and inshore waters (Kraan 2010).

9. Macroalgae provides a promising bioethanol feedstock owing to their high biomass yield with a superior production relative to various terrestrial crops (John et al. 2011).

In light of the considerations just mentioned, there is a need to develop large-sized culture areas in the open sea (off-shore) for the resources of biofuel production. In this context, the biofuel from the macro algae offers an excellent alternative to the currently used fossil fuels. Thus, the cultivation and engineering of the macro algae have drawn the world’s attention in view of their value as a substitute for the conventional fossil fuels which are fast becoming depleted.

Composition and Processing of Macroalgal Biomass

Macroalgal biomass has a great potential both in quantity as well as in quality for the production of variety of specific bioenergy components. Previous studies (Reith et al. 2005) have shown that growing macroalgae can be efficient and feasible if the production processes of the bioenergy and bio-based products are combined. Certain products from the algal industry have long been used for the production of various products, i.e., agars, alginates, and carrageenans (McHugh 2003). These polymers are storage materials located either in the cell walls or within the cells.

In general, chlorophyll and b are the major pigments in green macroalgae. Starch is the photosynthetic product in green algae, the cell walls of which are primarily made up of pectin and cellulose (Trono Jr. and Ganzon-Fortes 1988). The r-phycoerythrin is a major pigment in red macroalgae, the cell walls of which have minimum amounts of cellulose, while the maximum is gelatinous or amorphous sulfated galactan polymers i.e., funoran, agar, carrageenan, etc. Brown macroalgal colouration is related to the high amount of the xanthophyll pigments, particularly alginic acid and fucoxanthin (Ganzon-Fortes 1991), which are present along with cellulose and the other polysaccharides. The carbohydrates laminarin and mannitol are the food reserve materials that are particularly suitable for the production of ethanol (Davis et al. 2003). The content of carbohydrates in macroalgae varies widely among species and cultivar, and species selection can lead to evolution of strains having extremely high amounts of carbohydrate that can be utilized as an inventive bioethanol feedstock. The carbohydrate contents of some macroalgae are given in Table 1.

Some species of macroalgae gather a high amount of carbohydrates that are capable in the processes of microbial conversion as substrate, i.e., production of biofuels or the other desirable and attractive chemicals with high product price (Kraan 2010). Recently, Maceiras et al. (2011) discovered that triglycerides from a number of macroalgae such as Ascophyllum nodosum, Codium tomentosumEnteromorpha intestinalisFucus spiralisSaccorhiza polyschidesSargassum muticumUlva rigida, and Pelvetia canaliculataetc. could be used to produce biodiesel by a transesterification process. Horn et al. (2000) and Ross et al. (2008) reported that the water content in the macroalgae is higher than that in the terrestrial plants (80 to 85%), making macroalgae more suited for microbial conversion than for the thermochemical conversion or direct combustion processes. Macroalgae such as Sargassum spp.Gracilaria spp., Prymnesium parvumGelidium amansii, and Laminaria spp. are promising candidates for bioethanol production (Wi et al. 2009; Adams et al. 2009). The red alga, Gelidium J.V. Lamouroux has been prepared for the production of paper in which the waste products have been renewed into bioethanol (Seo et al.2010). Also, green algae of Ulva spp., with a high-grade polysaccharide, Ulvan (Lahaye and Ray 1996) have been used in the production of ethanol and methane (Adams et al. 2009). Meinita et al.(2011) have recently reported bioethanol production from Kappaphycus alvarezii. Similarly, Karunakaran and Gurusamy (2011) also reported bioethanol production from Eucheuma and Hypnea. In mid-2008, studies were initiated for the production of bioethanol and biofertilizer from K. alvarezii on a laboratory scale by an integrated 2-product strategy (Mody et al. 2009). Benjamin (1993) in his US patent explained the utilization of genetically transformed marine green macro algal (Enteromorpha) cells for the bioethanol production. Uchida and Murata (2004) described lactic acid and ethanol fermentation using various types of green, brown, and red algae. In addition, Adams et al. (2009) reported the effect of enzymatic pretreatment for their bioethanol production in a brown alga, Saccharina latissima (Laminaria saccharina).

Table 1. Carbohydrate Contents of Macroalgae (Dhargalkar and Pereira 2005)

In fact, only limited information has become available on the effectiveness of these processes with macroalgal carbohydrates (Horn et al. 2000), even though some breakthroughs have been newly made with respect to ethanol production from the brown macroalgae (Adams et al. 2009). Red algae produce high amounts of bioethanol-producing carbohydrates. Although macroalgae can look similar to the land plants, these organisms in fact, do not have the same lignin crosslinking molecules in their structures as cellulosic terrestrial plants because they grow in the water surroundings where they are able to grow erect despite their lack of lignin crosslinking (John et al.2011). Although macroalgae have low amounts of lignin, they have significant amounts of sugars that could be used in the fermentation process for the production of bioethanol (Wi et al. 2009). Nevertheless, in certain algae such as marine red algae, the content of carbohydrate is influenced by the occurrence of agar, a polymer of galactose and galactopyranose. Recent research has sought to improve methods of saccharification to release galactose from the agar and glucose from cellulose to produce ethanol through fermentation (Wi et al. 2009). The potential of macroalgae for ethanol production can be estimated based on the following postulations: a content carbohydrate 60 % of dry weight and a 90 % of conversion levels to ethanol through fermentation of 1 g of sugar can yield 0.4 g of ethanol. It will ideally give up 0.22 kg or 0.27 L ethanol from 1 kg dry weight of macroalgal biomass, equivalent to roughly 0.05 L ethanol per kg of wet weight (Kraan 2010).

Algae are also a potential source of commercial biogas products, such as biohydrogen and biomethane that can be used as gas fuels or for electricity generation (Mussgnug et al. 2010). Hydrogen produced by macroalgae is a popular attraction in the renewable energy scenario. Current research has revealed that Laminaria japonica (brown alga) and Gelidium amansii (red alga) are both potential biomass sources for the production of biohydrogen by anaerobic fermentation (Park et al. 2011). Macroalgae can produce biohydrogen under specific conditions. Ongoing discussions on the prospects of hydrogen production by algae have been well-documented (Prince and Kheshgi 2005; Rupprecht et al. 2006).

In a study of the feasibility of methane production from macroalgal biomass, case scenarios assumed yields of 11 dry t ha-1 y-1 based on data from commercial growers (Chynoweth 2002). Research to determine the technical and economic feasibility of biomethane production from marine biomass was conducted from 1968 until 1990 under the sponsorship of the U.S. Navy, the American Gas Association and Gas Research Institute, and the U.S. Department of Energy; such work was reviewed by Chynoweth (2002). The study compared the technical potential of different biomass sources (marine algae, wood and grass species, and municipal solid waste) to be used in the energy farms and concluded that the marine biomass offered the highest advantage. Marine algae, such as Gracilaria sp. and Macrocystis are excellent substrates for biomethane generation (Bird et al. 1990). The view expressed by many authors is that the best approach to biomethane production from macroalgae is the multipurpose use of algal biomass, for example gas evolution from the digestion of the residues from hydrocolloid extraction (Kerner et al. 1991). In this way, the coproduction of methane with the other products could bring down the production costs and could make biomethane production profitable. Methane, ethanol, and biohydrogen production from different macroalgal biomass sources are presented in Table 2.

Table 2. Different Strains of Macroalgae for Biofuel Production

From an economic point of view it is not viable to produce biofuels from macroalgae with the current technology, except if the production process is combined with another, such as pollutant removal or the production of bio-based products (Savage 2011; Pittman et al. 2011). It is estimated that macroalgae phycocolloids represent a world market of some US$ 600 Mio y-1 (Mc Hugh 2003). Using macroalgae as feedstock will add a new sector of this area for various commercial applications. However, production rates and costs are critical to the economic and commercial success of algal products; these issues are less commonly studied. Thus, more efforts are required to understand these processes with a view to identifying the potential of such macroalgae components for its application.

Microalgae

In general, microalgae are photosynthetic microorganisms that are found in both marine and freshwater habitats. Microalgae have been classified based on various characteristics such as pigmentation, photosynthetic storage product, the arrangements of photosynthetic membranes, and other morphological features. At present, microalgae species are divided into four groups, namely diatoms (Bacillariophyceae), green algae (Chlorophyceae), blue green algae (Cyanophyceae), and golden algae (Chrysophyceae) (Khan et al.2009). The dominating species of microalgae in commercial production include IsochrysisChaetocerosChlorellaArthrospira (Spirulina), and Dunaliella (Lee 1997). Chlorella species are capable of changing from phototrophic to heterotrophic modes of nutrition among microalgal species (Xiong et al. 2008; Xu et al. 2006). As heterotrophs, the algae rely on glucose or other utilizable carbon sources for carbon metabolism and energy. Some algae can also grow mixotrophically. The biomolecules such as carbohydrates, proteins, lipids, and nucleic acids are the common constituents in microalgae (Williams and Laurens 2010).

Importance of microalgal biomass

Research and commercial applications of microalgae have gained interest during the last few years. Owing to their rapid growth rate, i.e., 100 times faster than the land-based plants which can double their biomass in less than 1 day, microalgae appear to be an attractive renewable energy source (Tredici 2010). This is mostly due to their simple cellular system and big surface to quantity ratio that gave them the facility to utilize more amounts of nutrients from the source of water and hence, supporting their algae growth rate (Khan et al.2009). Many strains of microalgae are known to produce high quantities of lipids that can be converted into biodiesel. Biofuel production using microalgal farming offers the following advantages (Ahmad et al. 2011):

1. Increased efficiency or decrease in the cost. The sum of harvesting and transportation of microalgae costs can be relatively low compared to those of the other plant biomass resources. On the other hand, the production cycle does not directly affect the human food chain supply system, avoiding the food against fuel conflict.

2. Microalgae do not give any competition for land-based plants used for food production, fodder, and other value-added products (Huang et al. 2010).

3. Generally, microalgae can grow in fresh, brackish, or salt water environments or non-arable lands that are incompatible for growing other crops and conventional agriculture (Patil et al. 2008). In addition, they can be grown in photo-bioreactors (Janaun and Ellis 2010). For this reason – the nonselective growth – microalgae produce a greater yield per hectare with superior environmental attributes.

4. The most common microalgae contain oil ranges between 20 and 50% by dry weight of biomass, but superior productivities can be attained (Mata et al. 2010). Commonly, microalgae double their biomass within 24 h, but the exponential growth content can result in a doubling of their biomass in periods as short as 3.5 h (Chisti 2007).

5. Microalgae are able to produce various valuable supplementary products i.e., carbohydrates, proteins, biopolymers, and residual biomass, and these can be used for feed or fertilizer purposes. In addition, herbicides or pesticides are not required for the cultivation of microalgae (Rodolfi et al. 2008).

6. Consideration of microalgae as an efficient cellular system for harvesting solar energy for production of various organic compounds (Vonshak 1990).

7. Microalgae are able to fix carbon dioxide in the atmosphere, assisting the reduction of atmospheric carbon dioxide levels, which is recently considered a global crisis. In addition, production of microalgal biomass can affect the biofixation of waste carbon dioxide, reducing the releases of a major greenhouse gas (1 kg of dry microalgal biomass requires about 1.8 kg of carbon dioxide) (Chisti 2007; Rodolfi et al. 2008).

8. Microalgal lipids are typically neutral lipids. Owing to their high degree of saturation and fast accumulation in the cellular system at various stages of microalgal growth, lipids remain as a prospective replacement for diesel fuel (Danquah et al. 2009).

Besides lipid extraction, some microalgae (blue green) (which produce glycogen instead of starch) can produce biohydrogen under anaerobic conditions (Hankamer et al. 2007; Melis et al. 2000), and their fermentation can also be used for the production of methane. By extracting more than one type of microalgal biofuel as value-added products, the value of the biomass is increased while contributing additional offsets to the ecological impacts. As stated above, the combined biorefinery perception can be used to enhance ethanol substance from the algae (Danquah et al. 2009). This model can also be exploited in combination with the production of biohydrogen and biogas either by giving a valuable product before the techniques of fermentation or by using the products of gaseous fermentation to power the progress of producing those high value entities such as methane, biodiesel, and bio-hydrogen. Exploitation of micro-algae for combined applications of biofuels production is under research (Li et al. 2011; Chisti 2007).

Composition and processing of microalgal biomass to biofuel

Current research enterprises have shown that microalgal biomass appears to be one of the promising sources of renewable biodiesel, which is capable of facing the global demand. Oil content from the microalgal biomass can exceed 80% by dry weight (Rodolfi et al.2008). Oil ranges of 20 to 50% are quite common (Chisti 2007) (Table 3).

Oil production yield can be defined as the mass of oil produced per unit volume of broth of microalgal culture per day based on the growth rate of microalgae and the oil content of the biomass. Several species of algae produce high content of lipids as storage materials, as high as 50 to 60% of their dry weight. These lipid systems are chemically analogous to other crop-derived oil-seed lipids, rendering algae a promising source of biodiesel production (Griffiths and Harrison 2009). Various techniques have been applied for more efficient lipid extraction from microalgae. Most common methods are expeller/ oil press, ultrasound techniques, solvent extraction, and supercritical fluid extraction. The preferred characteristics of the extraction methods are that the process should be fast, non-damaging and effective to lipids extracted, and scaled up easily (Medina et al.1998). Extraction of lipid by using a modified Bligh and Dyer (Bligh and Dyer 1959) method is widely used (Mutanda et al. 2011). Extraction of microalgal fatty acids by using direct esterification, simultaneous extraction, and transesterification can be performed on various types of biomass, making it a versatile method for biofuels production. This is a multistep process and it needs a mixture of solvent extraction, ultrasonication, heating at high pressure, filtration, density separation of solvent, and liquids and oil recovery by the process of evaporation to dryness (Belarbi et al. 2000). Table 4 shows the advantages and limitations of various lipid extraction methods for microalgae oil.

Table 3. Oil Content of Microalgae by Chisti (2007) and Rodolfi et al. (2008)

Table 4. Advantages and Limitations of Various Lipid Extraction Methods for Microalgae Oil (Harun et al. 2010c).

These extraction methods are still on a laboratory scale and none of them has been demonstrated to be practical and economical for commercial production (Chen et al. 2009). Currently, most of the lipid extraction methods are facing many problems with high costs coupled with water removal and difficulties with disrupting the algal cellular system to make lipids efficiently accessible.

In spite of the high productivity, biodiesel from microalgae still has not yet become economical; algal biodiesel has been priced at US $1.25/lb, whereas petroleum-based diesel has been priced at US $0.43/lb (Li et al. 2011). The expenditure for the algae-derived biodiesel is proportional to the algal species-specific efficiency to carbon dioxide sequestration as lipids. Hence, microalgal prospecting would greatly impact the upcoming efficiencies and thus help reduce the production cost of algal biodiesel (Griffiths and Harrison 2009). Bioprospectors look for potential strains that are not only large lipid producers, but also show abundant growth and harvesting uniqueness (Mutanda et al. 2011). Several diatoms also have been examined for their lipid production, including Amphora (De la Pena 2007), Chaetoceros calcitrans (Rodolfi et al. 2008), Cyclotella cryptica (Sheehan et al. 1998), some species of Nitzschia (Griffiths and Harrison 2009), Phaeodactylum tricornutum (Ceron Garcia et al.2000), Thalassiosira pseudonana (Rodolfi et al. 2008), Chaetoceros gracilis, and Tetraselmis tetrathele (Araujo et al. 2011). Microalgae also have been studied for bioethanol production. Table 5 presents the bioethanol yield from various strains of microalgae.

Table 5. Bioethanol Production from Various Strains of Microalgae

The green algae Chlorococum spp. and Spirogyra spp. have been revealed to accumulate high contents of polysaccharides together in their complex cell walls and as starch. This accumulation of starch can be used in the bioethanol production (Harun et al. 2010a; Eshaq et al. 2011). Harun et al. (2010a) have stated that the green algae Chlorococum sp. produces 60% higher ethanol from samples that are pre-extracted for lipids against those that stay as desiccated undamaged cells. This implies that microalgae can be utilized for the production of both lipid biofuels and for ethanol biofuels from the similar microalgal biomass as a way to boost their overall economic value. Bioethanol has the prospect of being an alternative fuel, but it is highly important to ensure that the expansion of this fuel is not hindered by the raw material constraints (Harun et al. 2010b). In this context, the harvesting cycle of microalgae cells has a very short period (1 to 10 days) compared with the other feedstock (harvesting time once or twice per year), and thus can provide enough supplies to meet demands for the ethanol production (Schenk et al. 2008). Additionally, algae have the photon conversion ability and can synthesize and accumulate large amounts of carbohydrate biomass for the production of bioethanol from the cheapest source of raw materials (Subhadra and Edwards 2010). Hon-Nami (2006) has described the fermentation of Chlamydomonas perigranulata to produce ethanol, butanediol, acetic acid, and CO2, thus showing the multiutility of the algal biomass. Interestingly, Harun et al. (2010a) showed that the lipid-extracted micro-algae yielded 60% higher ethanol than that from the dried/intact microalgae, thus implying the significance of using the spent biomass for ethanol production.

Anaerobic fermentation

In recent times, microalgae have also become a subject of interest in the biogas production by the process of anaerobic fermentation. Biogas production is considered to lead to a net reduction for the emissions of greenhouse gas; this is because methane would otherwise be released into the atmosphere (Fredriksson et al. 2006). For this purpose, anaerobic digestion is an initial process that can solve the waste biomass issue as well as the energetic and economical balance of such a successful technology for algal biofuel production (Sialve et al. 2009). Chisti (2008) mentioned the energy recovery from the microalgal residues after biodiesel production, highlighting its importance to meet the current energy demands of the preceding processes. He also theoretically calculated that an average heating value of 9360 MJ/metric tons of microalgal residues was recoverable as methane. Furthermore, co-digesting the microalgal residues with a glycerol co-product obtained during the transesterification of algal oils in quantities equivalent to those produced was observed to increase the CH4 yields by 5 to 8% when compared to the digestion of the residues alone (Ehimen et al. 2008). Blue-green algae are also able to produce biohydrogen via an anaerobic process involving the oxidation of ferredoxin by the hydrogenase enzyme activity (Yacoby et al. 2011). Nevertheless, hydrogenases are directly involved with other metabolic processes for the detachment of electrons, and not all functions of hydrogenases activities alike. Thus, a significant volume of the recent research on microalgal photobiohydrogen production has been aimed at identifying the vigorous hydrogenase activities, accepting their interaction with ferredoxin and the other metabolic functions, and genetically changing these interactions to enhance the effectiveness for the production of biohydrogen (Yacoby et al. 2011; Wecker et al. 2011). Although hydrogen production from the algae is still a long way from its commercial viability, continued progress in this area indicates its ultimate potential.

The production of biogas efficiency has been revealed to be species-dependent and is based on the relative efficacy of cell deprivation and on the absence or presence of molecules that might prohibit the methanogenic archaea (Mussgnug et al. 2010). Production of biogas from algae may also involve an important function in phyco-remediation, as harmful algal blooms in ponds, lakes, or oceans can result in the release of poisonous secondary metabolites that can cause deleterious effects on these environments; clearing these algae for the production of biogas can minimize these impacts (Yuan et al.2011). Currently, the biogas production from algae is still incomplete, owing to the need to heat the digesters and the necessity for more land area and infrastructure to generate the same content of energy that can be obtained from the algal biodiesel (Collet et al. 2011). The value-added product can comprise biohydrogen produced anaerobically just before the process of anaerobic digestion for the production of biogas (Mussgnug et al. 2010). In addition, generation of electricity from biogas can be used to offset the requirements of energy for the microalgal anaerobic digestion during biogas production; agriculturally originated biogas can be used to give a CO2 stream for algal growth and the production of coproduct and biogas can be used to power the cultivation and lipid extraction methods for the biodiesel (Collet et al. 2011; Douskova et al. 2010; Harun et al. 2011).

Catalytic cracking and liquefaction for thermochemical conversion

Preliminary studies made by several researchers on Scenedesmus and Spirulina indicate that biomass with ~20% oil content is suitable for conversion into biogasoline through the catalytic cracking process, as the product is rich in hydrocarbons (Ueda et al. 1996; Biller and Ross 2011). Similarly, all forms of carbon present in this biomass can be converted into biocrude oil through a thermochemical liquefaction process. Thermo-chemical conversion technologies of biomass are certainly not the most important opportunity in recent times; combustion is responsible for over 97% production of the world’s bio-energy (Balat 2009). The primary thermochemical conversion processes are pyrolysis, gasification, and liquefaction. Biorenewable feedstocks can be converted into liquid or gaseous forms for the generation of electricity, heat, chemical, and gaseous or liquid fuels (Demirbas 2008). Although microbial or enzymatic transformations have more cost-reduction potentials in view of the recent developments and the constant efforts for their optimization, these routes are less prone to commercialization in the immediate future, thereby rendering the thermochemical conversion processes commercially viable, as these rely on processes that have been thoroughly studied over the years (Sims et al. 2010).

BIOFUEL APPROACHES

Algal Culture Systems

In general, the algal biomass grown with the industrial wastewaters can also be converted into biocrude oil using a thermochemical liquefaction process. Hence, growing algae in wastewaters for biofuel and bioenergy production seems a viable and eco-friendly option for the future. Two main culture systems are available for algal production. An open system generally combines waste treatment with algal production. This system employs the use of ponds, which range from the oxidation ponds to the high-rated algae ponds. An oxidation pond recycles nutrients through a bacteria-algae symbiotic process. The pond is one to two meters deep and unmixed. The algal yield in such a pond is thus low. In contrast, the high-rate algae pond (HRAP), which consists of an open raceway mixed by paddle-wheels, is very shallow and is capable of producing very high yields. High-rate algae ponds are suitable for the generation of algal biomass for high-quality animal feed and extraction of useful compounds such as protein and pigments. Research on the combined algal production and waste-treatment systems has been done in Israel (Shelef et al. 1980), India (Venkataraman et al. 1980), Thailand (Tanticharoen et al.1990), the United States (Christenson and Sims 2012; Ellis et al.2012; Rahman et al. 2012; Christenson and Sims 2011; Lincoln and Hill 1980), and some other countries.

As fresh water sources are scarce, utilization of poor quality wastewaters such as treated municipal sewage wastewater as low-cost nutrient growth medium for mass cultivation of biofuel algae appears a viable option for the future. In recent times, research into microalgal cultivation has gained importance because of application of this resource in the production of biofuels. Cultivation of microalgae in the open pond systems has been used since the 1950s (Borowitzka 1999), and raceway ponds are the most commonly used artificial systems. Open ponds provide a very efficient method of cultivating algae, but they become contaminated with the algal species very easily (Khan et al. 2009).

The major advantage of the open ponds is that they are very easy to construct and operate; in comparison to most closed systems, they are easy to clean up after cultivation and are ideal for mass cultivation of microalgae (Ugwu et al. 2008). This should be given consideration in view of the escalating equipment costs, particularly the use of the reactor-style systems that lack a reliable scale-up method. While considering the economic and the environmental aspects, a raceway pond coupled with a low cost harvesting technique would be a preferable choice to produce biodiesel.

While the demand for the production of biofuel is in part driven by ecological concerns, there is no doubt that constructing and operating an HRAP dedicated to producing algal biomass for biofuels can have an ecological impact. For example, resources of fresh water are consumed through evaporation, thus contributing to a water footprint (Park et al. 2011). Indeed, Clarens et al. (2010) summarized that production of algal biomass using freshwater and fertilizers would consume high energy, result in more greenhouse gas emissions, and use a lot of water compared to biofuel production from land-based crops, i.e., canola, switch grass, and corn. Production of algal biomass using wastewater HRAPs, by contrast, offers a far more interesting proposition from an ecological point of view. The impacts of the HRAP construction and operation are a necessity of providing the treatment of wastewaters and hence, the subsequent algal production represents a biofuel feedstock free of this ecological issue (Park et al. 2011).

Among the various cultivation systems involved in producing algal biomass, the aspect of harvesting biomass is an important economic issue. It was estimated that harvesting algae biomass can account for 20 to 30% of the total production cost (Gudin and Thepenier 1986). When, the algae grow phototrophically, their concentration is about 0.5 to 1.0 g L-1 for open ponds and around 5 to 10 g L-1 biomass concentration for closed systems (Chisti 2007). For the production of 1 g L-1 algal biomass, 1000 kg of water must be used to capture 1 kg of biomass.

Methods of algal biomass harvesting, such as filtration, centrifugation, sedimentation and flocculation, and floatation are being practiced either individually or in any combination. Several literature reviews have provided for the algae harvesting techniques (Mutanda et al. 2011; Grima et al. 2003; Chen et al. 2009; Harun et al. 2011). Among the various methods, centrifugation is a possible method suitable for higher-value products but is very expensive in an integrated system producing lower-value products, such as algal oils (http://www.ecs.umass.edu/biofuels). In the case of algal-derived biofuels, the low-cost promising method is gravity settling enhanced by flocculation, without benefit of chemical flocculants (Molina Grima et al. 2003). Other mechanisms exist, including the autoflocculation process, and it depends on the coprecipitation of calcium carbonate with microalgal cells and other precipitates that form in hard waters subject to high pH. Apart from settling, in some cases the biomass will float, either due to high oil content or by using a dissolved air flotation (DAF) process. Employing minor amounts of flocculants to assist in such a process could be cost effective, depending on the amount used. In general, the harvesting method of choice depends on algal species, the cultivation conditions, and the application of the product. For biofuels applications, low-cost algal harvesting techniques have not yet become established (Darzins et al.2010). If new algal harvesting techniques have been developed, they have not yet been assessed publicly and, therefore, are not documented in this review. Significant research effort will be needed to develop the cost-effective techniques.

Integrated palm oil mill effluent treatment and biofuel production

Algae are a substantial component of ecosystems ranging from marine and fresh water ecosystems to desert sands and from hot springs to ice. In recent years, microalgae have been identified using morphological and molecular tools for their several purposes reported by many authors (Jayappriyan et al. 2010, 2011). Worldwide, algae contain thousands of diverse strains. When combined with the recent advances in bioremediation, these strains can provide a good initial point for further improvement of microalgal production methods based on treatment of wastewaters. Being a developing country, Malaysia releases a significant amount of pollutants into water bodies. Modern industries, animal and agriculture husbandry, agrobased industries, and the activities of urbanization have all contributed to the devastation of the natural environment (Goh and Lee 2010). Algae-based solutions have offered certain benefits to the various countries. Malaysia is basically an agricultural country, its main revenue earners being palm oil and rubber. Major pollution problems have arisen from the agroindustries based on these two major crops as well as on the increased waste from farm animals. The combined wastewater discharges from the oil palm and rubber industries contribute an organic load of 0.5 million kg of Biochemical Oxygen Demand (BOD) per day (Phang 1987). Various treatment methods, including biological, chemical, and mechanical, have been developed over the last two decades, with some being successfully implemented (Phang 1987). Researchers at the University of Malaya and the other government institutions have demonstrated the feasibility of using microalgae to treat agroindustrial wastewaters. Significantly, combining microalgal production and wastewater treatment offers an advantage over the conventional treatment systems.

Realizing the vast potential of microalgae cultivation towards sustainable energy development, researchers in this area have noted a few key points in up-scaling the overall process. One of the difficulties in up-scaling microalgae culture to an industrial scale is the source of food nutrients in the culture medium. The requirement of high nitrogen content and the other related chemical fertilizers to cultivate microalgae on a large scale has moved the process towards a loss of the environment. Also, cultivation of microalgae can basically play an important role as a self-purification process of the wastewaters in a natural condition (Soeder 1980). Municipal wastewater using conventional treatment that involves primary and secondary bio-treatment helped to remove only a portion of the nitrogen and phosphorus contained in the wastewater (Orpez et al.2009). Therefore, culturing of microalgae in the wastewaters offers an inexpensive alternative to the conventional forms of tertiary treatment of wastewaters and at the same time consumes the nitrogen and the phosphorus contents in the wastewater to generate the microalgal biomass for the production of biofuel.

Recently, the concept of utilizing POME (Palm Oil Mill Effluent) as a nutrient source for the culture of microalgae in Malaysia has caught the attention of researchers. Because of its practical low cost and high value, most palm oil millers desire the culture of microalgae as a tertiary treatment before POME is released. Therefore, most of the nutrients such as nitrate and orthophosphate that are not eliminated during anaerobic digestion are further subjected to bioconversion in a microalgae treatment pond. After secondary treatment, the total nitrogen content of POME is still high and does not meet the discharge standard limit for wastewater which is 200 mg/L. In the meantime, the source of nitrogen, commonly existing in the nitrate form, plays an important function in promoting microalgal growth. In order to grow microalgae effectively, the basic nitrate concentration required should be in the range of 200 to 400 mg/ L (Li et al. 2008). Other minerals that are essential for microalgal growth, i.e. K, Ca, Mg, Fe, Zn, and P, are also present in POME (Habib et al. 1998). Hence, POME has emerged as an alternative choice for the nutrient removal to grow microalgae for the production of biomass and concurrently acts as a part of the wastewater treatment process and is used for biofuel production.

The University Kebangsaan Malaysia (UKM) has been involved in developing algae-based technology to treat the palm oil industry effluents with the collaboration of Simedarby (R & D), Malaysia since 2011. Various algal cultures were obtained from the Algaetech Company, Malaysia and maintained under laboratory conditions. They were allowed to grow in 2000 L of the POME as medium. Among them, Spirulina platensis showed higher growth rates and nutrient removal efficiency. A maximum growth rate of 1.1825 g/L was achieved by Spirulina platensis in anaerobically digested Palm Oil Mill Effluent on the 12th day in the culture flasks (Zainal et al. 2012). A pilot plant facility with raceway ponds is to be established at UKM for developing a continuous cultivation technology for algae in the treated (POME) wastewater for biofuel applications. The study also aims to evaluate the possibility of using the CO2-rich flue gas emissions from the biogas generator for growing the alga. Algal technology for treating effluents is to be tried in some Malaysian industries in the future. In Malaysia, various industries and research institutes have demonstrated mass cultivation and biomass production of various micro algae in different industrial waste waters. Recently, Algaetech Industry has demonstrated mass cultivation of microalgae in the open raceway pond and the possibility of conversion of the total lipid into biodiesel.

Global Status of Biofuel

Fossil fuels continue to be exploited extensively by developing countries despite objection from several ecological activist organizations. Development and the economic growth of a country depend considerably on how the demand for the biofuel is best met. The currently available feedstock involved in the biofuel production includes vegetable oils derived from the oilseed crops, e.g., sunflower, soybean, jatropha, oil palm or rapeseed, waste cooking oil and the animal fat, e.g., beef tallow and pork lard (Moser Bryan 2009). Currently, exponential research growth, development of technology, and demonstration enterprises have kick-started the investigation of algal biomass as an alternative resource for the biofuel industries. One such renewable energy is algal biofuel, which has shown a huge potential to serve as a replacement for petroleum-based diesel. Recently, a significant number of companies proposing to use algae for producing biofuels and abating the climate change through CO2 mitigation have emerged. Recently, two Canadian companies announced the formation of a new company with a proposal to convert CO2 to algal biomass. For example, researchers from the Tokyo University of Marine Sciences and Technology released the details of a proposal for large scale bioethanol-fuel production from macroalgae (Carlsson et al. 2007). Yamazaki (2007) accounted the initiative of Japanese companies on launching bioethanol fuel to the marketplace with the expectations of reducing CO2 emissions considerably.

Aizawa et al. (2007) reported that the “Oceans 2007” project has proposed to produce bioethanol by farming and harvesting Sargassum horneri. Stroazzo of Bio Fuel-System stated that production of biofuel from algae is a promising and ecofriendly approach. Radulovich (2008) also specified the use of macroalgae as a resource of fuel, apart from food, animal feed, and fertilizer. Kraan (2010) observed that Ireland is likely to become an important player in the future generation for the biofuel production with its wealthy, sustainable macroalgal sources. Mullins (2009) stated that a group at the Korea Institute of Technology in South Korea has developed a method to use macroalgae to produce bioethanol and avoid taking up land together. Seambiotic (2009), in collaboration with the Inventure Chemicals, successfully demonstrated the bioethanol production via fermentation of polysaccharides in algal biomass. In their plant, algae were cultivated in the fossil fuel power plants to eliminate CO2emitted as a source of inorganic carbon.

The production of ethanol in 2009 attained 73.9 billion liters, which illustrates a more than 400% increase compared with that in 2000 of 17 billion liters in the world. While it has been predicted that the global ethanol production would continue to rise until 2017, reaching a level twice that of 2007, it also predicted that the United States and Brazil would remain as the biggest ethanol producers through 2017 followed by China, India, and Thailand. In addition, the FAO analysis shows that with the exclusion of bioethanol from sugar cane in Brazil, biofuels are commonly not economically viable relative to the fossil fuels in the absence of subsidies. Also, as for global biodiesel production, the report projects a slightly higher growth rate than for bioethanol to achieve 24 billion liters by 2017 and that the EU, comprising over 50% of the global production, will continue to be the leading biodiesel producer in 2017 and will be followed by Brazil, Indonesia, the United States, and Malaysia, respectively (Renewable fuels association, 2010).

The production of algal biomass integrated with remediation is the greater option, since it will not influence the agricultural land and water (Christenson and Sims 2011, 2012). Cultivation of algae in waste waters will make the whole process system cheaper and more economically viable. Among these, many microalgal genera and species are remarkably rich in oils that can be converted into biodiesel with the available technology.

It is believed that investigations on identifying novel algal resources, both micro and macro types, would herald a remarkable energy revolution in the years to come. To our knowledge, large-scale commercial operations are yet to be established to date. It is recommended that the three main stakeholders such as the government, the researchers, and the industry could join in a consortium that will be able to constitute a closer frame-work so as to ensure a sustainable development.

RECOMMENDATIONS

Apart from the export potential, job opportunities to be created, and the environ-mental security, there is potential for securing a prestigious status of a global leader in the production of biodiesel production on a path toward achieving the status of a developed country. This could also serve to demonstrate the country’s capabilities in the international arena and thus, could help lift up the country’s self esteem. In the long run, this would surely lead to enhanced motivation or spur more interest in other countries to follow suit with similar objectives or goals and thus could help transform the country into a developed nation.

Therefore, given the intrinsic advantages of the microalgae, and now existing restraints of using other oil crops, microalgae growing in wastewater would facilitate a cost-effective production of the biodiesel. Moreover, the current highly sophisticated R&D centers are conducting extensive research on various aspects of biofuel production, and particularly biodiesel. During the last decade, in view of the importance placed on bioprospecting for better resources, especially in the agriculture sector, many well-equipped labs have been established in various countries, the world over.

This makes research organizations capable candidates to achieve projects on microalgal genetic engineering in order to address the rising biodiesel demand in the country. From the point of view of cultivation, it is imperative to note that apart from the free and unlimited access to salt water and plentiful sunshine, Malaysia and other tropical countries are known for their rich biodiversity of microalgae. Generally, technology development for large scale cultivation of macroalgae on the coastal regions for the production of bioenergy does not compete with that of food sources and does not alter the environment. The viability of algae mass cultivation can be judged from Malaysia’s wide seashore surrounded by several islands, which can offer the ideal habitats for the algae propagation. Additionally, it has been shown by various reports that countries with abundance of under-utilized rice land are suitable for cultivating algae. Those marginal lands are infertile because of salty water diffusion, and farmers are looking for other options. Cultivation of algae can provide one such alternative as the marine algae naturally grow well in the salt waters.

Research and Development on Gene Sequence for Biofuel Production in Algae

Conversion of polysaccharides from macroalgae to fermentable sugar and thus to ethanol and biogas has not yet been studied in detail (Goh and Lee 2010). Although there is a wide availability of macroalgae the world over, there are still no viable suggestions for the production of bioethanol from macroalgae. Successful mapping of the oil-algae genome could help recognize the genetic traits responsible for the production of oil, and it could find strains susceptible to contamination by the basal bacteria or fungi which could cause the algal biomass to deteriorate in the open raceway ponds. Moreover, the genetic information can also be applied to develop the harvesting regimes of the oil algae so that the biomass could be harvested at the appropriate period. Such a technological understanding is imperative to ensure a constant progress in the world’s biodiesel industry, as the land suitable for this purpose is so limited.

The successful decoding of the oil-algae genome is believed to have paved the way for a manipulation of the oil-algae genome for use in the future. The genetic manipulation in oil-bearing algae could provide the potential to produce high quantity of superior oil. With advanced biochemical and molecular techniques such as DNA recombination and site-directed mutagenesis, more groundbreaking outcomes could be expected to be employed in the near future (Eathington et al. 2007). These areas of research have laid a foundation for an understanding of the macroalgal biology. Despite the development and progress made in the functional genomics in terrestrial plants, macroalgae have received little attention worldwide and have not been incorporated in the approach to elucidate gene functions. It was not until the 1990s that studies on the genetic engineering of macroalgae were started. These studies have led the way with the improvement of the genetic transformation techniques on macroalgae (Cheney and Kurtzman 1992) and the characterization of genes involved in the synthesis of carbohydrates (Zhou and Ragan 1995). Macroalgal research has not received due interest and funding in the past, and the availability of the macroalgae genomic information is still meager in comparison to that of terrestrial plants. Given the fact that macroalgae share little similarity with the other organisms, the limited amount of genomic information available makes the mission of exposing the role of unknown genes from the macroalgae a difficult proposition.

CONCLUSIONS

Biofuel production and environmental effects have been issues of some concern worldwide. In this review, the authors have described the advantages of biofuel, and the kind of materials that can serve as a source for this purpose. This was followed by the argument that under the present global scenario, algae appear to be the most effective raw material for biofuel production. Furthermore, the importance of sustainable energy sources such as bioethanol, biodiesel, and biohydrogen were also discussed. Among these, biodiesel production from algae biomass still would be the major component. Nevertheless, diversified biofuels production from algae biomass is very important to improve overall energy balance. For example, higher net value could be achieved by using a combined operation in which algae-produced lipids are converted to diesel fuel and the cellulosic part of the algal biomass (after lipid extraction) is enzymatically converted to glucose, which is fermented to produce bioethanol and other byproducts. Apart from that, biofuel contributes to energy security and helps reduce CO2 emissions. A thorough understanding of the past may serve to overcome the past lapses toward building a better future. These recent biofuel discussions demonstrate two issues. First, they show the wide potential utility of these organisms that are capable of producing multiple products ranging from energy, chemicals, and materials to exploitation in the sequestration of carbon and remediation of wastewater. Second, they show the need for energetic support based on factual information to confirm decisions for the strategic improvement of algae and to counter those declarations made on a solely tentative basis to promote commercial investment.

ACKNOWLEDGEMENTS

The authors are grateful for the financial support of the various grants (UKM-YSD endowment; LRGS-TD-2011-UMP-PG-04; AP-2012-008; INDUSTRI-2012-040).

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Article submitted: April 1, 2013; Peer review completed: May 27, 2013; Revised version received: November 11, 2013; Accepted: November 12, 2013; Published: December 2, 2013.