Sugary biomass contains sucrose as a sugar source, a disaccharide consisting of glucose and fructose, which are both hexose monosaccharides (C6) (Fig. 2). Sucrose undergoes hydrolysis to release glucose to be converted into 1G ethanol via fermentation using a S. cerevisiae yeast (Oh et al. 2012).

Examples of sucrosic biomass include sugarcane and sweet sorghum, the lat­ter of which has a considerable concentration of free monosaccharide D-glucose when compared with sugarcane (Table 3). Sugarcane has a high content of sucrose which releases glucose after a hydrolysis step, and it is the most relevant feedstock for 1G ethanol production. However, sweet sorghum could be used as a comple­mentary crop during the sugarcane off season.

Analytical data are important for these feedstocks because we can obtain sugars content for bioethanol production. Then, we can monitor the process of conver­sion, their yields, and the product quality. It could be seen in the item 3.1.

After the process of extracting the oil from algae, the resulting product can be converted to biodiesel. The biodiesel produced from algal oil has physical and chemical properties similar to diesel from petroleum, to biodiesel produced from crops of first generation and compares favorably with the International Biodiesel Standard for Vehicles (EN14214) (Brennan and Owende 2010).

Contrasting to other sources of feedstock to produce biofuels, algae-based bio­fuels present several advantages. These advantages comprise:

1. Capability of producing oil during all year long; therefore, the oil productivity of microalgae is greater compared to the most efficient crops;

2. Producing in blackish water and on not arable land (Searchinger et al. 2008); not affecting food supply or the use of soil for other purposes (Chisti 2007);

3. Possessing a fast-growing potential and several species has 20-50 % of oil con­tent by weight of dry biomass (Chisti 2007);

4. Regarding air quality, production of microalgae biomass can fix carbon dioxide (1 kg of algal biomass fixes roughly 183 kg of CO2) (Chisti 2007);

5. Nutrients for its cultivation (mainly nitrogen and phosphorous) can be obtained from sewage; therefore, there is a possibility to assist the municipal wastewater treatment (Cantrell et al. 2008);

6. Growing algae do not require the use of herbicides or pesticides (Rodolfi et al. 2008);

7. Algae can also produce valuable coproducts, such as proteins and biomass; after oil extraction, the coproducts can be used as animal feed, medicines, or fertilizers (Spolaore et al. 2006; Brennan and Owende 2010), or fermented to produce ethanol or methane (Hirano et al. 1997);

8. Biochemical composition of algal biomass can be modulated by different growth conditions, so the oil yield can be significantly improved (Qin 2005); and

9. Capability of performing the photobiological production of “biohydrogen” (Ghirardi et al. 2000; Ferreira et al. 2013).

The above combination of the potential for biofuel production, CO2 fixation, wastewater treatment, and the possibility of production of biohydrogen highlights the potential applications of the microalgae cultivation.

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 (salt) water, on non-fertile land, and the oil yield production is far superior.

Biochemical conversion route makes use of biological/chemical agents, like microorganisms and enzymes, to break down the complex structure of the ligno — cellulose into its base polymers and further degrading them into sugar monomers (mainly glucose and xylose) (Pandey 2009). These sugar monomers can be sub­jected to microbial fermentation to produce bioalcohols (ethanol and butanol). The feedstocks that can be deconstructed using bioagents are mainly agricultural and for­est residues; however, they may also include industrial and municipal solid wastes.

The biochemical route mainly consists of four basic components: (1) feedstock pulverization, (2) pretreatment, (3) enzymatic hydrolysis, and (4) fermentation (Fig. 3). The complete process also includes feedstock harvesting, handling, recovery, and transportation; fractionation of the polymers; lignin separation; and recovery of end products (IEA 2008). The energy yield of liquid biofuels could be in the range of 2.3-5.7 GJ/tonnes of feedstock, considering 20 GJ/dry tonne of lignocellulose. The maximum energy efficiency that can be achieved is 35 % in the laboratory conditions;

however, under industrial conditions, it is yet to be known (Sims et al. 2010). As stated in the section above, other processes could be integrated such as combustion of lignin or conversion of some carbohydrates into other products of high value.

The downstream-processing step generates substantial amount of CO2, waste­water, and solid waste-containing lignin, residual carbohydrates, proteins, and cell mass. This represents about 1/3rd of the initial raw material (dry-weight basis) and can generate substantial heat and electricity upon combustion, thereby improving the overall process efficiency. The biochemical route seems to be quite promis­ing owing to its low-temperature requirements, cogeneration of heat and electricity from lignin combustion, and lower GHG emissions. At the moment, it is difficult to realize the full potential of biochemical route due to lack of data on its perfor­mance at demonstration or commercial scale units.

This study confined to catalyst-based degradation of plastics into liquid fuels by converting HDPE into a resource which can be used as feedstock for liquid bio­fuel synthesis. It emphasizes the methods using catalysts under optimum operat­ing conditions for converting HDPE into liquid and gaseous fuels for specific applications. However, the conversion methods of HDPE into fuel depend on the types of plastics to be targeted and the properties of other wastes that might be used in the process. Additionally, the effective conversion requires appropriate technologies to be selected according to local economic, environmental, social, and technical characteristics. In our study, we use HDPE as raw material, and prior to their conversion into fuel resources, HDPE was subject to various meth­ods of pretreatment to facilitate the smooth and efficient treatment during the subsequent conversion process. Because during liquid fuel production, HDPE containing liquid hydrocarbon can be used as feedstock for biofuel synthesis. It is well noted in literature that the type of plastic being used determines the pro­cessing rate as well as the product yield of hydrocarbon to be used as resource for biofuel production (Miskolczi et al. 2005). In our study, we discussed the potential application of acidic HZSM-5 and AlSBA-15 materials for catalytic degradation of HDPE into liquid hydrocarbon as feedstock for liquid biofuel using tubular batch reactor. The reaction was carried out at various catalyst load­ings between 5 and 15 % with 1:1, 2:1, and 3:1 HZSM-5 to AlSBA-15 ratios in order to optimize the reaction conditions to achieve higher hydrocarbon yield as feedstock for liquid biofuel. Catalyst characterization and variables affect­ing conversion of HDPE into liquid hydrocarbon yield have been discussed as below.

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.

Starch is a polysaccharide composed of glucose units (monomers). This poly­saccharide requires acidic hydrolysis to release the glucose monosaccharide to be fermented by S. cerevisiae yeast to produce 1G ethanol. The starch chemical structure is presented in Fig. 3. Examples of starch-containing plants include corn, potato, cassava, wheat, and barley (Table 4).

Fig. 4 Lignin structure (left) and its precursors (right): (I) p-coumaryl alcohol, (II) coniferyl alcohol, and (III) sinapyl alcohol. Author Silvio Vaz Jr

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).

The thermochemical conversion route is largely based on existing technologies that are in operation for several decades (IEA 2008). In the past, the focus was on conversion of coal to liquid fuels and chemicals; however, in the recent years, the focus has also been on the conversion of natural gas resources into fuels. The thermochemical route basically involves the production of syngas (synthesis gas), which should be purified before it can go into the Fischer-Tropsch (FT) process to synthesize liquid fuels for application in aviation and marine industries, and chem­icals chiefly synthetic diesel. Syngas (mix of CO and H2 with some CO2, meth­ane, and higher carbon compounds) is produced by a severe heat treatment process of dry lignocellulosic feedstock in a controlled atmosphere, so that gasification is
initiated. The main stages of thermochemical route involve: (1) biomass fuel con­ditioning, (2) gasification process, (3) gas purification, and (4) FT conversion.

The thermochemical route can provide a number of additional co-products in addition to biofuels. These co-products can be exploited as a feedstock for produc­tion of value-added chemicals. The products profile from FT conversion can vary significantly depending on the synthesis temperature. High temperature leads to production of synthetic gasoline and chemicals, whereas low temperature produces waxy products that can be further cracked to make naphtha, kerosene, or diesel fuel (Griffin and Schultz 2012). The advantage of thermochemical route over biochemi­cal route is that the former can essentially convert all organic component of the bio­mass into products. However, the major limitation of thermochemical route is the need of high-temperature gasifier that imparts high cost to the process.

Data on surface area and porosity are presented in Table 1. It is clear that the HZSM-5 zeolite possessed typical textural properties of microporous materials. The pore volume and size of the catalyst HZSM-5(14) were almost half as com­pared to those of HZSM-5 (80). This showed that a reduction in the aluminum content of the HZSM-5 catalyst affected the material by increasing the poros­ity while simultaneously reduced the total surface area. However, in the case of SBA-15 catalyst (AlSBA-15), significantly larger surface area than HZSM-5 was observed. This confirmed the mesoporous nature of AlSBA-15 that exhibited wider mesopores as suggested by data in Table 1. These findings were in accord­ance with those reported by Kilos et al. (2005) who also observed the mesoporous nature of aluminum-functionalized SBA-15 catalyst.

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).