10.2.1 Organisms

Microbial butanol synthesis was first noticed by famous French scientist Louis Pasteur in 1862 (Pasteur, 1862). While his organism Vibrion butyrique presumably represented a mixed culture, a pure culture was isolated a few years later by Albert Fitz (Fitz 1876, 1877, 1878, 1882). Around the turn of the twentieth century, further butanol-producing bacteria were isolated by many other scientists, amongst them are Martinus Beijerinck and Sergei Winogradsky. Probably, all of these isolates were members of the genus Clostridium, a term that was only used as a morphological description (from Greek kloster = small spindle) at that time. However, most of these strains were lost over the years (Durre, 2001; Durre, 2005a; Durre and Bahl, 1996).

In 1913, Charles Weizmann isolated a strain, which produced significantly higher butanol yields (Weizmann, 1915) and later became known as Clostridium acetobutylicum (McCoy et al., 1926). In succession, many similar strains were isolated and also designated as C. acetobutylicum. Only at the beginning of the 1990s, it was discovered that actually four different species (C. acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum and Clostridium saccharoperbutylacetonicum) were industrially used (Jones and Keis, 1995; Keis et al., 2001).

Other Clostridium species (see Table 10.1 and Fig. 10.1) are able to form minor amounts of butanol as well (Durre, 2005a; Durre and Bahl, 1996). However, aside from this genus, only Thermoanaerobacterium thermosaccharolyticum (formerly Clostridium thermosaccharolyticum; Collins et al., 1994), Butyribacterium methylotrophicum and the archeon Hyperthermus butylicus are known to produce 1-butanol (see Table 10.1 and Fig. 10.1). While the respective mechanisms in these organisms are still unclear (Brugger et al., 2007; Grethlein et al., 1991), butanol formation in clostridia has already been investigated extensively.

Under certain conditions, green algae and cyanobacteria can use water-splitting photosynthetic processes to generate molecular hydrogen. Biophotolysis-based hydrogen production can be carried out via direct or indirect means as identified by whether or not light is irradiated during hydrogen evolution (Benemann, 1998). A brief description of the principles, the systems (Fig. 13.2) and the main bottlenecks for the practical application of both processes are given below.

13.2 Direct (a) and indirect (b) biophotolysis of water.

Process Microorganisms Feedstock Advantages Disadvantages H2 synthesis rate mmol H2/L/h Direct biophotolysis Green algae Chlamydomonas reinhardtii Water, sunlight H2 production directly from water and sunlight Solar conversion energy increased by tenfold as compared to trees, crops High intensity of light 02 can be dangerous for the system Lower photochemical efficiency 0.07 Indirect biophotolysis Cyanobacteria Anabaena variabilis Water, sunlight H2 production from water Ability to fix N2 from atmosphere Removal of uptake hydrogenase enzymes because of H2 degradation 30% 02 present in gas mixture Sunlight requirement 0.355 Photo­ fermentation Photosynthetic bacteria Rhodobacter spheroides Biomass, sunlight A wide spectral light energy can be used Different organic wastes as substrates Inhibitory effect of 02 on nitrogenase Low light conversion efficiency 0.16 Biological water gas shift Photo-heterotrophic bacteria Rhodospirillum rub rum Rubrivivax gelatinosus CO Low temperatures and pressures High conversion efficiency Demand of CO source Inhibitory effect of 02 96 Dark fermentation Fermentative bacteria Enterobacter aerogenes Clostridium butyricum Mixed microbial cultures Biomass H2 production all day, without light independence Variety of carbon sources as substrates Valuable metabolites, as byproducts No 02 limitation Low obtained H2 yields 02 is a strong inhibitor of hydrogenase C02 present in the gas Requirement of further treatment of the fermentation effluent, before disposal 8.2-121 Microbial Electrolysis Cells Electrode reducing microorganisms Geobacter, Shewanella sps Biomass, electricity Higher yields No use of 02 Electricity supply Competitive methane generation 5.8

Direct biophotolysis

In this process, electrons are generated from water through photosynthesis and then transferred via an electron carrier — ferredoxin (Fd), to hydrogen producing enzyme — hydrogenase, to produce hydrogen. Microalgae, such as green algae and Cyanobacteria (blue-green algae), containing hydrogenases, have the ability to produce hydrogen. Well-known cyanobacteria that have been found to produce hydrogen in lab scale bioreactors are Anabaena sp. such as Anabaena cylindrical (Weissman and Benemann, 1977), Anabaena variabilis (Sveshnikov et al., 1997; Borodin et al., 2000) and Synechococcus (Howarth and Codd, 1985). Chlamydomonas reinhardtii is the representative of green microalgae for biohydrogen production (Tsygankov et al., 2006; Griesbeck et al., 2006; White and Melis, 2006). Other algal species such as Chlorococcum littorale and Platymonas subcordiformis have also been investigated for hydrogen production (Schnackenberg et al., 1996; Guan et al., 2004).

The main drawback of direct biophotolysis is that the process is limited because of the strong inhibition of hydrogenase by the oxygen produced. Thus, for the sustainability of hydrogen production, it is necessary to maintain the oxygen content at a low level, below 0.1% (Hallenbeck and Benemann, 2002). In practice, it is very difficult to maintain such low partial pressures of oxygen, without additional energy and cost demands. For example, neutral gases such as helium could be sparged in the reactor, in order to eliminate oxygen, but the supplemental cost of helium and hydrogen dilution, makes this solution unacceptable. Another approach involves the addition of oxygen absorbers (Hallenbeck and Benemann, 2002) but now this seems not practical at larger scale.

Other limitations, such as the low light conversion efficiencies and the requirement for large photobioreactors, make the process impractical for large — scale application as it becomes inefficient from an economical point of view. It ought to be mentioned that a number of approaches to improve H2 production by green algae are currently under investigation. These include genetic engineering of light gathering antennae (Polle et al., 2002), optimization of light input into photobioreactors (Gordon, 2002) and improvements to the two-phase H2 production systems used with green algae (Laurinavichene et al., 2002a; Tsygankov et al., 2002). Another challenge is the modelling and simulation of photolytic systems to support systems design and optimization.

Green seed canola oil is a low-quality green oil. This particular colour is due to the high chlorophyll content that is retained in the mature canola seeds due to exposure to sublethal frost (0-5°C) during seed development (Johnson-Flanagan et al, 1990). Compared to green seed canola oil, pure canola oil has a crystal yellow colour with low chlorophyll content and is produced from canola seeds with low green seed content.

The cost of processing green seed canola oil for edible purposes is high. Oil with high chlorophyll content cannot be used for manufacture of margarine since chlorophyll can inhibit the activity of hydrogenation catalyst (Abraham and de Man, 1986). Also, this oil cannot be used for edible purposes because the high chlorophyll content seriously affects the stability of the oil, causing rapid formation of oxidation products via the photosensitised singlet oxygen pathway (Rawls and Van Santen, 1970). The oxidative degradation of oil produces a number of volatile products that provide a bad odour to the oil. To remove chlorophyll, bleaching can be used; however, this can have a deleterious effect on the stability of a vegetable oil (Tautorus and Low, 1994). Processing conditions used for bleaching could produce new compounds from chlorophyll derivatives in the crude oil. Thus, green seeds are not recommended for feeding purposes. The percentage of green seeds is one of the major gradation factors for canola seeds.

However, tests to produce biodiesel from green seeds oils have been successfully performed. Kulkarni et al. (2006) found that the cloud point of green seed esters is lower than that of pure canola oil esters due to higher content of linoleic and linolenic acids. Furthermore, green seed esters have also been proposed as additive and the use of 1% (v/v) added to ultra-low sulphur diesel fuel to reduce the wear scar area and increase the lubricity has been recommended. Oxidation stability of methyl ester obtained from green seed canola oil is lower (4.9 hours at 110°C) than the European Standard EN 14214. Biodiesel originating from green seed canola oil shows good fuel quality parameters, but its oxidative stability needs to be improved to be considered a viable diesel fuel alternative (Kulkarni et al., 2006).

FAMEs production that meet specifications and standards can be easily obtained using refined vegetable oils or animal fats and the appropriate processing conditions. However, when alternative feedstocks are utilized including cooking oils or non-refined oils/fats, a post-treatment is required in order to reach standard properties.

Three different post-treatments are most generally employed in this regard: distillation, adsorption and filtration. The most effective is biodiesel distillation in order to remove non-volatile contaminants such as steryl glucosides, phospholipids, soaps, dimeric and polymeric materials and inorganic salts. However, the distillation has to be performed at ca. 200°C at 1 Mbar which is not an energy-friendly operation. The refining of biodiesel via vacuum distillation is illustrated by using used cooking oils (Ensungur, 2008; Zyaykina et al., 2009). In the refining step, a number of polar dimeric, polymeric and oxidation compounds have been formed together with the FFAs, di — and monoglycerides and trans fatty acids, while dimeric and polymeric FAMEs are generated during transesterification. Accordingly, the biodiesel obtained in this process will have a high viscosity and a very low oxidative stability. Used cooking oil containing 24% polar compounds was transesterified producing a biodiesel with a viscosity of 6.4 mm2/s. After distillation, the viscosity was reduced to 3.7 mm2/s. However, the oxidative stability was further reduced from 3.3 hours (before distillation) to 1.5 hours (after distillation).

Distillation is lowering the viscosity below the maximum limit but at the same time the oxidative stability is decreased as the natural anti-oxidants (tocopherols) are not distilled and remain in the distillation residue. Addition of synthetic anti­oxidants is necessary to reach to oxidative stability of 6 hours.

Distillation has however a favorable influence on the cold filtration properties of biodiesel (cold soak filter test and filter plugging point) due to removal of mono-, diglycerides and steryl glucosides during distillation.

In future, due to the expected high stringent standards, distillation is looking the most favorable process to produce biodiesel in compliance with all the standards and quality demands. Adsorption by magnesium silicate was also reported as an efficient procedure in order to upgrade the biodiesel quality (Bertram et al., 2009).

Table 5.4 shows the results of dry washing with 1% Magnesol®R60, at 70°C for 20 minutes, followed by filtration.

The most important result is that the heated biodiesel contained lower soap content and had higher oxidative stability. Similar results have been obtained for rapeseed oil and yellow grease feedstock. Biodiesel can also be purified by cellulose derivatives produced by Rettenmaier. Filtracel®EFC plus are silica gel encapsulated fibers combining the excellent filterability of cellulose filter aids with the excellent adsorption properties of silica gel in just one product.

It works as filter aid and adsorbent to remove soaps, trace metals, phospholipids and other polar oil contaminants, being more efficient than bleaching earth. Some Filtracel grades have been additionally activated by citric acid which chelates non-hydratable soaps, phospholipids and metals by converting them into a hydratable form for adsorption. These cellulose derivatives are acting as desiccant and also remove hazy cloudiness. Biodiesel is chilled to crystallize free sterol glucosides, which are removed by filtration.

There are many microalgae, yeasts (e. g. Candida, Cryptococcus, Lipomyces, Rhodotorula, Rhodosporidium, Trichosporon), fungi (e. g. Mortierella, Cunninghamella) and bacteria that can accumulate intra-cellularly high amounts of SCO that has fatty acid composition similar to vegetable oils (Meng et al., 2009). Microorganisms can be characterized as oleaginous in the case that they can accumulate SCO to more than 20% of their total cellular dry weight (Ratledge, 1991). SCO could be used either for value-added applications (e. g. food additives) or commodity uses (e. g. biodiesel production). The industrial application of SCO for biodiesel production is dependent on the development of a fermentation process that provides high carbon source to SCO conversion yields, high productivities, high lipid content in cellular biomass and high S CO concentrations. The previous criteria constitute a useful tool so as to select the appropriate microorganisms that will facilitate the industrial implementation of biodiesel production from SCO. For instance, microalgae may accumulate high amounts of microbial lipids but they cannot compete with oleaginous yeast and fungi because their cultivation requires a big area and long fermentation duration. Furthermore, bacteria may achieve high growth rates but the majority of bacterial strains accumulate relatively low amounts of SCO (up to 40% of total cellular dry weight) (Meng et al., 2009). Some yeast strains (e. g. Rhodosporidium sp., Rhodotorula sp., Lipomyces sp.) may accumulate intra-cellularly around 70% (w/w) of SCO (Guerzoni et al, 1985; Li et al, 2007; Angerbauer et al., 2008; Meng et al., 2009).

Table 8.1 shows that mainly yeasts and some fungi may offer appropriate cell factories for the production of SCO. Table 8.1 clearly demonstrates that cell densities up to 185 g/L with a lipid content up to 67.5% (w/w) have been achieved mainly in fed-batch cultures or continuous fermentations with recycling (Yamauchi et al., 1983; Pan et al., 1986; Ykema et al., 1988; Meesters et al., 1996; Li et al., 2007). In many cases, SCO has similar fatty acid composition as in the case of vegetable oils used for biodiesel production. SCO is mainly composed of triacylglycerols — TAGs — with a fatty acid composition rich in C16 and C18, namely palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1) and linoleic (18:2) acids (Meesters et al., 1996; Ratledge and Wynn, 2002; Li et al., 2007; Meng et al., 2009). The SCO produced by C. curvatus has similar composition to palm oil (Davies, 1988). The SCO produced by Yarrowia lipolytica contains stearic, oleic, linoleic and palmitic acid (Papanikolaou et al., 2002a).

There is a remarkable plethora of (pure or raw agro-industrial) substrates that can be used by oleaginous microorganisms for microbial growth and accumulation of microbial lipids (Table 8.1). Production of SCO implicates utilization of pure sugars as substrates (e. g. analytical glucose, lactose, etc.) (Moreton, 1985; Moreton and Clode, 1985; Aggelis et al., 1996; Papanikolaou et al., 2004a, 2004b; Li et al., 2007; Zhao et al., 2008; Fakas et al., 2009a), sugar-based renewable materials or sugar-enriched wastes (Ykema et al., 1989, 1990; Davies et al., 1990; Papanikolaou et al., 2007a; Fakas et al., 2006, 2007, 2008a, 2008b, 2009a), vegetable oils (Bati et al., 1984; Koritala et al., 1987; Aggelis and Sourdis, 1997), crude-waste industrial hydrophobic materials (e. g. industrial free-fatty acids, waste fats, crude fish oils, soap-stocks etc) (Guo et al., 1999; Guo and Ota, 2000; Papanikolaou et al., 2001, 2002a, 2007b; Papanikolaou and Aggelis, 2003a, 2003b), pure fatty acids (Mlickova et al. 2004a, 2004b) or glycerol (Meesters et al., 1996; Papanikolaou and Aggelis 2002; Mantzouridou et al., 2008; Andre et al., 2009; Makri et al., 2010). This indicates that it is feasible to utilize various natural resources for the production of SCO providing the opportunity to develop processes producing SCO-derived biodiesel either integrated in existing food industries or as individual production plants (e. g. in agricultural areas so as to utilize various lignocellulosic feedstocks).

Starch-based waste or by-product streams (e. g. wheat flour milling by-products, waste bread, flour-based waste or by-product streams from the confectionary industry) generated by the food industry or collected as disposed food by dedicated companies could be used for the production of glucose-based fermentation media. Wheat flour milling by-products has been considered for the production of biofuels and platform chemicals (Neves et al., 2007; Dorado et al., 2009) and therefore could be regarded as a potential feedstock for the production of SCO-derived biodiesel. In the case of SCO production, certain oleaginous microorganisms have the ability to consume both glucose and xylose. This

Table 8.1 SCO production from various microorganisms, carbon sources and cultivation modes Microorganism Cultivation Carbon Total dry MO content Productivity Reference mode source weight (g/L) (%, w/w) (g L-1 h-1) Yeast species Yarrowia lipolytica single-stage Glucose 9.2 25 0.08 Aggelis and continuous Komaitis, 1999 Yarrowia single-stage Crude glycerol 8.1 43 0.11 Papanikolaou and lipolytica continuous Aggelis, 2002 Yarrowia shake flask Stearin 15.2 52 N. A. Papanikolaou lipolytica etai, 2007 b Candida sp. 107 single-stage continuous Glucose 18.1 37.1 0.4 Gill etai, 1977 single-stage continuous Glucose 13.5 29 0.16 single-stage continuous Sucrose 16 28 0.18 Candida curvata single-stage Lactose 18 31 0.22 Evans and continuous single-stage continuous Xylose 15 37 0.27 Ratledge, 1983 single-stage continuous Ethanol 11.5 35 0.2 Apiotrichum curvatum batch Glucose 14.5 45.6 N. A. Hassan etai., 1993 Apiotrichum curvatum batch Whey 21.6 36 0.119 Ykema et ai, 1988 recycling 85 35 0.372 continuous 20 36 0.382 partial recycling 91.4 33 0.995 Cryptococcus curvatus fed-batch Glycerol 118 25 0.59 Meesters etai., 1996 Lipomyces starkeyi shake flask Glucose & Xylose 20.5 61.5 N. A. Zhao et ai, 2008 Lipomyces starkeyi shake flask Glucose & 9.4 68 N. A. Angerbauer

153

24.1

19.5

17.1 16.9

21.5 36.4

28.6

22.7 23.6

15.8 106.5

9.60

25 185

15

13.5

4.1

27

10.4

8.4

27

62

indicates that it will be feasible to consume the major carbon sources in wheat flour milling by-products (i. e. glucose from starch and xylose from hemicelluloses). Waste bread and other starch-based food could be collected prior to disposal by dedicated companies and could be used for the production of SCO derived biodiesel. Waste bread has been evaluated for the production of bioethanol (Ebrahimi et al., 2008). Furthermore, waste or by-product streams from the confectionary industry that contain mainly starch and sucrose as carbon sources could be considered as potential feedstocks for SCO production.

Other waste streams from the food industry that could be used for the production of SCO-derived biodiesel are whey and molasses. Whey constitutes a significant waste stream from the dairy industry and its valorization is an important environmental target. The yeast strain Cryptococcus curvatus can accumulate intra-cellularly a SCO content of around 60% (w/w) of the total cell dry weight during fermentation on whey or other agricultural and food processing wastes (Ratledge 1991; Meesters et al., 1996). In addition, molasses (a by-product from sugar refining) has been used as fermentation medium in shake flask cultures for the production of SCO by the yeast Trichosporon fermentans to produce 36.4 g/L total dry weight with an SCO content of 35.3% (w/w) (Zhu et al., 2008).

As indicated in Table 8.1, certain oleaginous microorganisms can utilize glycerol for the production of SCO (Meesters et al., 1996; Papanikolaou and Aggelis, 2002). Therefore, crude glycerol generated from biodiesel production plants could be recycled for the production of SCO-derived biodiesel. More importantly, the ability of some oleaginous microorganisms to consume various sugars derived from lignocellulosic biomass (e. g. xylose, mannose, galactose, cellobiose) could lead to the utilisation of lignocellulosic biomass for the production of SCO-derived biodiesel (Zhu et al., 2008; Huang et al., 2009).

Biorefineries should depend entirely on crude biological entities for the formulation of fermentation media that will contain all the necessary nutrients for microbial growth and SCO accumulation. In order to implement this principle, protein-rich industrial waste streams should be used for the production of fermentation media enriched in organic sources of nitrogen (e. g. amino acids, peptides), phosphorus, minerals, vitamins and trace elements. Such nutrient supplements for fermentation processes could be produced from oilseed residues generated after oil extraction in the first-generation biodiesel production plants (e. g. protein-rich rapeseed or sunflower cakes), meat-and-bone meal, sewage sludge, protamylase (residual stream enriched in amino acids and peptides that is generated during the industrial production of starch from potatoes), corn steep liquor and residual yeast from potable or fuel ethanol production plants. Protein and other nutrients are also contained together with carbon sources in various food waste streams (e. g. waste bread, whey). Therefore, in many cases, a single waste stream from the food industry could be sufficient for the production of nutrient-complete fermentation media for SCO production. It should be stressed that organic N-sources may enhance lipid accumulation (even two or three times higher than the amount of lipids accumulated with inorganic N-sources) in certain oleaginous microorganisms (e. g. Rhodosporidium toruloides, Trichosporon cutaneum and T. fermentans) (Evans and Ratledge, 1984a, 1984b; Zhu et al., 2008).

The conversion of waste streams into fermentation media would require the development of advanced upstream processing strategies that exploit the full potential of complex biological entities. Similar upstream processing schemes have been developed in the case of cereal conversion into bioethanol, biodegradable plastics and platform chemicals (Arifeen et al., 2007; Koutinas et al., 2007b; Du et al., 2008; Xu et al., 2010). In addition, pre-treatment technologies that have been developed for the generation of fermentation feedstocks for bioethanol production could be adapted in the case of SCO-derived biodiesel production (Lloyd and Wyman, 2005; Zhu et al., 2009).

Based on the maximum theoretical conversion yields of glucose to SCO (0.33 g/g) and bioethanol (0.51 g/g) and the lower heating values (LHVs) for SCO — derived biodiesel (37.5 MJ/kg) and bioethanol (26.7 MJ/kg), then the LHV per kg glucose that could be generated via fermentative production of SCO and bioethanol is 9% higher in the case of ethanol. However, the overall energy balance (output/ input) could be favourable in the case of SCO-derived biodiesel because it is expected that the energy required to produce biodiesel after SCO fermentation would be lower than the energy required to purify bioethanol from fermentation broths. This will also result in surplus lignin that will be used for chemical production when lignocellulosic biomass is used as raw material. In the case of bioethanol production, all lignin is required for energy generation for the plant. In addition, biodiesel production from SCO would create a sustainable supply of glycerol that is regarded as an important building block for the chemical industry. For instance, we could combine biodiesel production from SCO with biodegradable polymer (e. g. polyhydroxyalkanoates) and platform chemical (e. g. 1,3-propanediol, succinic acid, itaconic acid) production from crude glycerol generated during biodiesel production (Jarry and Seraudie, 1997; Papanikolaou et al., 2000; Lee et al., 2001). We should also highlight the well-understood efficiency of diesel engines which lead to a lower level of CO2 emitted per kilometre travelled.

S. D. MINTEER, St Louis University, USA

Abstract: Bioethanol and biobutanol are the two most commonly discussed bioalcohols for fuel purposes. However, other alcohols can be produced from biomass. This chapter will introduce other bioalcohols and discuss their production. These bioalcohols include: biomethanol, biopropanol, bioglycerol, bioethylene glycol, as well as branched chain bioalcohols and other theoretical biofuels. It will compare the advantages and disadvantages of employing different fuels on the basis of both availability and ease of production as well as chemical, biological, and physical properties of these fuels.

Key words: biomethanol, glycerol, biopropanol, bioalcohols, biofuel production.

11.1 Introduction

When researchers think bioalcohols, bioethanol and biobutanol are the first alcohols that come to mind, because they are the most prevalent and the most frequently researched.1-4 These are commonly considered the alcoholic fuels in the United States, because of the ability to produce them from corn and corn by-product, which is a large agricultural product in the Midwestern United States. However, other bioalcohols can be produced and they each have their own advantages and disadvantages which will be described below.

In general, we consider an alcohol to be a bioalcohol if it is produced from biomass. There are many forms of biomass, including: wood and wood residue; agricultural crops and waste by-products; municipal solid waste, animal waste, and sewage; waste from food processing; and algae and aquatic life.5 This chapter will discuss not only the bioalcohols that can be produced from biomass, but also the traditional methods and types of biomass employed to produce the bioalcohols. This is important when considering biofuels and their use as a renewable energy source. Different countries have different biomass sources and, therefore, there will not likely be a single bioalcohol/biomass solution for renewable energy for all countries and all applications. Other issues to consider when choosing a bioalcohol fuel are the toxicity of the fuel or fuel by-products, the volatility of the fuel, and the energy density of the fuel.

Biochemical production of other bioalcohols 259

In developed and developing countries facing fluctuations of oil prices, the improvement of energy security and supply is increasingly becoming a fundamental reason for implementing biofuels policies. Rich and industrialised countries driving their economies on fossil fuels and oil products and derivates are experiencing shortage of finite resources with a consequent high risk of depletion and exhaustion. In addition, intensification of trade in oil commodities creates trade unbalances for those countries which are strongly dependent on imported energy commodities such as the European Union, United States, China, Japan and India.

In a number of countries, regulation is currently being adopted or under scrutiny to favour energy supply and safety. The following description will focus on the European Union, United States and Brazil. In the European Union, a new set of energy regulations are changing current and future scenarios of energy use and supply. The Commission Directive 2009/28/EC on the ‘promotion of the use of energy from renewable sources’ which abolishes the previous Biofuels Directive (Commission Directive, 2003/30/EC) and the Commission Directive 2001/77/EC on electricity from renewables. The new legislation body put in place an exclusive framework for renewable energy production within Member States. In particular, the Directive 2009/28/EC sets reference values of energy from renewables computed from estimates of gross final demand by 2020.

These reference values correspond to the achievement of the European Union ‘20-20-20’ strategy which is a fundamental voluntary policy adopted in March 2007 by the European Commission to further attain the goals of the Kyoto Protocol. The 20-20-20 policy establishes by 2020 to reach a target of 20% reduction of greenhouse gases (GHGs) by using 20% renewables. Given this ambitious scenario, Member States are required to set their shares of energy from renewables and create measures to promote the development of a competitive energy market ensuring access to electricity network from renewables. The Directive also promotes biodiversity protection of threaten species in those lands where biodiesel and bio-liquids production would have negative impacts on flora and fauna. Raw materials used in biodiesel and bio-liquids production should therefore achieve the status of ‘sustainable’, by competent bodies, before being processed.

In the longer term, the 2007 Renewable Energy Road Map (European Commission, 2007) specifies the adoption of a minimum ten per cent consumption of biofuels in the transport sector. Biofuels use in the transport sector would contribute to 14% of total market fuels (corresponding to about 43 million tonnes of equivalent oil) and the share may increase from either current bio-ethanol production in Sweden or biodiesel production in Germany and other European Union countries or other feedstock such as ethanol from straw, rapeseed oil, palm oil and second-generation biofuels mainly obtained from wood processes (De Lucia, 2010).

Over the last decades, Brazil has become one of the major biofuels producers. Although regulation on biodiesel entered into force in 2004, Brazilian production of biofuels is mainly centred on ethanol from sugar cane. Contrarily to biodiesel, ethanol is being processed since 1975 which makes Brazil the second-largest producer of transport fuels over a 30-year period. The abundance of land and proper climate conditions for sugar cane production and the possibility of transport subsidies ensuring full ethanol distribution within the country are important factors for the evolution of such industry. Several reasons have been adopted in favour of governmental support for biofuels in Brazil. These vary from purely economic-profit oriented ones to those including environmental concerns, energy security and rural development. Energy safety nonetheless was encouraged since the oil crisis during the 1970s when Brazil had to overcome national debt crisis by borrowing foreign capital. Ethanol production was then seen as a safe way to reduce import and interest costs. Simultaneously to the expansion of the ethanol industry, major employment creation occurred in the biofuels sector favouring the expansion of unskilled workers in rural areas and the formation of more than 60 000 small-sized farmers countrywide (Moreira, 2006).

The success of the Brazilian experience also lies behind a direct or indirect connection with several synergies such as those with other economic sectors. In this case, established relationships with the sugar and electricity and heat production markets are relevant. The sugar market played a primary role in driving the ethanol growth within and outside the country. On the supply side, the degree of price elasticity between sugar and ethanol (e. g. 0.20, Elobeid and Tokgoz, 2008) and the international volatility of sugar prices pushed Brazilian farmers toward ethanol production. Productivity of the ethanol sector also rose substantially to more than 100% (Moreira, 2006) during the 25 years period from 1975 to 2000. The electricity and heat production industry were also fundamental to boosting biofuels production as these served both the internal and foreign markets with using by-products from sugar cane. By the end of 2010, the amount of electricity from biomass mainly obtained from sugar mills is expected to be around 7.8 GW (Empresa de Pesquisa Energetica, 2008). The Brazilian government played nonetheless an essential role for the enhancement of the biofuels industry. In particular, it provided incentivising measures (see also Section 2.5) throughout the entire biofuels chain production (including support to technological advances in the sector) and to final end-users. Most of all, the establishment of a transparent institutional framework has guaranteed full competitiveness within markets. However, it was not until recent years, where consumer habits for fuel-switching engine cars increased rapidly, that ethanol production took off considerably. In 2006, 75% of new car models were produced with fuel-switch technology engine. New sugar mills implementations are expected to be operative by 2010 (Empresa de Pesquisa Energetica, 2008) and generate diversified energy and food output: from electricity grids, to biodiesel plants, to rotation plantations for food crops.

Under Obama’s presidency, the United States (joint world leader of biofuels production with Brazil) is currently experiencing a revision of its Renewable Fuel Standard (RFS) policy (Environmental Protection Agency, 2010) adopted under the Energy Policy Act (EPA) in 2005. Recent economic recession and other factors (i. e. the existing mismatch between biofuels distribution requirements and current infrastructures for petroleum industry) are preventing the United States reaching its congressional goals of the Energy Independence and Security Act (U. S. Senate, 2007). This requires 100 million gallon biofuels from biomass by 2010 and 36 billion gallon per year by 2022. Sustainability of supply chain is being threatened by high transaction costs in meeting the requirements between feedstock production and research and rural wealth. Likewise, although the accomplishment of expected results from currently funded projects, lack of integration at all levels of government is also causing delays achieving national biofuels targets (Environmental Protection Agency, 2010). The existence of a 15 billion gallon cap on ethanol biofuels from cellulosic by 2022 is posing further challenges to EPA’s current policies for distribution, transportation and storage of current and future ethanol production. The need for a new strategy is desirable. The new 2010 and beyond EPA programme on renewable fuels released on 10 February 2010 by the President’s Biofuels Interagency Working Group (2010) intends to adopt a strategic approach to optimise and integrate biofuels production development at all levels. This would mean not only to ensure coordinated measures for research, demonstration and commercialisation phases, but also guarantee coherence and efficiency of management across government funding, farmers and companies.

To ensure management efficacy in the biofuels industry, the creation of a small management team was proposed to help establish deliverables and corrective measures to keep projects on track, monitoring results throughout the entire biofuels supply chain and report progress works to the Biofuels Interagency Working Group. The reinforcement of the biofuels supply chain management is also established by the involvement of federal departments such as the Office of Science for research issues; the Feedstock Development and Production units at the USDA addressing environmental, economic and education concerns for biofuels chain; Department of Energy Efficiency and Renewable Energy to assist the setting up and development of pilot projects; and other departments at EPA and USDA for monitoring and regulatory procedures, sustainability issues, policy support and technical assistance. It is vital for integrating various efforts put in place from this multitude of agencies and departments and also for the success of deliverables and targets to ensure a continuum in the biofuels chain management. EPA’s strategy is also pursuing first — and second-generation biofuels development together with boosting third-generation biofuels advances through financial support actions, feasibility studies, technological improvements and new markets for corn-based ethanol production. Finally, a fundamental aspect of the EPA’s biofuels management is an integrated approach to economic, environmental and social concerns.

Feedstock for bioethanol production is essentially composed of sugar cane (Fig. 4.7) or molasses (by-product of sugar mills) and sugar beet (Fig. 4.8) (UNCTAD, 2006). Two-third of the world sugar production is from sugar cane and one-third is from sugar beet. Sugar cane is grown in tropical and subtropical countries, while sugar beet is only grown in temperate climate countries.

While Brazil is the world’s largest producer, in European countries, Spain is the largest producer of bioethanol, and beet molasses are the most utilised sucrose — containing feedstock (Cardona and Sanchez, 2007). Sugar beet crops are grown in most of the European Union (EU) member states, providing 90% of the total EU demand of sugar. The advantages of sugar beet are a lower cycle of crop

production, higher yield and high tolerance of a wide range of climatic variations, low water and fertiliser requirement. Compared to sugar cane, sugar beet requires 35-40% less water and fertiliser (Balat et al., 2008).

Cellular biomass of oleaginous yeasts and filaments fungi (Miao and Wu, 2006) has also been evaluated as a cheap source of renewable raw materials for biodiesel production. In addition to being cheap, using microalgae to produce biodiesel will not compromise production of food and other products derived from crops. Above that, oil crops, waste cooking oil and animal fat cannot realistically satisfy the demand required to achieve the target of replacing all current transport fuel consumed with biodiesel. This scenario changes dramatically, if microalgae are used to produce biodiesel. It has been reported that for microalgae of 30% oil content per weight of biomass, the oil yield per hectare is estimated at 58.7 m3 per hectare (Chisti, 2007). This is almost ten times the yield of palm oil, and the difference becomes even higher if compared to microalgae of 70% oil content per weight of biomass. It appears therefore that microalgae are the only source of biodiesel that has the potential to completely displace fossil diesel. Microalgae commonly double their biomass within 24 h. However, during exponential growth period, doubling times are as short as 3.5 h (Chisti, 2007). In addition, oil content in microalgae may exceed 80% by weight of dry biomass (Spolaore et al., 2006).

Besides microalgae, other oil producing heterotrophic oleaginous microorganisms have been used to produce biodiesel (Ratledge and Wynn, 2002). Nevertheless, heterotrophic production is not as efficient as using photosynthetic microalgae, because the renewable organic carbon sources required for growing heterotrophic microorganisms are produced ultimately by photosynthesis, usually in crop plants, which brings us back to square one.

The use of oils from microalga, Chlorella protothecoids for large-scale biodiesel production using immobilized Candida sp. lipase has been reported (Li et al, 2007). Algal oils have been largely produced through substrate feeding and heterotrophic fermentation. In their work, Li et al. (2007) achieved an increase in the lipid content up to 48% of the cell dry weight of the microalga. The oils were then used as raw material to produce biodiesel using immobilized Candida sp. lipase.

Historically, the production of ethanol was developed thousand years ago when it was produced as wine from grapes. Ethanol was also produced from grains. For many years ethanol has been produced by the catalytic addition reaction of water to ethene which is a fraction from oil refining. But a sustainable ethanol production needs renewable raw materials (feedstock) and cost-efficient methods to be able to replace ethanol made from a fossil precursor. There are some differences in the processing technology between first generation feedstocks (sugar feedstock, starchy feedstock) and second generation feedstocks (lignocellulosic feedstock).

Generally, commercial bioethanol production requires several steps:

• Preparation of the feedstock to achieve maximum yield of the feedstock and also its sugar content.

• Preparation (actually size reduction) of the feedstock to achieve the right (optimal) physical size and form of the raw material in the ethanol production process. This also reduces the transport cost of the feedstock.

• Pre-treatment of the feedstock to release cellulose, starch or sucrose from lignin, fibre and other biological parts of the raw material.

• Hydrolysis of the feedstock to achieve partial or complete hydrolysis of the simple and complex polymeric molecules to produce sugar units. This hydrolysis might be either thermochemical hydrolysis or a combination of thermochemical and biochemical hydrolysis.

• Fermentation of the sugar units from the hexose fraction to ethanol by yeast.

• Fermentation of the sugar units from the pentose fraction to ethanol by other microorganism or enzymes.

• Several purification and distillation steps.

A schematic figure of different steps in bioethanol production is shown in Fig. 9.5.

Preparation of the feedstock is an important part of ethanol production since a substantial part of the ethanol production cost is the price of the feedstock, depending on what feedstock is used. Therefore, it is essential to optimise the yield of the feedstock with a high amount of fermentable sugar content. Preparation of the first generation feedstock usually includes cutting the material to proper size and form, e. g. sugarcane chopped and milled (dry or wet milling) and corn or woody materials chopped as chips. An optimum size of the feedstock reduces the transport cost and thereby production cost of bioethanol. Size reduction also increases the contact surface of the feedstock with the pre-treatment catalysts.

In the pre-treatment, the raw material is subjected to a mechanical or thermochemical treatment to make the carbohydrate polymers (cellulose and hemicellulose) available for hydrolysis. In the hydrolysis process, the polymers are hydrolysed into fermentable sugar units. Depolymerisation of lignocellulosic feedstock releases both pentoses and hexoses, depending on what type of feedstock is used. When the sugars have been released, fermentation takes place. The fermentation is an anaerobic catabolism of sugar by one or several microorganisms. After the fermentation step the ethanol concentration varies between 4% and 15% depending on what kind of feedstock is used (first or second generation) and what process. The ethanol is then purified by filtration and/or distillation steps. The ethanol concentration is increased to a maximum of 95% after distillation and after the absolutisation (drying) step it is more than 99% pure ethanol. The distillation and absolutisation steps require lots of energy. There are both similarities and differences between ethanol production techniques using first generation feedstocks and second generation feedstocks, and therefore the processing technology for ethanol production is separated into different sections.