Glycerol-free biofuels in a market flooded by the overproduction of glycerol from biodiesel utilization can be very convenient and advantageous. Ecodiesel®, DMC-Biod® and Gliperol® could be another good alternative for the future. They integrate glycerol as a by-product (MG, DMC or triacetin, respectively)
forming single homogeneous mixtures, thus avoiding the generation of waste or by-products in their preparation processes. Their preparation processes do not require any additional separation processes. MG, DMC or triacetin may be perfectly incorporated (and thus burned) with the mixture of FAMEs (or FAEEs) in diesel engines. In terms of green chemistry, glycerine incorporation into biofuels also increases the efficiency of the process (nominally from the current 90-100%), without causing substantial changes in the physical-chemical properties of biofuels. The atomic efficiency also experiences the corresponding improvement, given that the total number of atoms involved in the reaction is part of the final mixture that forms the biofuel.

The application of immobilized lipases, as heterogeneous enzymatic catalyst, may constitute a competitive procedure in the future, with respect to the current process based on basic homogeneous catalysis, because these biocatalysts are able to generate a novel family of biofuels that reduce the complexity of the process (avoid wash processes to remove the residual glycerine), increase the process yields and minimize waste generation. In addition, enzyme production processes are conducted in conditions that are comparatively more gentle (or green) to those conventionally utilized for the production of biodiesel (pH, temperature, pressure, etc.). With regard to combustion properties, relevant for the application of these biofuels in diesel engines, no important differences with respect to petroleum diesel have been found. Even better, properties including pour and cold points and lubricity are improved.

Finally, a very critical shortcoming, such as the use of water to clean/remove glycerol traces in biodiesel production, is also avoided by using these biofuels. This problem is a major issue in many southern European countries (e. g. Portugal, Italy, Spain, Greece) where draught can be a severe problem during summer.

In summary, biofuels integrating glycerol into their composition should be an urgent priority for the near future, as until now, none of them are legalized by the European Union despite several procedures being available to produce them.

Solvent toxicity proved to be the most severe limitation during the ABE fermentation process. To overcome this issue, more tolerant strains were designed (see Section 10.5) and integrated recovery techniques were applied (see Section 10.6).

While acetone and ethanol are only moderately toxic, butanol has disastrous effects on bacterial cells even at low concentrations. Already at a concentration of 1.1% (120 mM) butanol, the growth rate of C. acetobutylicum is decreased by 50% and at 1.5% (165 mM) butanol, growth is almost completely inhibited (Baer et al, 1987; Vollherbst-Schneck et al, 1984). This effect is caused by an increase in membrane fluidity (Baer et al, 1987, 1989; Ingram, 1976; Vollherbst-Schneck, 1984) and inhibition of membrane proteins such as transporters (Bowles and Ellefson, 1985; Moreira et al., 1981; Ounine et al., 1985) and ATPases (Terracciano and Kashket, 1986).

Fermentation reactions can be carried out at mesophilic (25-40°C), thermophilic (40-65°C), extreme thermophilic (65-80°C), or hyperthermophilic (>80°C)

Table 13.4 Actual wastes/wastewaters used for fermentative hydrogen production Type of waste/ wastewater Microorganisms Operation mode H2 production rate Maximum H2 yield References Rice winery wastewater Mixed culture Continuous 9.33 L/gVSS/d 3.81 L/L/d 2.14 mol/mol hexose Yu etal., 2002 Sugar factory wastewater Mixed thermophilic culture Continuous 4.4 L/L/d 2.6 mol/mol hexose Ueno ef a/., 1996 Potato processing wastewater Mixed mesophilic culture Batch 2.8 L/L wastewater Van Ginkel etal., 2005 Olive pulp Mixed mesophilic culture Continuous 0.26 L/L/d 0.19 mole/kg TS Koutrouli etal.,2009 Cheese whey Mixed mesophilic indigenous microbial culture Continuous 2.51 L/L/d 0.9 mol/mol hexose Antonopoulou etal., 2008a Dairy wastewater Mixed mesophilic culture Continuous 1.59 mmol H2/L/d Venkata Mohan etal., 2007 Molasses Mixed mesophilic culture Continuous 4.8 L/L/d Ren ef at., 2007 Food waste — sewage sludge Mixed mesophilic culture Batch 2.67 L/gVSS/d 122.9 mL/g COD carbohydrate Kim ef at., 2004 Food waste Mixed thermophilic culture Batch 0.28 8 L/gVSS/d 1.8 mol/mol hexose Shin ef al., 2004 OFMSW Mixed mesophilic culture Batch 0.4 L/g VSS/d 0.15 L/g OFMSW Lay ef al., 1999 OFMSW: organic fraction of municipal solid wastes

Crops/residues Microorganisms Operation mode H2 production rate Maximum H2 yield References Corn starch Mixed mesophilic cultures Continuous 2.57 L/L/d 0.51 mol/mol hexose added Arooj et at., 2008 Wheat starch Mixed mesophilic cultures Continuous 1.26 mol/mol hexose Hussy et ai, 2003 Sweet sorghum extract Indigenous microbial mesophilic culture Continuous 8.52 L/L/d 0.86 mol/mol hexose Antonopoulou et ai, 2008b Sweet sorghum extract Rumicococcus albus Batch 2.61 mol/mol hexose Ntaikou et al., 2008 Sweet sorghum residues Rumicococcus albus Batch 2.59 mol/mol hexose Ntaikou et ai, 2008 Wheat straw Caldicellulosiruptor saccharolyticus Batch 3.8 mol/mol glucose (44.7 L/kg dry biomass) Ivanova et ai, 2009 Maize leaves Pretreatment: enzymatic hydrolysis Caldicellulosiruptor saccharolyticus Batch 3.6 mol/mol glucose (81.5 L/kg dry biomass Ivanova et ai, 2009 Corn stalks Pretreatment: acid hydrolysis Mixed mesophilic cultures Batch 0.1824 L/d 0.150 L/kg TVS Zhang et ai, 2007a Corn stover Pretreatment: acid hydrolysis Thermoanaerobacterium thermosaccharolyticum W16 Batch 3.305 L/d 2.24 mol/mol hexose Cao et ai, 2009 Sugarcane bagasse hydrolysate Pretreatment: acid hydrolysis Clostridium butyricum Batch 1.611 L/L/d 1.73 mol/mol total sugar Pattra et ai, 2008

temperatures. Hydrogen production could be achieved either by using pure cultures of hydrogen producing bacteria grown in the dark on carbohydrate-rich substrates or by mixed acidogenic microbial cultures, selected by natural environments such as soil, wastewater sludge, and compost. At a full-scale application, a mixed culture system would be cheaper to operate, easier to control, and would have a broader choice of feedstocks (Valdez-Vazquez et al., 2005). In Tables 13.4 and 13.5 the different feedstocks used by pure or mixed microbial cultures in lab — scale experiments are presented, since data from full scale applications are not available so far.

Hydrogen production is a specific mechanism to dispose of excess electrons through the activity of the enzyme hydrogenase in bacteria. Bacteria that possess such capability include strict anaerobes (Clostridia, methylotrophs, rumen bacteria, methanogenic bacteria, archaea), facultative anaerobes (Escherichia coli, Enterobacter, Citrobacter), and even aerobes (Alcaligenes, Bacillus). Figure 13.4 presents the morphology of fermentative bacteria selected from a hydrogen producing reactor, at pH 5.5. Among the hydrogen-producing bacteria, Clostridium sp. and Enterobacter, are the most widely studied. Species of genus Clostridium such as C. butyricum (Chong et al., 2009), C. acetobutyricum (Lin et al., 2007), C. beijerinckii (Lin et al., 2007), C. thermolacticum (Collet et al.,

2004) , C. tyrobutyricum (Jo et al., 2008), C. thermocellum (Levin et al., 2006) and C. paraputrificum (Evvyernie et al., 2000) are examples of strict anaerobic and spore-forming microorganisms, generating hydrogen gas during the exponential

growth phase. In parallel, facultative anaerobes such as the species of genus E. coli and its modified strains (Manish et al., 2007) and the species of genus Enterobacter, such as E. aerogenes (Tanisho and Ishiwata, 1994; Yokoi et al., 2001) and E. cloacae (Kumar and Das, 2001) have also been used for hydrogen production. In recent years, extensive research has also been carried out in hydrogen production at high temperature, using thermophilic or hyperthermophilic bacteria, since the increase of temperature favours the reaction kinetics. The thermophiles include Caldicellulosiruptor saccharolyticus (van Niel et al., 2002), Thermoanaerobacterium sp. such as T. thermosaccharolyticum (O-Thong et al., 2008) and Thermotoga sp. such as Thermotoga maritima (Schroder et al., 1994) and Thermotoga elfii (de Vrije et al., 2002).

Degradation of glucose (or its isomer hexoses or its polymers, starch, glycogen and cellulose) by mixed microbial culture, under anaerobic conditions is accompanied by the production of hydrogen and various metabolic products, mainly volatile fatty acids ((VFAs) acetic, propionic, and butyric acid), lactic acid, and alcohols (butanol and ethanol), depending on the microbial species present and the prevailing conditions. The hydrogen yield can be correlated stoichiometrically with the final metabolic products, through the reactions describing the individual processes of acidogenesis:

For complex substrates, the hydrogen production could also be expressed in terms of hydrogen productivity (HP) which is defined as the percentage of influent substrate electrons which are distributed to hydrogen gas (gaseous and dissolved phases) (Kraemer and Bagley, 2005). It is obvious that the production of acetic and butyric acids favors the simultaneous production of hydrogen, with the fermentation of glucose to acetic acid giving the highest theoretical yield of 4 mol of H2/mol of glucose (HP = 33%) (reaction [13.7]) and the conversion to butyric acid resulting in 2 mol of H2/mol of glucose (HP = 17%) (reaction [13.8]), while the production of propionic acid consumes hydrogen (reaction [13.9]).

From the reactions [13.7], [13.8] and [13.9], it is obvious that the metabolism should be shifted towards acetate and/or butyrate production in order to achieve a high hydrogen yield. Clostridia sp. produce a mixture of acids, with butyrate in excess of acetate, upon biological degradation of glucose (Mizuno et al., 2000; Fang and Liu, 2002). In practice, the production of more metabolic products (lactate or ethanol), accompanied by a negative or zero hydrogen yield, results in lower overall yields of hydrogen (HP: 10-20%). Moreover, the metabolism towards acetate may occur via different, non-hydrogen-yielding pathways. In mixed fermentation processes, the microorganisms may select different pathways while converting sugars, as a response to changes in their environment (pH, sugar concentration, etc.). The absence or presence of hydrogen-consuming microorganisms in the microbial consortium also affects the microbial metabolic balance and consequently, the fermentation end products.

In order to harness hydrogen from a fermentative hydrogen production process, the mixed cultures need to be pre-treated in order to suppress as much hydrogen­consuming bacterial activity as possible, while still preserving the activity of the hydrogen-producing bacteria. The pre-treatment method is achieved mostly by relying on the spore-forming characteristics of the hydrogen-producing Clostridium, which is ubiquitous in anaerobic sludge and sediment (Brock et al, 1994). Treating an anaerobic sludge under harsh conditions, Clostridium would have a better chance to survive than the non-spore-forming bacteria, many of which are hydrogen consumers (Lay, 2001). Effective pre-treatment processes include heating (100°C, 15 minutes), acidic (pH = 3, adjusted with ortho-phosphoric acid, 24 hours) or basic treatment, aeration, chemicals addition (chloroform, acetylene), and application of an electric current (3-4.5 V). Another approach involves the use of the indigenous mixed microbial culture already contained in a wastewater through its activation for one day at mesophilic temperatures, a practice that has been applied and proposed by Antonopoulou et al. (2008a; 2008b). The most widely used pre-treatment method for enriching hydrogen — producing bacteria from

mixed microbial inocula is heat — pre-treatment, which combines the simplicity with the effectiveness, securing that Clostridium sp. will survive.

The most promising way to meet the sustainability goals for the future (including the reduction in GHG emissions, reduced dependence on fossil fuels, etc.) is to promote the utilisation of low-carbon technologies to convert biomass into a
variety of chemicals, biomaterials and energy, maximising the value of the biomass while minimising waste.

This integrated approach has been defined as ‘the biorefinery concept’ and has recently received a great deal of attention in many parts of the world.16,17 The biorefinery of the future will be analogous to today’s petrorefineries18,19 in such a way that many different industrial products will be generated from biomass (Fig. 1.1). These include low-value, high-volume products, such as transportation fuels (e. g. biodiesel, bioethanol, etc.), commodity chemicals and materials, as well as high-value, low-volume products or speciality chemicals, such as cosmetics and nutraceuticals.

Energy and, most precisely, biofuels are the main driver for developments in this area, but other relevant products are expected to be developed as biorefineries become more and more sophisticated with time.

Several types of biorefineries have been described in the literature, mainly phase I, II and III biorefineries, depending on the variety of feedstocks, processes and products obtained in the facilities. Biofuels are part of the products obtained from the treatment of a wide variety of biomass feedstocks, actually playing a major role in the economics of the process. This highly interesting topic will be fully tackled and expanded in the Appendix chapters (Chapters 21 and 22), in which types of integrated biorefineries, processes for biofuels production and by-products and related subjects will be revised.

Last but not least, engine tests are of utmost importance to test the feasibility of biofuels implementation in the future. In this way, Chapter 23 accounts for a nice contribution, combining a revision with experimental results on the implementation of biofuels (both pure and as blends) in engine tests.

1.1 Comparison of petro — versus biorefinery (from Introduction to Chemicals from Biomass, Edited by James Clark and Fabien Deswarte; Copyright John Wiley & Sons, 2008; reproduced with permission).

Terminalia catappa is popularly known in Brazil as ‘castanhola’ (dos Santos et al., 2008). The tree is tolerant to strong winds and moderately high salinity in the root zone (Fig. 4.6). It grows principally in freely drained, well-aerated and sandy soils.

The oil is obtained from the kernels of the fruit (that is non-edible and considered a waste), with yields around 49% w/w (Abdullah and Anelli, 1980). Biodiesel production, using either basic or acid catalysts, has been studied, concluding that basic catalysts performed more efficiently producing a yield of ca. 93% biodiesel (dos Santos et al., 2008).

Lipases are classified according to the sources from which they are obtained, such as microorganism, animal and plant. Lipase can easily be produced in high yields, by fermentation processes and few basic purification steps, from microorganisms such as fungi (e. g., Candida antarctica) or bacteria (e. g., Pseudomonas fluorescens). Lipases from animal or plant sources are rarely used in industry, and hence, the focus of this section will be on lipases from microbial sources, which have real industrial potential. Some lipases show position specificity towards the substrate, whereas others do not. Pure lipases extracted from different sources have been successfully used in the production of biodiesel; however, Candida antarctica B lipase, immobilized on acrylic resin, commercially known as Novozym 435, has been by far the most commonly used enzyme for the production of biodiesel. A comparative study on the type of free lipases from different sources revealed that

P. fluorescens lipase has the highest enzymatic activity (Iso et al., 2001; Kaieda et al., 2001). Generally, lipases from fungal sources show better transesterification activity of triglycerides compared to those from bacterial sources (Al-Zuhair et al., 2008). Table 6.1 shows examples of lipases from different sources previously used in biodiesel production.

Sugarcane production requires a tropical climate and Brazil has the largest sugarcane cultivation (about 27% of global production) and was the first and biggest producer of bioethanol in the world for many years. Sugarcane (Saccharum spp.) contains about 15% sucrose (saccharose) which is a disaccharide of hexose units (one molecule of glucose and one molecule of fructose). The chemical bonds can be broken relatively easily (e. g. by yeast Saccharomyces cerevisiae) resulting in glucose which is free and available for fermentation in the ethanol production process. The sucrose is extracted from the sugarcane by pressing the already chopped and shredded cane. The remaining solid biomass from the pressing (bagasse) is fibrous and usually used as a fuel in the sugar mill. Several steps are involved in isolating sugar as a pure solid, including several crystallisation steps, however these purification steps are not necessary in ethanol production. The sugarcane must be processed a short time after harvesting (normally within 48 hours of harvesting) to achieve the maximum yield of ethanol avoiding the possible oxidation and degradation of the sugar units.

Sugar beet (Beta vulgaris L.) is a plant whose roots contain large amount of sucrose (about 17%). Sugar beet generates good yields (more than 50 tonnes/ha) but compared to sugarcane is an energy — and chemical-intensive crop. Sugar beet cannot be cultivated more than once every three years on the same field because of the potential survival of pests in the soil. After the washing of sugar beet, the beet is sliced, pressed and the sugar content separated from water by several decolourisation and separation techniques. Mostly, European countries like France and Russia together with the USA produce most of the sugar beet in the world, e. g. the ten biggest producer countries in Europe produced 242 million metric tons of sugar beet in 2005.

The anaerobic consortium consists of several microorganism groups with different physiology that coexist syntrophically or antagonistically, resulting in a different response to environmental changes. As a consequence, when the activity of one of the microorganism groups is inhibited, the growth rates of other microorganisms are affected, changing the population balance, often causing a decrease in process efficiency or even failure. It has been recognised that the most important factors affecting the anaerobic digestion process are the pH, the temperature, the nature of the feedstock (composition, nutrients), the presence of toxic or inhibitory substances and the organic loading rate.

12.2.1 The pH

The pH affects the dissociation of weak acids and bases, and therefore, the formation of undissociated acids and bases which can easily penetrate the cellular membrane changing the internal pH of the cells. The pH also influences the function of the extracellular enzymes and has an impact on the hydrolysis rate. In most cases, the anaerobic transformation of organic matter is achieved most efficiently at a neutral pH. Many species though can grow at lower or higher pH values.

Low values of pH and the concomitant intermediate acid accumulation are more inhibitory to the methanogens than the acidogenic bacteria. Acidogens can grow and continue to produce acids at low pH values (5-6), intensifying the inhibitory conditions to the methanogens. However, it is known that methanogenesis can occur in extreme environments where very low or high pH values prevail such as swamps, hot springs, etc.

It is common to the acidogens that produce a mixture of metabolic products to switch their metabolism towards the formation alcohols to avert any further pH decrease (Huang et al, 1986; Gottschal and Morris, 1981; Lowe and Zeikus, 1991).

As shown in Fig. 3.1, the life cycle of biofuels encompasses planting, growing and harvesting of biomass (if applicable), conversion to the biofuel and its use, also including all transportation steps used in the system. Each stage in the life cycle is associated with several sustainability issues, depending on the type of the feedstock and biofuel. Some of these issues are listed in Table 3.1 (Brigenzu et al., 2009; IDB, 2009; The Royal Society, 2008). Several of the environmental and social issues, particularly those associated with land use, food security and health impacts, have been discussed in chapter 2. Here, the focus is on the life cycle environmental impacts and economic costs.

Pennisetum purpureum, also known as elephant grass (Fig. 4.14), is a species of grass native to the tropical grasslands of Africa. It is a tall (2-4.5 m) perennial plant with a very high productivity, both as a forage grass for livestock and as a biofuel crop. It is usually harvested before winter, so it can be burnt in power plants (Langeland et al., 2008).

Helianthus tuberosus

Helianthus tuberosus, also called Jerusalem artichoke, sunroot, sunchoke, earth apple and topinambur, is a species of sunflower native to the eastern United States that can grow up to 3 m high (Fig. 4.15). Due to its high alcohol yield (5000-6000 l/ha in Spain), Jerusalem artichoke also has an unused great potential as a producer of ethanol fuel from stems. Tubers are an important source of fructose for industry (Huxley et al., 1992; Davidson et al, 2006).