Among the oleaginous microorganisms reported in the literature, filamentous fungi show the highest lipid accumulation after yeast, besides the capacity to produce a wide range of products, i. e., enzymes, antibiotic, and chemicals (Karimi and Zamani 2013). Some of the main differences between filamentous fungi and other oleaginous species (yeast, microalgae, and bacteria) on the production of oils are based on the capability of filamentous fungi to build pellets in submerged cultures, due to filamen­tous growth during fermentation. Moreover, the viscosity of the broth is reduced, thus improving the mixing and mass transfer performance. Finally, due to the formation of pellets, they are easy to harvest from broth by simple cell filtration, which reduces the cost compared with traditional methods like centrifugation (Xia et al. 2011).

To decrease the cost of the process, methanolysis from fungal biomass has been proposed as an alternative to the oil extraction process. Through the use of metha­nol and a catalyst, usually H2SO4 or HCl, some authors reported a yield of FAME conversion of 91 %, being the cetane number 56.4, thus making this technique an attractive alternative for the biodiesel industry (Liu and Zhao 2007).

The stored lipids in filamentous fungi contain a high percentage of saturated (Venkata Subhash and Venkata Mohan 2011) and polyunsaturated fatty acids (Mitra et al. 2012), accumulated during the stationary phase in special organelles, named lipid granules. Like bacteria, filamentous fungi may also consume a wide range of carbon sources, including lignocellulosic biomass (Table 6), thus provid­ing inexpensive raw material for biodiesel.

Although lignocellulose comprises hemicellulose, cellulose, and lignin, only hemicellulose and cellulose may be consumed as feedstock for biological conver­sion. For this purpose, to make carbohydrates accessible to microorganisms, lig — nocellulose needs a pretreatment before hydrolysis (Zeng et al. 2013). Zikou et al. (2013) used a mixture of xylose and glucose, which are abundant sugars from ligno — cellulosic biomass, to produce y-linolenic acids (GLA) by Zygomycetes T. elegans. Results showed that the best combination of xylose to glucose is 1:1, achieving 12.6 g/L lipids and 936 mg/L GLA. Instead, when glucose was used as the sole medium, the values were 15 g/L and 1,014 mg/L, respectively. M. isabellina was also tested, and a positive influence of the increment of these sugars separately in the medium over the accumulation of lipids was found (Ruan et al. 2012). The same filamentous fungus was used for the production of oil when rice hulls hydrolyz — ate, which is a lignocellulosic material, was used as a substrate. Authors proposed a mathematical model to simulate the consumption of sugar and nitrogen, the fat-free biomass formation, and the accumulation of lipids (Economou et al. 2011). Khot et al. (2012) isolated fungi of tropical mangrove wetlands, but only five out of 14 showed lipid accumulation above 20 % dry cell biomass. Fungi from this ecosystem were also used for the production of lignocellulosic enzymes. The oil of three out of the previous five was transesterified, the biodiesel properties predicted, and it was found that the most appropriate fungus was IBB M1, known as A. terreus strain. Another important issue to be fixed when lignocellulosic biomass is used consists in the inhibitory effects of the lignocellulose-derived compounds over oil accumulation

(continued)

D. E. Leiva-Candia and M. P. Dorado

(lignin aldehydes, furan aldehydes, and weak acid). When M. isabellina was used to determine the inhibitory effect of these compounds, the lignin derivative was found to be the main inhibitor considering lipid accumulation, while acetic and formic acid doubled the lipid accumulation with respect to the control test (Zeng et al. 2013). It was concluded that the most suitable combination of fungus and lignocellulosic material substrate for fungal oil production was provided by the strain M. isabellina when it consumed non-detoxified lignocellulosic hydrolyzate, due to both the high oil content and the simplified process of fermentation (Zheng et al. 2012b).

In terms of environmental preservation, the bioremediation of soils contami­nated by hydrocarbons is an important issue. For this purpose, the use of A. terreus has been investigated to transform petroleum hydrocarbons in oils to be used in the biodiesel industry. Results showed that the use of hydrocarbons as carbon source provides sevenfold higher lipid accumulation compared to the use of glucose as sub­strate (Kumar et al. 2010). Crude glycerol is a by-product of the biodiesel industry, which has recently been released in high quantities due to the increasing biodiesel demand. It usually comprises residues of alcohol (methanol or ethanol) and a basic catalyst. This by-product has been tested as a carbon source for Mucor sp., C. echi — nulata, M. ramanniana, T. elegans, Z. moelleri (Chatzifragkou et al. 2011; Bellou et al. 2012), and M. isabellina (Chatzifragkou et al. 2011). Chatzifragkou et al. (2011) used the fungi mentioned above and compared lipid accumulation with that of yeasts. Results showed that all fungi were able to accumulate higher amount of oil than yeasts under nitrogen-limited conditions. Bellou et al. (2012) focused their research on the production of PUFA produced by filamentous fungi. In the majority of the tested fungi, authors observed that PUFA was mainly accumulated in myce­lial membranes during mycelial growth. However, one of the studied filamentous fungi (Mortierella ramannniana) depicted the opposite trend. In this sense, PUFA continued decreasing after the end of the growth phase, thus suggesting PUFA is involved in primary metabolism of this microorganism (Bellou et al. 2012).

Filamentous fungi have been genetically engineered focusing on lipid produc­tion, giving relevance to metabolic routes governing fatty acid synthesis and lipid storage. Unique metabolic features have been identified in Mortierella alpina and Mortierella circinelloides, particularly with respect to NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotype (Vongsangnak et al. 2013). The gene coding for acetyl-CoA carboxylase (ACC) was isolated from Mucor rouxii. This gene is able to increase by 40 % the total fatty acid content of non-oleaginous microorganism (Ruenwai et al. 2009). Wynn et al. (1999) studied the significant role of malic enzyme on lipid accumulation. Authors used a fungus with low lipid accumulation (M. circinelloides) and found out that the enzyme disappeared 15 h after the depletion of the nitrogen source, which was coincident with the end of lipid accumulation. Instead, when a high-lipid accumula­tion fungus like Mortierella alpine was used, the enzyme was held 60 h after the completion of the nitrogen source, which lasted longer than the lipid accumulation.

The accumulation of lipids from filamentous fungi is increasingly attractive because of the high oil yields, versatility of the microorganisms to use different car­bon sources (including wastes like lignocellulosic material), and the possibility to

be grown in submerged cultures, which give the opportunity to easily collect the biomass. In this context, genetic engineering may be a magnificent tool to help in the inclusion of these microorganisms to provide an alternative oil to the biodiesel industry. Although most research in this area is focused on the production of high — value-added products such as enzymes and polyunsaturated fatty acids, among many others, the production of microbial oil could provide an extra value to the process.

Data on the effects of reaction temperature on degradation of HDPE into various products using HZSM-5 (80) catalyst are shown in Table 3 while Table 4 presents the yields of degradation products using AlSBA-15 catalyst. It is clear that gen­erally the yield of volatile products increased with increasing reaction tempera­ture. It was well noted that by increasing the temperature, cracking probability was increased to result in higher yield of volatile products. At higher tempera­ture, the reaction was generally accelerated, and the accessibility of the reaction products resulting from interaction with external active sites and the reactant was improved. Thus, cracking reactions to give smaller molecular-sized sub­stances were improved. On other hand, the small internal pores of the HZSM-5 and AlSBA-15 catalysts could create hindrance toward the formation of liquid

Fig. 4 Effects of different catalysts on liquid products yield at 623 K with 10 % catalyst loading

products with relatively larger molecular sizes. The effect was more severe in the case of zeolite catalyst with internal pores in the micropore size range. However, it was also noted that the HZSM-5 (Si/Al = 80) catalyst had better capability to con­vert liquid products into gaseous products especially at high temperatures. Similar observations have been reported in literature (Mastral et al. 2006). For exam­ple, it was found that the liquid yield decreased with increasing temperature and higher temperature evidently caused decreases in boiling points of liquid products (Hernandez et al. 2006).

Findings made in this study were in good agreement with those reported by Mastral et al. (2006). They found that zeolitic materials were suitable to be used in

Fig. 5 Effects of reaction temperature on gas products yield by 10 % loading of HZSM-5(80) catalyst

catalytic degradation of polyethylene due to their acidity and structural suitability. As observed in this study, the highest gas yield was achieved by increasing the experi­mental temperature from 350 to 400 °C. Our results regarding the effects of tempera­ture on AlSBA-15 catalyst were also in good agreement with those of Sinfronio et al. (2006) who used Al-MCM-41 as the mesoporous catalyst. Based on these observa­tions, it could be concluded that the most suitable temperature range for maximum liquid fuel yield using HZSM-5 catalyst was 350 °C while it was 400 °C for AlSBA — 15 catalyst. However, higher coke deposition could sometimes correspond to the increasing reaction temperatures, and formation of waxy compound might prevent the accurate calculation of the yield of liquid and gaseous products.

Data regarding the effect of reaction temperature on the gaseous product yield using HZSM-5(80) catalyst are presented in Fig. 5. It is shown that 500 °C gave the most uniform products distribution with highest composition showed by C3 (34.5 %) and the lowest by C5 (11.2 %). By carrying out the reaction at 400 °C, remarkable reduction on the C1 while at the same time an increase in the propor­tion of carbon chain C5 was observed.

In order to compare the effect of temperature when a mesoporous catalyst was used, similar experimental run was carried out using AlSBA-15 catalyst. Figure 6 presents data that were obtained using the mesoporous catalyst. In this case, 350 °C showed the highest gaseous products yield with the highest composition showed by C4 while for the other two reactions, i. e., 400 and 500 °C, C3 predominated. Generally, temperature does not have dominant effects on the gaseous products dis­tribution. However, the difference in gaseous products distribution was significant when comparing results obtained with microporous HZSM-5 (80) and mesoporous AlSBA-15 catalysts. AlSBA-15 catalyst under same 10 % catalyst loading did not produce detectable C1 gas products. This mesoporous catalyst also led to increases in C5 (11.6 and 12.7 %) as compare to those of HZSM-5 microporous catalyst (4.4 and 7.0 %) for the reaction temperatures of 350 and 400 °C, respectively.

However, degradation of liquid products using HZSM-5 (80) showed a decreas­ing trend for increasing carbon chain from C8 to C25+. As shown in Fig. 7, the highest proportion of liquid products for all four reaction temperatures was in

Fig. 6 Effects of reaction temperature on gas products yield by 10 % loading of AlSBA-15 catalyst

Fig. 7 Effects of reaction temperature on liquid products yield by 10 % loading of HZSM-5 (80) catalyst

the carbon chain range of C8-C12. The lowest proportion of carbon chain range was the heaviest carbon chain, i. e., C25+ for all reaction temperatures studied. Increasing reaction temperature had the tendency to produce higher amount of shorter carbon chain molecules while simultaneously reducing the longer carbon chain molecules in the products mixture.

Figure 8 presents the data obtained using AlSBA-15 catalyst under the effect of varying temperatures. Generally, the results showed similar downward trend as observed in the case of using HZSM-5 (80) catalyst. The highest proportion was recorded by carbon chain range of C8-C12 for all three reaction tempera­tures. The second highest composition was recorded by the carbon chain range of C13-C16. This composition also dropped steadily as the reaction temperature increased. The lowest fraction of carbon chain range for the overall liquid prod­uct was C25+. By comparing the effect of varying reaction temperatures to both microporous HZSM-5 (80) and mesoporous AlSBA-15 catalysts, it was concluded that generally liquid degradation products for AlSBA-15 consisted of shorter

carbon chain range molecules. This could be seen for carbon ranges of C8-C12 and Сіз-Сі6 at 350 °C. AlSBA-15 catalyst produced nearly 78.2 % of overall liquid composition while it was only around 63.5 % for HZSM-5(80) catalyst.

Despite its vocation as a potential source of biofuels, many challenges have hindered the development of biofuels technology from microalgae to become commercially viable.

Among them, and based on recent literature, we elect as the most important:

1. The selection of species must balance the requirements for biofuel production and extraction of valuable by-products (Ono and Cuello 2006);

2. Achieving greater photosynthetic efficiency through the continuous develop­ment of production systems (Pulz and Scheibenbogen 1998);

3. Developing techniques for growing a single species, reducing evaporation losses, and diffusion of CO2 (Ugwu et al. 2008);

4. Few commercial cultivating “farms,” so there is a lack of data on large-scale cultivation (Pulz 2001);

5. Impossibility of introducing flue gas at high concentrations, due to the pres­ence of toxic compounds such as NOx and SOx (Brown 1996);

6. Choosing algae strains that require freshwater to grow can be unsustainable for operations on a large-scale and exacerbate freshwater scarcity (Mcgraw 2009);

7. Current harvest and dewatering are still too energy intensive (Chen et al. 2009);

8. Some recent life cycle analyses (LCAs) project algae biofuels as having poor energy or greenhouse gas benefits (Benemann 2012; Clarens et al. 2010);

9. Depending on the processes, PBR systems can consume more energy than they produce (Slade and Bauen 2013);

10. Another disappointment that will likely arise is the scarcity of sites with favorable climate, land, water, and CO2 resources, all required in one place (Benemann 2012; Clarens et al. 2010; Slade and Bauen 2013);

11. CO2 supply is relatively expensive, due to high capital and operational costs for piping CO2 to, and transferring it into, the ponds (Benemann 2012).

12. Large-scale cultivation of algal biomass will require a lot of nitrogen and phosphorus; at a small-scale, recycling nutrients from wastewater could potentially provide some of the nutrients required (Slade and Bauen 2013).

Finally, to reach positive energy balance, it will be needed technological advances and highly optimized production systems. The amount of GHG decreases when the microalgae yield increases, primarily because CO2 is the main raw material utilized in photosynthesis during the growth of microalgae. Therefore, it is impor­tant to achieve high yields of biomass and oil in the cultivation plant. The miti­gation of environmental impacts, and in particular water management, presents both challenges and opportunities, many of which can only be resolved at the local level. Existing cost estimates need to be improved, and this will require empiri­cal data on the performance of systems designed specifically to produce biofuels (Slade and Bauen 2013).

Traditionally, yeasts have been used in the food and beverage industry, so the major­ity of yeasts have been adapted to meet these procedures. The ability to accumu­late lipids above 20 % of its weight is achieved by only 5 % of the known yeasts (Beopoulos et al. 2011). Lipid accumulation in oleaginous yeast occurs under excess of carbon sources, being scarce the nitrogen source, so the carbon excess is channeled into triglycerides (Ageitos et al. 2011). Similar to other microorganisms, yeast is able to consume different sources of carbon and nitrogen, from waste to laboratory-pure sources. However, to take advantage of this technology, the use of widely available waste is a key parameter. According to this, the main by-products of the rapeseed oil-based biodiesel industry, glycerol (carbon source) and rape — seed meal (nitrogen source), were used as culture medium for the oleaginous yeast Rhodosporidium toruloides Y4 and the accumulation of oil was analyzed. Results showed that the accumulation of oil reached up to 19.7 g/L, higher than 16.2 g/L achieved when a medium composed of glycerol and yeast extract as nitrogen source was used. Besides, the oil fatty acid composition comprised a high content of mon­ounsaturated fatty acids, which makes it suitable for biodiesel production (Uckun Kiran et al. 2013). Many authors have proposed the use of glycerol as carbon source to grow different oleaginous yeasts, i. e., Cryptococcus curvatus (Liang et al. 2010), Rhodotorula glutinis (Saenge et al. 2011), Rhodotorula graminis (Galafassi et al. 2012), and R. toruloides (Xu et al. 2012). In all cases, it was considered a suitable carbon source for lipogenesis. Also, the hydrolyzate from lignocellulosic materials has been considered an interesting substrate due to the availability and economic feasibility (Yu et al. 2011; Gong et al. 2012; Uckun Kiran et al. 2012).

The culture conditions, such as C/N ratio (close to 100), substrate, culture mode, microelements, and inorganic salts, are crucial in lipid accumulation (Ageitos et al. 2011). While the ratio C/N plays the most important role in lipid accumulation, the culture mode is also of special interest. For this reason, Zhao et al. (2011) used dif­ferent feeding strategies with yeast R toruloides Y4 and concluded that the fed-batch strategy exhibited the largest oil accumulation potential under large-scale production plant, while keeping the residual glucose concentration to 5 g/L of carbon source and the fed-batch cycles were multiple times repeated. Authors removed the majority of the mature culture at the end of each cycle, keeping 900 ml of the culture in the bioreactor. Then, fresh media were added and a new cultivation cycle was initiated. As a result, the highest amount of lipids reported in the literature, 78.7 g/L, was achieved (Table 7).

The main disadvantage of oleaginous yeast is the extraction of the oil, due to the resistance of the cell walls to different solvents. In most cases, a chloroform methanol stream has been used, although this solution is not environmentally friendly because of the toxicity of reagents. An interesting alternative is provided by an enzyme-assisted method, consisting in a microwave-aided heating pretreat­ment, further enzymatic treatment with the recombinant P-1,3-glucomannanase and plMAN5C, and later oil extraction with ethyl acetate. The percentage of extraction with this method is close to 96.6 % of the total oil (Zeng et al. 2013).

Table 7 shows the fatty acid composition of yeast oil. Although it varies depending on the species and substrate, it is mostly composed of palmitic and oleic acid, the lat­ter being preferred for the biodiesel industry due to its high unsaturation degree (Pinzi et al. 2011). Wahlen et al. (2012) compared biodiesel properties, performance, and emissions in a diesel engine, biodiesel being produced from soybean, algae, bacteria, and yeast oil. Only small differences in terms of exhaust emissions were detected, as biodiesel from yeast oil emitted lower hydrocarbon but higher NOx emissions.

4 Conclusion

Many studies have demonstrated that the use of oleaginous macro — and microor­ganisms has a great interest to the biodiesel industry, as an alternative to first — and second-generation biodiesel. Although each species has its own characteristics that make it suitable to the production of biodiesel, insects posses the ability to recycle organic waste like manure and produce high amount of good-quality oil, while micro­organisms may be fermented on conventional bioreactors, which is a very attractive feature. In the improvement of these technologies, genetic engineering provides a key tool, besides the increase of knowledge about organisms, i. e., culture media and growing conditions. Moreover, the oil composition of oleaginous organisms may be genetically modified to meet the ideal biodiesel requirements, but also it can be modi­fied in pursuit of the best combination of substrate, species, or culture mode. It may be concluded that yeast is the preferred oleaginous microorganism among those ana­lyzed in this chapter, due to its rapid growth, ability to be scaled up, production of lipids, and suitable fatty acid composition to be transesterified into biodiesel.

Acknowledgments This research was supported by the Spanish Ministry of Education and Science (ENE2010-15159) and the Andalusian Economy, Innovation and Enterprise Council, Spain (TEP-4994).

Catalyst loading is another important parameter that can significantly affect the yields of degradation products. In this study, three different levels of catalyst load­ing, i. e., 5, 10, and 15 % were investigated for effects on the degradation of HDPE. In this study, the reaction time was fixed at 3 h for HZSM-5 (Si/Al = 80) catalyst. Meanwhile, in case of AlSBA-15 catalyst, initially, the reaction was carried out at a catalyst loading of 5 % for 4 h due to slower reaction to achieve significant level of degradation. As shown in Table 5, a catalyst loading at 10 % yielded the highest amount of light liquid while it produced the least amount of volatile gaseous product as compared to the other two catalysts loadings. The solid coke formation was found to be about 7 % for catalyst loadings of 5 and 10 %. Its yield was slightly higher with 15 % catalyst loading as 7.7 wt. % of the overall weight of residue was obtained.

The trace for waxy compound with 5 % catalyst loading amount was detected, but it was negligible and difficult to be accurately measured. The possible reasons of low conversion of degradation product to liquid were due to the concentration of 5 wt. % catalyst which was considered low to provide sufficient number of active sites for the degradation of large- to moderate-sized polymer chains. However, the optimum catalyst loading was found to be at 10 wt. %. It was further noted that larger amount of catalyst loading might promote faster reaction with higher forma­tion of coke in the residue of degradation products. This might be the reason for the catalyst inactivity throughout the degradation process (Ochoa et al. 1996).

In Fig. 9, the highest amount of liquid degradation product was obtained at 15 wt. % of catalyst loading (23.5 wt. % of the feed polyethylene). It also pro­duced moderate amount of volatile gaseous degradation products (52.2 wt. %) as compared to that obtained using HZSM-5(80). Furthermore, it could prove the theory that zeolitic catalysts such as HZSM-5 promote the production of

gaseous products during degradation of polyethylene. At the same time, it was not considered to be an ideal catalyst for the purpose of maximizing the degradation into liquid yield. However, one of the major disadvantages of using higher cata­lyst loading to increase the liquid product yield was that the formation of higher amount of solid coke in the residue might lead to the poor overall catalyst activity.

It was also observed that at 5 % of catalyst loading, no liquid or gaseous prod­ucts were successfully collected due to the formation of large amount of solid waxy compounds leading to a blockage at the reactor outlet. This was due to the rapid solidification of melted polyethylene degradation products when they were exposed to lower temperature at the reactor outlet. The small amount of catalyst loading was also unable to reduce the activation energy of the degradation process to enable the degradation mechanism to take place rapidly. However, significant formation of solid waxy compound was successfully inhibited at 10 wt. % of cata­lyst loading. Consequently, sufficient amount of liquid and gaseous degradation product was successfully collected. As illustrated in Fig. 9, the maximum liquid yield could be obtained at 15 % of catalyst loading. At the same time, less amount of solid waxy compound might be formed.

Fig. 10 Composition of gas products at 673 K using HZSM-5(80) catalyst over different catalyst loadings

Fig. 11 Composition of gas degradation products using various loadings of AlSBA-15 catalyst at 673 K

Figure 10 shows the data on the gaseous products using HZSM-5(80) catalyst. The highest composition was reported by C4 for 5 and 15 % of catalyst load­ings (27.8 and 33.6 %, respectively). Meanwhile for 10 % of catalyst loading, the highest proportion of C3 carbon chain was 38.4 %. At 5 % of catalyst loading, the amount of catalyst active site was deemed insufficient to cause significant deg­radation of polyethylene. Thus, higher amount of longer carbon chain molecules were produced as they could undergo further cracking reactions into smaller mol­ecules (Pierella et al. 2005). The results also indicated that increasing amount of catalyst would create better uniformity in the products distribution as seen in the case of 15 % of catalyst loading.

Likewise, data regarding catalyst loading and its effects on the degradation of HDPE to gaseous products using AlSBA-15 catalyst at 400 °C are shown in Fig. 11. The highest yield was recorded by the carbon chain of C4 with 15 % of catalyst loading (38.2 %). At 10 % of catalyst loading, its C3 carbon chain composition in the product was 42.1 %. It was also confirmed in earlier findings that the gas prod­uct was more concentrated in the middle of the carbon chain range such as C3 and

Fig. 12 Composition of liquid degradation products by various loadings of HZSM-5 (80) catalyst at 673 K

Fig. 13 Composition of liquid degradation products using various loadings of AlSBA-15 catalyst at 673 K

C4 (Hua et al. 2001). However, no product was detected for Ci products. Similar to earlier case, the use of higher catalyst loading produced more uniform products dis­tribution as in case of HZSM-5 catalyst. By comparing the degradation results using HZSM-5(80) and AlSBA-15 catalysts, it could be concluded that microporous cata­lyst HZSM-5(80) produced more shorter-chain carbon products. At the same time, mesoporous catalyst AlSBA-15 yielded more longer-chain carbon products.

Figure 12 shows the effect of catalyst loading on the distribution of degradation liquid products using HZSM-5(80) as catalyst at 400 °C. The highest composition was recorded with 5 % of catalyst loading to give a C21-C24 carbon chain range of 25.1 %. For the use of 10 and 15 % of catalyst loadings, the compositions of C8—C12 carbon chain range were 34.2 and 30.4 %, respectively. The longest carbon chain range C25+ was the minor product leftover after the degradation process. For this catalyst, it could also be seen that highest product yield was accu­mulated at the lighter end of the C8-C12 and C13-C16 carbon chain ranges.

By comparing to the earlier findings made using HZSM-5(80) catalyst (Koc and Bilgesu 2007), AlSBA-15 mesoporous catalyst showed a more non-uniform distribution (Fig. 13). The highest composition was recorded by a carbon chain

range of C8-C12, i. e., at 10 % catalyst loading (34.2 % yield) while at 15 % catalyst loading, 31.1 % yield of C13-C16 substances was obtained. Similarly, the heaviest carbon chain range, i. e., C25+ again recorded the smallest composition. The difference between the higher end and lower ends was bigger compare to the findings made using HZSM-5(80) microporous catalyst.

As of today, it has been shown that it is scientifically and technically possible to derive the desired energy products from algae in the laboratory. The question lies, however, in whether it is a technology that merits the support and development to overcome existing scalability challenges and make it economically feasible (Mcgraw 2009). Additionally, the basic economic motivation for biofuels is that they are a convenient, low-priced, domestically producible, and a substitute for oil; an energy source that is getting costlier; and it is mostly imported from politically volatile regions (Castanheira and Silva 2010). Economic feasibility is believed to be currently the main hurdle to overcome for this technology. Current costs associ­ated to both the state of the science and technologies are sizeable and represent a main factor hampering development.

High costs often prevent the market diffusion of novel and efficient energy technologies. As microalgae biofuel is not a mature technology, it becomes important to provide a revision of technological innovation and diffusion aspects to enlighten some available options that may help overpass the barriers found by innovative technologies (Ribeiro and Silva 2013).

It is widely recognized that modern economic analysis of technological innova­tion originates fundamentally from the work of Schumpeter (1934), who stressed the existence of three necessary conditions for the successful deployment of a new tech­nology: invention, innovation, and diffusion. His seminal work has been constantly referred (Soderholm and Klaassen 2007), and each of the keywords represents differ­ent aspects; in particular, invention includes the conception of new ideas; innovation involves the development of new ideas into marketable products and processes; and diffusion, in which the new products and processes spread across the potential market.

Emergent technologies are relatively expensive at the point of market intro­duction but eventually become cheaper due to mechanisms such as learning-by­doing, technological innovation and/or optimization, and economies of scale. The combined effects of these mechanisms are commonly referred to as technological learning. Over the last decades, learning theories in combination with evolutionary economics have led to the innovation systems theory that expands the analysis of technological innovation, covering the entire innovation system in which a tech­nology is embedded. In particular, “an innovation system is thereby defined as the network of institutions and actors that directly affect rate and direction of techno­logical change in society” (Junginger et al. 2008).

In the emerging energy technologies field, there is a strong need to influence both the speed and the direction of the innovation and technological change. With that in mind, policy makers are putting their efforts on lowering the costs of renewable energy sources to support the development of renewable technologies, either through direct means such as government-sponsored research and devel­opment (R&D), or by enacting policies that support the production of renewable technologies. It is well documented (Johnstone et al. 2010; Popp 2002) that both higher-energy prices and changes in energy policies increase inventive activity on renewable energy technologies (Popp et al. 2011).

As noted by Popp et al. (2011), the higher costs of renewable energy technologies suggest that policy intervention is necessary to encourage investment. Otherwise, in the lack of public policy favoring the development of renewable energy, production costs remain too high and they do not represent an option in replacing fossil fuels.

Policies to foster innovation should not only focus on the creation and sup­ply of new technologies and innovations, but also on the diffusion and take-up of green innovations in the market place. Such policies need to be well designed to ensure that they support, do not distort the market formation, and should be aligned with competition policies and international commitments (OECD 2011).

With this purpose, several government policies have been introduced in the energy markets worldwide in an effort to reduce costs and accelerate the market penetration of renewables. Although the effectiveness of alternative policies to encourage innovation still needs to be tested empirically, it is expected that these policies will stimulate innovation in renewable energy (US DOE 2010).

In the next section, some of the policies that could enhance the development of microalgae biofuels are, therefore, revised.

The overall schematic diagram of the apparatus used in this study is shown in Fig. 1. The experiment was carried out in a Pyrex glass reactor (volume 75 cm3) under atmospheric pressure at an isothermal temperature of either 350, 400, 450, or 500 °C. Nitrogen gas was continuously passed at a flow rate of 30 cc/min to purge the remain­ing air in the reactor to ensure inert atmosphere. In a typical run, 10 g of HDPE sam­ple and 5-10 % by weight of catalyst were blended together before being fed into the reactor. The reactor was heated to 120 °C in 60 min and held for 60 min at 120 °C. The nitrogen flow was then cut off, and the temperature was increased from 120 °C at a heating rate of 30 °C/min up to the desired temperature. The temperature of the polymer was measured with a thermocouple (Type J). The outlet of the reactor was connected to a water-cooling condenser maintaining at 20-25 °C. The gases (Cl-C5) were separated from the liquid oils (C5-C25) and then analyzed using a gas chromato­graph. The products from the reactor were collected over a period of 3-4 h.

Lauro A. Ribeiro, Patricia Dias, Luis Felipe Nascimento and Patricia Pereira da Silva

Abstract Despite the challenges, depending on the local conditions and practices, renewable energy sources are already a significant contribution to the energy mix. Although this is true for electricity generation, the same does not apply for the trans­portation sector, where the available renewable sources are limited and still have a modest impact in the overall consumption. In this context, advanced biofuels such as microalgae are worldwide believed to be a better choice for achieving the goals of incorporating non-food-based biofuels into the biofuel market and overcoming land — use issues. Compared to other biofuel technologies, the most favorable factors for the cultivation of microalgae for the production of biofuels are they can be grown in brackish water and on non-fertile land, and the oil yield production is far supe­rior. Main challenges are currently the feasibility of large-scale commercialization, since the majority of economic and financial analyses rely on pilot-scale projects. Environmental issues are most likely to diverge opinions from experts. This chapter presents a review of microalgae cultivation (species, usage, processes, and culture) and biofuel production, highlighting advantages and challenges of algae biofuel.

L. A. Ribeiro (*)

School of Sciences and Technology, University of Coimbra and INESCC, R. Antero de Quental, 199, 3030-030 Coimbra, Portugal e-mail: lribeiro@inescc. pt

P. Dias • L. F. Nascimento

Management School, Federal University of Rio Grande do Sul, Av. Washington Luiz, 855, 90010-460 Porto Alegre, Brazil e-mail: patricia. dias@ufrgs. br

L. F. Nascimento

e-mail: nascimentolf@gmail. com

P. P. da Silva

School of Economics, University of Coimbra and INESCC, R. Antero de Quental, 199, 3030-030 Coimbra, Portugal e-mail: patsilva@fe. uc. pt

A. Domingos Padula et al. (eds.), Liquid Biofuels: Emergence, Development and Prospects, Lecture Notes in Energy 27, DOI: 10.1007/978-1-4471-6482-1_12, © Springer-Verlag London 2014

1 Introduction

Innovative technologies and sources of energy must be developed to replace fossil fuels and contribute to the reductions of emissions of greenhouse gases associated with their use. Biofuels are particularly important as an option by means of trans­portation that lack of other fuel options (especially trucks, ships, and aircrafts). However, alternative sources of biofuel derived from terrestrial crops such as sugarcane, soybeans, maize, and rapeseed impose pressure on food markets, con­tribute to water scarcity, and precipitate forest devastation. In this way, the sustain­ability of biofuels will depend on the development of viable, sustainable, advanced technologies that do not appear to be yet commercially viable.

In this perspective, algal biofuels are generating substantial awareness in many countries. In the United States, they may contribute to achieve the biofuel produc­tion targets set by the Energy Independence and Security Act of 2007. Likewise, in the European Union (EU), they may assist to the achievement of goals established in the recent Renewables Directive. In order to address the technical-economic barriers to the further development of this type of bioenergy, it is thus necessary to contribute with a study that incorporates biomass feedstock availability assess­ment, production, management, and harvesting in support of the upscaling of this promising technology.

Biodiesel and bioethanol are the two liquid biofuel options currently looked upon with more attention and under more vigorous development, since they can be used in today automobiles with little or no modifications of engines, for replac­ing diesel and gasoline, respectively. The Directive 2009/28/CE also targets the transportation sector fuels; in particular, each member state should reach a mini­mum 10 % share of renewable energy by 2020. Complementary, the Directive also states that this must be possible by using electricity and sustainable biofuels (i. e., based on a sustainable production). It also mentions that correct sustainability cri­teria should be adopted for biofuels, so that the rising world demand for biofuels does not destroy or damage land biodiversity and establish many others’ recom­mendations to ensure total sustainability of biofuels. An interesting point of this Directive is that, it recommends member states to incentive and support the use of biofuels that add supplementary diversifying benefits, such second — and third-gen­eration biofuels (e. g., biodiesel from microalgae or bioethanol from lignocellulosic materials). Some changes were recently proposed to the Directive 2009/28/CE (EC 2012), in particular dealing with the calculation of the carbon footprint, namely how to account for the ILUC (indirect land-use changes), and setting new goals deemed more adequate to promote the growing European biofuels industry.

In this context, the overall purpose of this literature review is to provide an inte­grated assessment of the potential of microalgae as a source to produce biofuels, while confronting it with competing emerging biofuel technologies. It is intended to provide a comprehensive state of the art technology summary for producing fuels and coproducts from algal feedstocks and to draw some insights into the fea­sibility and techno-economic challenges associated with scaling up of processes.

Impressive biofuel support policies have in recent times been adopted in both the USA (with projected production of 60 billion liters of second-generation biofuel by 2022) and the European Union (with 10 % renewable energy in the transport sector by 2020). Due to the magnitude of the two markets and their sizeable biofuel imports, the US and EU mandates could become an important driver for the global development of advanced biofuels, since current scenarios from the International Energy Agency (IEA) evidence a shortfall in domestic production in both the US and EU that would need to be met with imports (US DOE 2010; EU 2010).

Although algae biofuels are not yet fully competitive in the biofuel market, many venture capital firms had made recent investments in algae fuel ventures (Oligae 2010). Accordingly, a set of policies to assist the development of microal­gae technology is being created and constantly improved. These policy incentives aim at increasing renewable energy deployment, in latu sensu, and subsequently will promote development in the algae industry.

In this context, the US Department of Energy published on May 2010 impor­tant information for the US policy trends in the “National Algal Biofuels Technology Roadmap” (US DOE 2010). This document represents the output of the National Algal Biofuels Workshop held in Maryland in 2008 and was intended to provide a comprehensive road map report that summarizes the state of algae biofuels technology and documents the techno-economic challenges that have to be met and taken into account before algal biofuel can be produced commercially.

Afterward, the US Environmental Protection Agency (U. S. EPA) suggested revisions to the National Renewable Fuel Standard program (RFS). The proposed changes intended to address changes to the RFS program as required by the Energy Independence and Security Act of 2007 (EISA). The revised statutory requirements establish new specific volume standards for cellulosic biofuel, biomass-based diesel, advanced biofuel, and total renewable fuel that must be used in transporta­tion fuel each year. The regulatory requirements for RFS will apply to domestic and foreign producers, and importers of renewable fuel (US EPA 2013).

While cellulosic ethanol is expected to play a large role in meeting the 2007 American Energy Independence and Security Act (EISA) goals, a number of next-gen­eration biofuels show significant promise in helping to achieve the 21 billion gallon goal. Of these candidates, biofuels derived from algae, particularly microalgae, have the potential to help the US meet the new renewable fuels standard (RFS) while at the same time moving the nation ever closer to energy independence (US DOE 2010).

To accelerate the deployment of biofuels produced from algae, the American President Obama and the US Secretary of Energy Steven Chu announced on May 5, 2009, the investment of US $800 Millions on new research on biofuels in the American Recovery and Renewal Act (ARRA). This announcement included funds for the Department of Energy Biomass Program to invest in the research, development, and deployment of commercial algal biofuel processes (US EPA 2013). The funding will focus on algal biofuels research and development to make it competitive with traditional fossil fuels as well as the creation of a smooth tran­sition to advanced biofuels that use current infrastructure.

Meanwhile, in order to promote the use of energy from renewable sources, the European Parliament published on April 2009, the Directive 2009/28/EC, which estab­lishes a common framework for the promotion of energy from renewable sources, as well as it establishes sustainability criteria for biofuels and bioliquids (EU 2009).

By the end of 2010, a communication from the European Parliament has set the strategy for a competitive, sustainable, and secure energy future by 2020. The stra­tegic energy technology (SET) plan sets out a medium-term strategy valid across all sectors. Yet, development and demonstration projects for the main technologies (e. g., second generation biofuels) must be speeded up (EU 2010). The European SET plan lists several energy technologies, which will be required to bring together economic growth and a vision of a decarbonized society. It states that advanced biofuels, namely microalgae, are supposed to play a significant role. EU energy policy aims to represent a green “new deal,” which will hopefully enhance the com­petitiveness of EU industry in an increasingly carbon-constrained world. However, in our dataset, it was possible to include only three European studies. In the forth­coming years, it is expected a rise in the volume of European available data, due to both the strong European transport energy policy drivers and scenarios made avail­able by the IEA regarding Energy Technologies Perspectives 2010. In this sense, incentives and targets are to be met as well as the witnessing of a higher prolifera­tion of pilot-stage algae installations in this highly oil-dependent continent.

The microalgae are photosynthetic organisms can grow in a wide variety of environments and conditions, including freshwater, salty, and brackish water (Benemann 2012). Their mechanism of photosynthesis is similar to higher plants, with the difference that the conversion of solar energy is generally more efficient because of their simplified cellular structure and more efficient access to water, CO2, and other nutrients.

Its uniqueness that separates them from other microorganisms is due to presence of chlo­rophyll and having photosynthetic ability in a single algal cell, therefore allowing easy operation for biomass generation and effective genetic and metabolic research in a much shorter time period than conventional plants (Singh and Sharma 2012).

In addition, the cultivation requirements are quite small, as most species only need water, CO2, and some essential nutrients such as nitrates, phosphates, and potas­sium, without needing the use of pesticides or fertilizers (Groom et al. 2008; Singh and Sharma 2012). Microalgae can produce lipids, proteins, and carbohydrates in large amounts over short periods of time. For these reasons, microalgae are capa­ble of producing 30 times as much oil per unit of land area compared to terrestrial oilseed (Sheehan et al. 1998). And these oil can be processed into both biofuels and valuable coproducts (Singh and Sharma 2012).

The microalgae cultivation can be heterotrophic or autotrophic. The hetero­trophic method is a biochemical conversion that relies on input feedstock derived from an upstream photosynthetic source. This approach uses closed bioreactor systems in a biochemical conversion process without light inputs. This dark fer­mentation process is based on the consumption of simple organic carbon com­pounds, such as sugars or acetate. The cultivation of algae using cellulosic sugars produced from wood and agricultural wastes or purpose-grown energy crops is an area of active research and development (Buford et al. 2012).

In the other hand, the autotrophic cultivation requires only inorganic com­pounds such as CO2, salts, and a source of light energy for their growth. This photosynthetic conversion involves two main methods: open ponds and closed photobioreactors (PBRs). The biomass produced in these autotrophic processes includes lipids that can be converted to fuels (Brennan and Owende 2010; Buford et al. 2012).

According to Benemann (2012), algae have been essentially produced in open ponds with the main strains currently being cultivated are Spirulina, Chlorella, Dunaliella, and Haematococcus. Most designs include mixing systems that use paddle wheels and carbonation techniques to supply and transfer CO2 (in-ground carbonation pit, bubble covers, and in-pound sumps1).

Microalgae are also grown in tanks and small-scale PBRs, in hundreds of dif­ferent systems around the world, producing from small amounts to huge sums of

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biomass annually. In this closed autotrophic approach, algae grow with sunlight or artificial lighting (Benemann 2012; Buford et al. 2012). Different types of PBRs have been designed and developed for cultivating algae that can be horizontal, vertical, tubular, flat, etc. (Benemann 2012; Singh and Sharma 2012). Each of these PBRs has their own advantages and disadvantages. Several studies are being developed which may overcome their limitations in the years to come (Singh and Sharma 2012).