Descripción del equipo

Ethanol (C2H5OH) is the second member of the aliphatic alcohol series. Ethanol is also known as ethyl hydrate, ethyl alcohol or informally as alcohol (Figure 4.2). It is a volatile liquid with many uses including as a psychotropic drug, a solvent, and as a fuel among other industrial and domestic applications. It is a psychoactive drug consumed recreationally and is the primary alcohol found in alcoholic beverages (O'Leary, 2000).

Figure 4.2: Structure of ethanol (www.commons.wikimedia.org )

Renewable ethanol is generally considered to be an environmentally beneficial energy source because its emissions on combustion are less toxic than those of petrol; its production recycles the CO2 produced from its combustion and because it reduces dependence on fossil fuel (Balat, 2011). It was first used in 1897 in the internal combustion engine by Nikolas Otto (Rothman et al., 1983) and can be used in various blends in engines that use petrol (Balat et al., 2008). If its production process is powered with renewable energy, then there is no net addition of CO2 to the atmosphere. With the inevitable depletion of petroleum resources and the drive for sustainability discussed in Section 1.1.3, coupled with its ease of manufacture, renewable ethanol has been increasingly favoured and is currently considered the cleanest liquid fuel alternative (Lin and Tanaka, 2006). It is now the most widely used biofuel in transportation globally (Balat, 2011).

99 4.1.1.1 Physical Properties of Ethanol

Ethanol is a volatile colourless liquid which is completely miscible in water and organic solvents and is very hygroscopic. It has a refractive index of 1.36, a pleasant odour and a burning taste. Ethanol has a density of 0.79 g/cm³, a melting point of -114^oC and boiling point of about 78.4^oC. It is very flammable and burns with a smoke-less blue flame. It is commonly available as a 95% azeotrope but can also be obtained as absolute (100%) ethanol (O'Leary, 2000).

4.1.1.2 Chemical Properties of Ethanol

Ethanol has a molecular weight of 46 g/mol. It has about 2/3 the energy density of petrol but a higher octane rating (a measure of fuel quality), leading to improved efficiency and performance. Since it is an oxygenated fuel (35% oxygen) it burns with fewer particulates and nitrous oxide emissions upon combustion (Balat et al., 2008).

Ethanol participates in various chemical reactions including oxidation to ethanal and then ethanoic acid; dehydration to ethane; esterification with organic acids and halogenation among several others (O'Leary, 2000).

4.1.1.3 Ethanol Biosynthesis

Ethanol fermentation is a biological process that involves the decomposition of one mole of glucose into two moles of product. Ethanol is produced by several microorganisms referred to as ethanologens which are mainly yeasts such as Saccharomyces cerevisiae, Scheffersomyces stipitis, Kluyveromyces marxianus, Schizosaccharomyces pombe and Candida shehatae. It converts hexoses such as glucose and fructose to pyruvate via glycolysis which is then eventually converted into ethanol (Cardona et al., 2009; Greetham et al., 2014).

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A simplified illustration of ethanol biosynthesis pathway in S. cerevisiae is summarised in equation below.

Zymomonas mobilis rapidly ferments glucose into ethanol via the Entner-Duodoroff pathway (Macedo and Brigham, 2014).

4.1.1.4 Commercial Ethanol Production

Ethanol is the oldest synthetic organic chemical produced by man (Gupta and Demirbas, 2010). It is produced by two major industrial routes. Ethanol required for industrial use is prepared by the hydration of steam, whilst that required for food and fuel uses is mainly produced by fermentation (O'Leary, 2000). Recently ethanol production has been increasing annually and has reached the highest recorded levels in the United States. The Global Renewable Fuels alliance (GRFA) an international federation representing most of the world’s renewable fuels production reported that 88 billion litres of bioethanol were produced in 2013 (Baker, 2014). Brazil and the USA produce over 80% of the world’s ethanol annually (Demirbas, 2009) by the fermentation process (Figure 4.3).

Glucose Glyceraldehyde

-3-Phosphate Pyruvate Acetaldehyde Ethanol

Glycolysis

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Figure 4.3: World leading producers of bioethanol (AFDC, 2016)

Brazil has conducted a bioethanol program since 1931. By 2011 it produced over 62,000 m³ of ethanol from sugarcane daily and in 2012 it set a rate of 20-25% anhydrous ethanol to gasoline blend (Koizumi, 2014). The USA produces over 144,000 m³ daily (Kamm, 2014;

Koizumi, 2014) mainly from maize.

Several countries have enacted policies to support and increase the uptake of biofuels in transport. Renewable chemicals are also being supported with improved access to capital and improving legislative support in the USA (Young and Jalbert, 2016).

Microorganisms

Yeasts have several advantages for the industrial production of ethanol, for instance they are able to tolerate high concentrations of up to 150 g/l of ethanol in the fermentation broth before product inhibition sets in (Cardona et al., 2009). Also, S. pombe is able to tolerate high concentrations of salts and solids in the medium while K. marxianus can grow under thermophilic conditions which make them ideal candidates for simultaneous cellulose hydrolysis and fermentation as cellulases function at higher temperatures (Ballesteros et al., 2004). The S. cerevisiae yeast strains are the most widely employed ethanologen. They are capable of converting hexose sugars to carbon dioxide under aerobic conditions to produce mainly biomass as in the production of baker’s yeast, but produce mainly ethanol under anaerobic conditions (Cardona et al., 2009).

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A key limitation of S. cerevisiae is that it is unable to assimilate the pentoses liberated from the hydrolysis of hemicellulose such as xylose and to a lesser extent, arabinose.

Consequently, several yeasts which can assimilate both hexoses and pentoses such as S.

stipitis and C. shehatae are being utilised. However, these organisms will also more readily utilise and exhaust the hexoses, then switch to the pentoses after a short lag phase. The ethanol productivity and tolerance are however considerably lower than in S. cerevisiae and their cultures require oxygenation.

Bacterial ethanologens have also been investigated and these include the thermophiles Clostridium thermohydrosulfuricum, C. thermosaccharolyticum and C. thermocellum (Figure 4.4). These organisms have several advantages including the ability to transform pentoses into ethanol at high temperatures; saccharolytic properties which make them capable of converting untreated wastes such as lignocellulose into ethanol (Macmillan, 1997 in Cardona et al., 2009).

Figure 4.4: Various ethanol producing microorganisms. A: S. cerevisiae; B: Z. mobilis; C: S.

stipitis; D: C. thermosaccharolyticum E: C. thermocellum

(Image credits A: www.bbsrc.ac.uk B: www.ejbiotechnology.info C: genome.jgi.doe.gov; D:

www.aem.asm.org; E: www.lookfordiagnosis.com)

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The main limitation of these organisms is their low ethanol tolerance of under 30 g/l; in addition, they produce several by-products and thus have low ethanol yields. However, the bacterium Zymomonas mobilis, like S. cerevisiae, produces ethanol in quantities approaching the theoretical maximum in its native form (Darku and Richard, 2011) and both organisms are homo-ethanol fermentative. Z. mobilis is a facultatively anaerobic, Gram-negative bacterium which can utilise both hexoses and pentoses under anaerobic conditions, has a high tolerance of over 100 g/l, requires no addition of oxygen, is quite amenable to genetic manipulations, and ferments at a higher temperature. Its drawbacks include that it can only utilise a narrow range of substrates limited to simple sugars such as glucose, fructose and sucrose; the formation of levan which increases viscosity during fermentation of sugarcane syrup; and the production of sorbitol which decreases conversion efficiency (Cardona et al., 2009; Macedo and Brigham, 2014).

Genetic modification techniques are now being used to try and optimize the fermentative abilities of various bacterial and yeasts, with the greatest successes being seen in the modification of Gram-negative bacteria. One method involved the integration of pyruvate decarboxylase (pdc) and alcohol dehydrogenase genes II (adh II) from Z. mobilis into Escherichia coli followed by introduction of the fumarase reductase gene to enable it produce up to 95% of theoretical maximum yields under anaerobic conditions. Similarly, the integration of the PET (Production of Ethanol) operon (comprising pdc and adh II co-expressed under the native lac promoter) into E. coli resulted in high productivities of up to 0.72 g/l/h and hyper-tolerance to ethanol inhibition (Dien et al., 2003). S. cerevisiae has also been modified to be able to utilise xylose by the introduction of xylose isomerase genes into its genome (Macedo and Brigham, 2014).

Feedstock

Globally, the production of ethanol is carried out by the fermentation of raw materials rich in carbohydrates. There are three major classes of these feedstocks namely sugar-based feedstock such as sugar cane, beet, sweet sorghum; starchy feedstocks including maize, sorghum, cassava and wheat; and lignocellulosic feedstocks such as straw and wood.

Sugar-based materials have a unique advantage over other feedstock in their simplicity, they do not require a prior hydrolysis step as sucrose can be broken down and utilised by yeasts.

Sugarcane is the main feedstock used in global ethanol production. It is more widely

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employed in tropical countries. For example, about 79% of Brazil’s ethanol is produced from fresh sugarcane juice while sugarcane molasses accounts for the rest and for the bulk of India’s ethanol production (Cardona et al., 2009). Beet sugar is also used in ethanol production in temperate regions of the European Union such as in France which is Europe’s largest bioethanol producer; and in the United States. Sweet sorghum is similar to sugar cane in that it contains a sugar-rich juice in its stalk and is also used in tropical regions. Sweet sorghum also has the additional benefit of drought resistance (Demirbas, 2009). The main draw-back of sugar-based bioethanol substrates is the fact that they compete directly with the food uses of these crops; and their perishability.

Starchy feedstocks include grains such as maize which is used to make up to 90% of all the bioethanol in the United States (Balat et al., 2008), and wheat which is widespread in Europe.

In addition to the juice, sweet sorghum also contains a grain head which is starch-rich and which is usually hydrolysed and fermented too, or used in the production of animal feed. The starch is hydrolysed into glucose syrup which is then fermented into ethanol. Amylases and amyloglucosidases are usually used to enzymatically saccharify the starch to sugars (liquefaction) prior to fermentation by microorganisms (Demirbas, 2009). Alternatively, dilute acid could also be used to achieve the hydrolysis of the starch polymer.

Maize ethanol production in the U.S. causes more soil erosion and uses more nitrogen fertilizer than any other crop grown; as is the case with sugar cane production in Brazil (Pimentel, 2003). Furthermore, starch-based bioethanol production is expensive because of the cost of cooking the starchy material before liquefaction (Balat et al., 2008) and the high cost of liquefying and saccharifying enzymes, prior to fermentation. In addition, this 1^st gen process competes with food production and negatively impacts food prices, as a result of which the process is also discouraged as elucidated in Sections 1.3 and 1.4 and is being phased out in favour of 2^nd gen processes.

Second generation bioethanol addresses the challenges of the use of food sources. The use of two lignocellulosic materials, agricultural residues and wasted crops, could potentially yield up to 491 billion litres of bioethanol per year (Kim and Dale, 2004). However, the use of lignocellulosic feedstock is still very limited because of the aggressive and expensive pretreatment processes required to overcome the recalcitrance of lignocellulose and the low yields obtained (Gupta and Demirbas, 2010).

105 4.1.1.5 Industrial Applications of Ethanol

Ethanol is a versatile organic chemical and it is an integral part of humans’ daily lives. It is employed in a wide range of uses including domestic and industrial applications.

Transportation Fuels

As discussed in preceding sections, the most important industrial/commercial application of ethanol is as a transportation fuel. Ethanol was by far the most widely used transportation biofuel in the last decade (Figure 4.5), and the IEA estimates that by 2030 biofuels use will have increased but that bioethanol will remain the dominant biofuel (Eisentraut, 2011).

Figure 4.5: Global biofuel production 2000-2010 (Eisentraut, 2011).

Most of Brazil’s bioethanol is used domestically to substitute 40% of local petrol consumption while another 20% is exported (Balat et al., 2008). Ethanol is used in combinations with petrol known as “blends”. The most common blend in the USA is the 10%

bioethanol to 90% petrol blend known as E10 or “gasohol”; while gasohol in Brazil is 24%

bioethanol to 76% petrol (Balat et al., 2008). Higher blends above E10 such as the E85 usually require engine modifications or are used exclusively in Flex-Fuel Vehicles (FFVs),

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cars with internal combustion engines designed to run on more than one fuel. Ethanol is also used in bipropellant rockets.

Alcoholic Beverages

The most common nonfuel use of ethanol is in alcoholic beverages such as wines, spirits and beers. It is the only alcohol that can be safely drunk in moderate quantities (Ramsden, 2001).

Solvent

Ethanol is the second most widely used solvent in industrial and consumer products after water as it can dissolve a range of both polar and non-polar substances. It is a widely used solvent for paints, varnishes, cosmetics, toiletries and drugs (O'Leary, 2000; Ramsden, 2001) and being volatile, it evaporates and easily, leaving the solute behind.