Environmentally Sustainable Biofuels – The Case for Biodiesel, Biobutanol and Cellulosic Ethanol

Abstract Due to diminishing petroleum reserves and the deleterious environmental consequences of exhaust gases from fossil-based fuels, research on renewable and environmentally friendly fuels has received a lot of impetus in recent years. With oil at high prices, alternate renewable energy has become very attractive. Many of these technologies are eco-friendly. Besides ethanol, other alternatives are: biodiesel made from agricultural crops or waste cooking oil that is blended with diesel; biobutanol; gas-to-liquids (GTL) from the abundance of natural gas, coal, or biomass; oil trapped in the shale formations such as found in the western United States, and heavy oil lodged in Canadian tar sands. In this chapter, we examine advances made in environmentally friendly fuels such as biodiesel, biobutanol, and cellulosic ethanol in recent years. Keywords Biodiesel · Cellulosic ethanol · Biobutanol · Lipase · Microalgae · Microbial · Enzymatic

1 Introduction
According to the Energy Information Administration [1], currentestimates of worldwide recoverable reserves of petroleum and natural gas are estimated to be 1.33 trillion barrels and 6,186 trillion cubic feet, respectively. The world consumes a total of 85.4 million barrels per day of oil [2] and 261 billion cubic feet per day of natural gas [3]. The US consumes 24.6% of the world’s petroleum (2), 26.7% of the world’s natural gas (3), and 43% of the world’s gasoline (1). At current consumption levels, worldwide reserves of oil will be exhausted in 40 years, and reserves of natural gas in 60 years.

P.T. Vasudevan (B) Department of Chemical Engineering, University of New Hampshire, Durham, NH 03824, USA e-mail: vasu@unh.edu

O.V. Singh, S.P. Harvey (eds.), Sustainable Biotechnology, DOI 10.1007/978-90-481-3295-9_3, C Springer Science+Business Media B.V. 2010


P.T. Vasudevan et al.

Along with diminishing petroleum reserves, the price of oil and natural gas has increased dramatically. A barrel of crude oil reached a record high price of $147.27 in July 2008, which is an increase of 1,190% over the $12.38 per barrel price in July 1998 [4]. Due to the rapid increase in the price of oil, the price per gallon of regular unleaded gasoline increased from $1.08 in July 1998 to $4.09 in July 2008 [5], representing an increase of 379%. As the price of petroleum increased, so did corporate profits. Exxon/Mobil reported a second-quarter profit of $11.68 billion in August 2008, when gas prices were the highest [6]. The concentrations of heat-trapping greenhouse gases in the atmosphere have significantly increased over the pastcentury due to the burning of fossil fuels, such as oil and coal, combined with deforestation. As a result, the average temperature of the Earth’s surface is increasing at an alarming rate [7]. The issue of climate change is one of the key challenges facing us and it is imperative that steps are taken to reduce greenhouse gas emission. The combination of diminishing petroleum reserves (it is generally believed that we reached a global “peak” oil or a global Hubbert’s peak in 2006 [8]), and the deleterious environmental consequences of greenhouse gases has led to an urgent and critical need to develop alternative, renewable and environmentally friendly fuels. Examples include biodiesel, biobutanol, and cellulosic ethanol; the topics of this chapter. Biodiesel is a renewable, non-toxic [9], biodegradable alternative fuel, which can be used in conjunction with or as a substitute for petroleum diesel fuel. Biodiesel is made entirely from vegetable oil or animal fats by the transesterification of triglycerides and alcohol in the presence of a catalyst. An advantage is that compression-ignition (diesel) engines, manufactured within the last 15 years, can operate with biodiesel/petroleum diesel at ratios of 2% (B2), 5% (B5), or 20% (B20), and even pure biodiesel (B100), without any engine modifications. Biodiesel contains no polycyclic aromatic hydrocarbons, and emits very little sulfur dioxide, carbon monoxide, carbon dioxide, and particulates, which greatly reduces health risks when compared to petroleum diesel. Butanol is a four-carbon alcohol that can be produced frompetroleum or biomass, and is currently used as an industrial chemical solvent. Biobutanol is an advanced biofuel that has an energy density, octane value, Reid vapor pressure (RVP), and other chemical properties similar to gasoline [10]. Without any engine modifications, it can either be blended at any ratio with standard grade petroleum gasoline or used directly as a fuel. Biobutanol can be produced from the fermentation of sugars from biomass or by the gasification of cellulosic biomass. Compared to gasoline, the combustion of butanol reduces the amount of hydrocarbons, carbon monoxide, and smog creating compounds that are emitted [11]. Cellulosic ethanol is ethyl alcohol, a two-carbon straight-chained alcohol, which is produced from wood, grass, or other cellulosic plant material, particularly the non-edible portions. Ethanol produced from renewable sources can be used as a high-octane biodegradable motor fuel, and is clean burning. It can be used in current automobile engines in blends up to 10% with gasoline (E10) without any engine modifications, and in higher percentages (E85 and E100) in Flex Fuel Vehicles (FFVs). Biomass consists of cellulose, hemicellulose, and lignin, which



requires pretreatment before processing. Enzymatic saccharification followed by fermentation and fermentation using cellulolytic microorganisms are the two main processing techniques used for the production of cellulosic ethanol. In this chapter, we will examine the current state of the art in the production of biodiesel, biobutanol and celluloseethanol, respectively.

2 Biodiesel 2.1 Background
Over the past decade, interest in biodiesel use has grown due to the increasing price of petroleum and the effect of carbon emissions on climate change. Biodiesel is a non-toxic and biodegradable alternative fuel, which can be used in conjunction with or as a substitute for petroleum diesel fuel. The first account for the production of biodiesel was in 1937 by the Belgian professor G. Chavanne of the University of Brussels, who applied for a patent (Belgian Patent 422,877) for the “Procedure for the transformation of vegetable oils for their uses as fuels” [12]. The chemical structure of biodiesel is that of a fatty acid alkyl ester, which is clean burning [13]. Biodiesel contains no polycyclic aromatic hydrocarbons, and emits very little sulfur dioxide, carbon monoxide, carbon dioxide, and particulates, which greatly reduces health risks when compared to petroleum diesel. The first diesel engine was created in 1893 by a German mechanical engineer, Rudolph Diesel. The diesel engine is an internal compression-ignition engine that uses the compression of the fuel to cause ignition, instead of a spark plug for gasoline engines. As a result, a higher compression ratio is required for a diesel engine, which for the same power output (when compared to a gasoline engine), is more efficient and uses less fuel. The higher compression ratio requires the diesel engine to be built stronger so it can handle the higher pressure; consequently, the longevity of a diesel engine is generally higher than its gasoline equivalent. Thesevehicles therefore require less maintenance and repair overall, thus saving money [14]. In the European markets, over 40% of new car sales are diesel. This is due to a large influx of highly efficient diesel engines used in small cars. An advantage of biodiesel is that current compression-ignition (diesel) engines, 15 years old or newer, can operate with pure biodiesel, or any blend, with no engine modifications. Older engine systems may require replacement of fuel lines and other rubber components in order to operate on biodiesel. The current infrastructure for petroleum diesel fuel can be utilized for biodiesel, thus reducing costs and widespread implementation criteria. The Environmental Protection Agency (EPA) in 2006 limited sulfur emission in diesel fuels to 15 ppm. New trucks and buses with diesel engines, from model year 2007, are now required to use only ultra low sulfur diesel (ULSD) with new emissions control equipment. The higher sulfur levels aided in diesel fuel lubrication; however, biodiesel is oxygenated and therefore is naturally a better lubricant and has similar material compatibility to ULSD. Many



countries are utilizing biodiesel’s lubrication properties to blend with ULSD so that expensive lubricating additives are not needed [15]. The production of biodiesel is from the transesterification of triglycerides or by the esterification of fatty acids, which are both found in grease, vegetable oils, and animal fat. The transesterification of the triglycerides with a short chain alcohol (such as methanol, ethanol, propanol, orbutanol) along with a catalyst, results in fatty acid esters (biodiesel) and glycerol as a by-product. The generalized transesterification reaction is given by the following stoichiometry 1[triglyceride] + 3[alcohol] ↔ 3[fatty acid ester (biodiesel)] + 1[glycerol] The fatty acids are almost entirely straight chain, mono-carboxylic acids that typically contain 8–22 even number carbons. Fatty acids are obtained mainly from soybean, palm kernel, and coconut oils and from the hydrolysis of hard animal fats. The esterification of the fatty acids with a short chain alcohol along with a catalyst, results in a fatty acid ester (biodiesel) and water as a by-product. The generalized esterification reaction is given by the following stoichiometry 1[fatty acid] + 1[alcohol] ↔ 1[fatty acid ester (biodiesel)] + 1[water

2.2 Feedstock
The large-scale production of a renewable and environmentally sustainable alternative fuel faces several technical challenges that need to be addressed to make biodiesel feasible and economical. The two main concerns with any renewable fuel are raw materials and the technologies used for processing. Advances in genetic modification and other biotechnologies are resulting in new or modified feedstocks that have significantly increased the yields of alternative fuels, such as genetically modified Clostridium to improve alcohol production [16]. Technological advancements are also being made to convert the feedstocks into fuels by improving techniques or developing completely new and environmentally friendly approaches to biofuel production. There are manyfeedstocks for biodiesel production such as virgin oils, biomass, algae, and waste oils, to name a few. Feedstocks also vary with climate and location and what might be a great source in one place may not be a good source in another. A considerable amount of research has been done using edible sources of virgin oils from vegetables, like soybean, rapeseed, sunflower seed, and canola oils, to produce biodiesel. However, oil with water or high free fatty acid content can result in the formation of soap as a by-product. Therefore, additional steps must be taken to prevent soap formation, which requires the utilization of more resources. The production of biodiesel has increased demand for soybean oil from 1.56 billion pounds in 2005–2006, to 2.8 billion pounds in 2006–2007 [17]. The increasing demand for virgin vegetable oil stocks has lead to an increase in price of these oils. The profitability of biodiesel relies heavily on the cost of its feedstock. The costs of




soybean oil can account for up to 75% of the final cost per gallon of biodiesel. This has resulted in crops being sold as fuel crops, reducing the food supply and leading to an increase in food prices around the world. To help with this issue, many oil-bearing non-edible plants have been investigated for the production of biodiesel. These are mainly tree species that can grow in harsh environments, such as Jatropha curcas, Pongamia pinnata, Castor, Mohva, Neem, Sal, etc. Jatropha curcas has the most significant potential due to its characteristics and growth requirements[16, 18]. It requires very little fertilizer and water (as little as 25 cm a year), is pest resistant, and can survive in poor soil conditions such as stony, gravelly, sandy or saline soils. Most important, it is fast growing, and can bloom and produce fruit throughout the year with a high seed yield. Optimized production has been found to yield an average of more than 99% of Jatropha biodiesel [19], which has comparable fuel properties to that of diesel from petroleum. It is expected that some varieties of Jatropha can produce as much as 1,600 gal of diesel fuel per acre-year compared to the wild variety that produces about 200 gal/acre-year [20]. Jatropha trees can capture four tons of carbon dioxide per acre and the fuel emits negligible greenhouse gases. There is a growing interest in using algae as a feedstock for biodiesel production within the United States. Algae have become an appealing feedstock due to their aquatic environment providing them an abundant supply of water, CO2 , and other nutrients. This results in a photosynthetic efficiency that is significantly higher than the average land based plants [21]. However, the power required to use artificial lighting to grow an aquatic species, such as microalgae, for the production of a biofuel would greatly reduce the overall efficiency of the process [22]. As the algae convert carbohydrates into triglycerides, the reproduction rate slows down so that the higher oil storing strains of algae reproduce at a much slower rate than lower oil storing strains [23]. This was shown by the Department of Energy’s (DOE) AquaticSpecies Program, which found the overall yield to decrease as the algae’s oil storage increased. Recently, Vasudevan and Briggs [21] summarized research on biodiesel production in a review article. According to them, a crude analysis of the quantum efficiency of photosynthesis can be done without getting into the details of the Calvin cycle; rather simply by looking at the photon energy required to carry out the overall reaction, and the energy of the products. In general, eight photons must be absorbed to split 1 CO2 and 2 H2 O molecules, yielding one base carbohydrate (CH2 O), one O2 molecule, and one H2 O (which, interestingly, is not made of the same atoms as either of the two input H2 O molecules.) With the average energy of “Photosynthetically Available Radiation” (PAR) photons being roughly 217 kJ, and a single carbohydrate (CH2 O) having an energy content taken to be one-sixth that of glucose ((CH2 O)6 ), or 467 kJ/mole, we can calculate a rough maximum efficiency of 26.9% for converting captured solar energy into stored chemical energy. With PAR accounting for 43% of incident sunlight on earth’s surface [24], the quantum limit (based on eight photons captured per CH2 O produced) on photosynthetic efficiency works out to roughly 11.6%. In reality, most plants fall well below this theoretical limit, with global averages estimated



typically at between 1 and 2%. The reasons for such a difference generally revolve around rate limitations due to factors other than light (H2 O and nutrient availability, for example), photosaturation(some plants, or portions of plants receive more sunlight than they can process while others receive less than they could process), and photorespiration due to Rubisco (the protein that serves ultimately as a catalyst for photosynthesis) also accepting atmospheric O2 (rather than CO2 ), resulting in photorespiration. In the US, the average daily incident solar energy (across the entire spectrum) reaching the earth’s surface ranges from 12,000 to 22,000 kJ/m2 (varying primarily with latitude). If the maximum photosynthetic efficiency is 11.6%, then the maximum conversion to chemical energy is around 1,400–2,550 kJ/m2 /day, or 3.8 × 1012 J/acre-year in the sunniest parts of the country. Assuming the heating value of biodiesel to be 0.137 GJ/gal, the maximum possible biodiesel production in the sunniest part of the US works out to be approximately 28,000 gal/acre-year, assuming 100% conversion of algae biomass to biodiesel, which is infeasible. It is important to keep in mind that this is strictly a theoretical “upper limit” based on the quantum limits to photosynthetic efficiency, and does not account for factors that decrease efficiency and conversion. Based on this simple analysis though, it is clear that claims of algal biodiesel production yields in excess of 40,000 gal/acreyear or higher should be viewed with considerable skepticism. While such yields may be possible with artificial lighting, this approach would be very ill-advised, as at best only about 1% of the energy of the energy used to power the lights would ultimately be turned into a liquid fuel (clearly, one needsto look at the overall efficiency). This upper limit also allows us to assess how truly inefficient many crops are when viewed strictly as biofuel producers. With soybeans yielding on average 60 gal of oil (and hence biodiesel) per acre-year, the actual fuel production is staggeringly small in comparison to the amount of solar energy available. This should further make it clear that using typical biofuels for the purpose of electricity generation (as opposed to the transportation sector) is an inefficient means of harnessing solar energy. Considering that photovoltaic panels currently on the market achieve net efficiencies (for solar energy to electrical energy) on the order of 15–20%, with multi-layer photovoltaics and solar thermal-electric systems achieving efficiencies of twice that in trial runs, biomass to electricity production falls far behind (considering typical plant photosynthetic efficiencies of 1–2%), with conversion of that biomass energy to electrical energy dropping the net efficiency to well under 1%. Currently, the research for algae growth for fuel production is being done using photobioreactors. Unfortunately, current designs demand a high capital cost, which makes large-scale production uneconomical until a low cost design or new method of production is discovered. Storing energy as oil rather than as carbohydrates slows the reproduction rate of any algae, so higher oil strains generally grow slower than low oil strains. The result is that an open system (such as open raceway ponds) is readily taken over by lower oil strains, despite efforts to maintaina culture of higher oil algae. Attempts to grow higher oil extremophiles, which can survive in extreme conditions (such as high salinity or alkalinity) that most other strains cannot tolerate, have yielded poor results, in terms of the net productivity of the system. While an



extremophile may be able to survive in an extreme condition, that doesn’t mean it can thrive in such conditions. Many research groups have therefore turned to using enclosed photobioreactors of various designs as a means of preventing culture collapse or takeover by low oil strains, as well as decreasing the vulnerability to temperature fluctuations. The significant downside is the much higher capital cost of current photobioreactor designs. While such high costs are not prohibitive when growing algae for producing high value products (specialty food supplements, colorants, pharmaceutical products, etc.), it is a significant challenge when attempting to produce a low value product such as fuel. Therefore, substantial focus must be placed on designing much lower cost photobioreactors and tying algae oil production to other products (animal feed or fertilizer from the protein) and services (growing the algae on waste stream effluent to remove eutrophying nutrients, or growing nitrogen fixing algae on power plant emissions to remove NOx emissions). An additional challenge, when trying to maximize oil production with algae, is the unfortunate fact that higher oil concentrations are achieved only when the algae are stressed – in particular due to nutrientrestrictions. Those nutrient restrictions also limit growth (thus limiting net photosynthetic efficiency, where maximizing that is a prime reason for using algae as a fuel feedstock). How to balance the desire for high growth and high oil production to the total amount of oil produced is no small task. One of the goals of DOE’s well-known Aquatic Species Program was to maximize oil production through nutrient restriction; however their study showed that while the oil concentration went up, there was a proportionally greater drop in reproduction rate, resulting in a lower overall oil yield. One approach to balancing these issues has been successfully tested on a small commercial scale (2 ha) by Huntley and Redalje [25], using a combination of photobioreactors and open ponds. The general approach involves using large photobioreactors for a “growth stage”, in which an algal strain capable of high oil content (when nutrient restricted) is grown in an environment that promotes cell division (plentiful nutrients, etc.) – but which is enclosed to keep out other strains. After the growth stage, the algae enter an open raceway pond with nutrient limitations and other stressors, aimed at promoting biosynthesis of oil. The nutrient limitations discourage other strains from moving in and taking over (since they also require nutrients for cell division). Waste oils, such as restaurant grease and spent frialator oil, can also be used in the production of biodiesel. This eliminates the “food or fuel” debate that affects virgin edible oil sources. These waste oils normally cost money forrestaurants and other establishments to dispose off. This can have a negative feedstock cost which reduces the overall cost of production. However, like virgin oils, traditional processes of converting waste oils to biodiesel can result in soap formation due to the presence of water and free fatty acids. The waste oils usually contain particulates that require filtration or separation prior to processing. Demand for waste oil as a biodiesel feedstock has already resulted in companies now paying restaurants for their waste vegetable oil (WVO). Quantities of WVO are limited (it is estimated to be about 1.1 billion gallons per year in the US), but it is certainly a good option for producing biodiesel.




2.3 Comparison of Technologies
A conventional base-catalyzed reaction is used in the majority of transesterification processes to produce biodiesel. Sodium hydroxide is used as the catalyst when methanol is the acyl acceptor, and potassium hydroxide is used when ethanol is the acyl acceptor, due to solubility considerations [15]. The ethyl esters have a slightly higher energy value than the methyl esters due to the presence of the additional carbon atom, and ethanol can be more easily produced from renewable sources, such as corn. Typical reactions take place with a high molar ratio of alcohol to oil of about 6:1 with methanol, and 12:1 for ethanol [15]. The excess alcohol allows for complete conversion of the triglycerides to the fatty acid esters. An advantage of base-catalyzed transesterification is the relatively short reaction time toachieve conversion levels of 98% or greater, compared to other processes. The reaction is a direct process, needing no intermediate steps, and operates at a relatively low temperature and pressure of about 66a—Š C and 1.4 atm, respectively. However, a major disadvantage of the base catalyzed process is the formation of soap when water or free fatty acids are present in the feedstock. Thus the feedstock should be anhydrous but the process still requires a large amount of base to be added to neutralize the fatty acids [15]. Soap formation results in additional downstream separation problems combined with a reduction in the fatty acid ester yield. The process also requires two steps and uses large amounts of chemicals as catalysts. Acid-catalyzed transesterification is a viable alternative, in which sulfuric acid is typically used. One advantage over the base-catalyzed method [26] is that it is not as susceptible to soap formation. The resulting downstream product is easily separated and produces a relatively high quality glycerol byproduct. The process also requires only one step, compared to two steps in the base-catalyzed process. However, acid-catalysis reactions are slower and result in lower yields than basecatalysis, ranging from 56.8 to 96.4% depending on the feedstock [27]. A major disadvantage to either base or acid transesterification process is the disposal of the glycerol byproduct. Glycerin is already inexpensive, easily available, and is used in a wide array of pharmaceutical formulations. The major issue is with the purity of the glycerin; the byproduct glycerinfrom the production of biodiesel is 80–88% while industrial grade is 98% or higher [15]. The low market value of glycerin does not make purification economical. Many researchers are investigating innovative chemical and biological processes for the conversion of glycerin into value-added products including antifreeze agents, hydrogen, and ethanol [28]. A relatively new and promising development in the production of biodiesel is via enzymatic transesterification with lipase as the catalyst. Several microbial strains of lipases have been found to have transesterification activity; Pseudomonas cepacia [29], Thermomyces lanuginosus [30], and Candida antarctica [31] are a few that have been reported. The products of an enzyme-catalyzed reaction can easily be collected and separated. Unlike alkali-based reactions, enzymes can be recycled since they are not used up and require much less alcohol to perform the reaction. However, enzyme reactions take much longer to complete and can have lower yields due to inhibition of the enzyme caused by glycerol formation. Methanol, the acyl




acceptor, can also strip the essential water from the active site of the enzyme, resulting in deactivation of the enzyme. Enzymes are also expensive and require treatment such as immobilization, purification, pre-treatment, and modification [32]. New technologies are being developed to produce biodiesel that do not form glycerol as a byproduct. The hydrocracking process uses hydrocracking, hydrotreating, and hydrogenation reactions to convert a wide range offeedstocks to biodiesel with yields of 75–80% [15]. This process is currently being utilized in petroleum refineries and uses a conventional commercial refinery hydrotreating catalyst. However, the hydrocracking process requires hydrogen, which is primarily obtained from natural gas. To reduce the costs of hydrogen, the process could be easily integrated with a refinery. The production of biodiesel has significantly increased over the past few years. The National Biodiesel Board reports an increase in production from 250 million gallons in 2006 to 450 million gallons in 2007, an increase of 55.6%. European countries produced 5.7 million tons of biodiesel in 2007 ( 1.5 billion gallons), which is an increase of 16.8% from 2006 according to the European Biodiesel Board. Germany is the World leader in biodiesel production and produced 2.9 million tons ( 790 million gallons) in 2007, which is over 50% of the European biodiesel market.

2.4 Summary
Biodiesel is a clean-burning fuel that is renewable and biodegradable. A recent United Nations report urges governments to beware of the human and environmental impacts of switching to energy derived from plants. There should a healthy debate about turning food crops or animal feed into fuel and the consequences of the switch to biofuels needs to be carefully thought out. The focus of biodiesel production needs to be on sources like waste oil and grease, animal fats, and non-edible sources. Current research has focused on these areas as well as on algae-based biofuels. Many technical challenges remain and these include development ofbetter and cheaper catalysts, improvements in current technology for producing high quality biodiesel, use of solvents that are non-fossil based, conversion of the byproducts such as glycerol to useful products such as methanol and ethanol, and development of low cost photobioreactors.

3 Biobutanol 3.1 Background
Over the past few years, butanol made from biomass, popularly known as biobutanol, has gained a lot of attention as a biofuel. Butanol is an alcohol-based fuel that contains four carbons and has chemical properties similar to that of gasoline, thus making it an attractive substitute or additive. Biobutanol can be produced from the

fermentation of sugars from biomass or by the gasification of cellulosic biomass. It can be blended in any ratio with gasoline and be used in existing automobiles without any need for engine or fuel line modifications. It is an attractive substitute to gasoline because its BTU content is 110,000 BTU’s per gallon, which is very close to the 115,000 BTU per gallon of gasoline, resulting in little change to fuel economy. The Reid vapor pressure (RVP) of butanol (0.33 psi) is low compared to ethanol (2 psi) or gasoline (4.5 psi), resulting in lower evaporative emissions. The octane values and energy density of butanol are also closer to gasoline than is ethanol. Ethanol is 100% soluble in water whereas the solubility of butanol is 9.1% at 25a—Š C [10]; this results in less water absorbed and rust dissolved into the fuel from tanks and pipelines. An added benefit to the low solubility is reducing thespread into groundwater in case of a spill. However, biobutanol is not a perfect fuel and has several disadvantages. Butanol is more toxic to humans and animals than lower carbon alcohols. The LD50 oral consumption for a rat for butanol is 790 mg/kg compared to 7,060 mg/kg for ethanol [13]. However, it is well known that gasoline contains chemicals such as benzene, which is toxic and carcinogenic. There have been no definitive tests as to whether butanol will degrade the materials in an automobile over time, but current evidence suggests that this is unlikely [10]. Environmental Energy, Inc. tested a 1992 Buick Park Avenue by driving it 10,000 miles on 100% butanol [33]. No modifications were done to the car and it passed all emission tests performed in 10 states with an average increase in gas mileage of 9%. Compared to gasoline, combustion of butanol reduces the amount of hydrocarbons, carbon monoxide, and smog-creating compounds that are emitted [33]. Butanol is used as an industrial solvent and the market demand is about 350 million gallons a year worldwide, with the United States accounting for 63%. The production of butanol via fermentation is the second oldest fermentation process, next only to ethanol. Since the 1950s however, production of butanol via fermentation has not been an economically viable alternative due to the historic low cost of petroleum. A new push for renewable alternative fuel sources has been fueled by the increasing cost of petroleum combined with the generation of more greenhouse gases. These two reasons and the development of new technologiesform the underpinnings of the reemergence of the butanol fermentation process.

3.2 Comparison of Processes
The oldest method of butanol production is the acetone-butanol-ethanol (ABE) bacterial fermentation by Clostridium acetobutylicum, which dates back to Louis Pasteur in 1861 [13]. The bacterial microorganism, C. acetobutylicum, was first isolated by Weizmann [13]. In the ABE fermentation process, C. acetobutylicum produces acetic, butyric, and propionic acids from glucose that can be generated from various biomass sources. Potential feedstocks include corn, molasses, whey permeates, or glucose. An enzyme catalyzed reaction of acetoacetyl-CoA transfers



CoA to acetate forming acetyl-CoA. Through a series of metabolic reactions, butyryl-CoA is produced from acetyl-CoA, which is then converted to butanol in the solventogenic pathway [33]. Acetyl-CoA can also produce ethanol and acetone from acetoacetyl-CoA. A typical process produces acetone, butanol and ethanol in the ratio 3: 6:1. The butanol yield from the ABE fermentation of glucose is relatively low, about 15–25 wt% typically [33]. This is due to the buildup of acetic, butyric, and propionic acids along with the products acetone, butanol, and ethanol, during the fermentation process. The solvents are toxic to C. acetobutylicum. The butanol destabilizes the cell membrane of the microorganisms ultimately resulting in cell death. Higher yields can be achieved by continuously removing the harmful solvents, mainly butanol, and/or by genetically modifying strains ofmicroorganisms that can tolerate higher concentrations of butanol [33]. A butanol-tolerant mutant strain of C. acetobutylicum has been developed and designated as SA-1 [34]. This strain shows a 121% improvement in butanol tolerance over the typical strain used in ABE fermentation. The enhancement of the strain results in an overall increase in butanol production of 13.2%. Additional advantages of the mutated strain are an increase in growth rate, more pH resistance, more effective utilization of carbohydrates, and reduction in acetone concentration by 12.5–40% [34]. Other studies using genetic and metabolic engineering have modified strains, which have resulted in an increase of about 320% in the final butanol concentration [35]. The antisense RNA process helps down-regulate genes for butyrate formation by acidogenesis and increases the butanol yield through solventogenesis. The process has resulted in strains with butanol yields of 35% [36]. Tetravitae Bioscience has combined a patented mutant strain of C. beijerinckii and a continuous, integrated fermentation process that utilizes gas stripping. C. beijerinckii is a species of rod-shaped anaerobic bacteria that is known for the synthesis of organic solvents, and uses a broader substrate range and better pH range than C. acetobutylicum. The solvent genes of C. beijerinckii are located on the chromosome, which is more genetically stable than on the plasmid for C. acetobutylicum. The gas stripping process prevents the butanol concentrations from reaching toxic levels by sparging oxygen-free nitrogen or fermentation gasesthrough the fermentation solution and the ABE captured in the gas are condensed [13]. The exhaust gas is then recycled back to the reactor to collect more ABE for removal. Advantages of this method are the low energy requirements, the fact that it does not remove important acid intermediates, and that it allows for efficient recovery of butanol [37]. Environmental Energy Inc. (EEI) and Ohio State University (OSU) have developed a two-step anaerobic fermentation process in a joint project to produce butanol from biomass. The first process converts the feedstock carbohydrates into butyric acid through acidogenesis using C. tyrobutyricum. The second step converts the butyric acid, using C. acetobutylicum, into butanol, which results in a significant improvement from conventional processes. The butanol solution requires purification from a recovery unit after the second step reactor. EEI’s process uses a purification process that takes advantage of the azeotrope formed by butanol


(55%) and water (45%), which is used to minimize the energy required for distillation. These processes utilize OSU’s proprietary fibrous-bed bioreactor (FBB) that has demonstrated improvements in long-term production with a scalable packing design. The packing consisted of a spiral-wound, fibrous matrix that allows for a high surface area with large enough voids to allow for a high cell density. Immobilizing the cells in the FBB minimizes the energy consumption required by the cells [33]. British Petroleum (BP) has partnered with DuPont to commercialize biobutanol usingadvanced metabolic pathways for 1-butanol. They have announced plans to produce 30,000 tons per year of biobutanol at the British Sugar facility in Wissington, UK. This will help meet the United Kingdom s Renewable Fuels Obligation set for 2010. Along with 1-butanol, they plan on developing biocatalysts to produce higher octane isomers such as 2-butanol and iso-butanol, and to increase the interest and utility as a fuels additive or substitute [38]. BP and Dupont plan on initially marketing biobutanol to the current market as an industrial solvent and then implement a larger commercialization into fuel blending by 2010 [38]. A different approach to producing butanol utilizes a thermochemical route for the gasification of biomass by a syngas catalyst. W2 Energy Inc. is working to produce biobutanol from a Gliding Arc Tornado plasma reactor (GAT) for biomass gasification. The GAT is a non-thermal plasma system, which utilizes reverse vortex flow that allows for a larger gas residence time and ensures a more uniform gas treatment. An advantage to the GAT system is that because of the thermal insulation, it does not require high-temperature material, thus reducing costs [39]. The gasification of biomass is accomplished by the solid biomass undergoing a thermochemical reaction under sub-stoichiometric conditions with an oxidizing fuel. The biomass’s energy is released in the form of CO, CH4 , H2 , and other combustible gases (syngas) [40]. The syngas consists of basic elementary components, which can be made into butanol using various petrochemical techniques. Other advances ingasification technology have been made by the National Renewable Energy Laboratory’s (NREL) Battelle Labs.

3.3 Summary
Biobutanol is a renewable, biodegradable, alternative fuel, which can be used neat or blended with gasoline. Properties such as energy density, octane value, and Reid vapor pressure (RVP) are similar to gasoline; hence current vehicles can use biobutanol without any engine modifications. Biobutanol can be produced from biomass by the fermentation of sugars and starches or by thermochemical routes using gasification. The emergence of butanol as a fuel is growing with companies such as BP, DuPont, EEI, Tetravitae Bioscience, and W2 Energy Inc. investing in new technology as well as in manufacturing. Worldwide commercialization of biobutanol can replace or enhance blends of gasoline to reduce the dependence on petroleum as well as reduce greenhouse gas emissions.


4 Cellulosic Ethanol 4.1 Background
Henry Ford test drove his first prototype automobile called the Ford Quadracycle in July 1896 that ran on pure ethanol. He told the New York Times in 1925 that “The fuel of the future is going to come from fruit like that sumach out by the road, or from apples, weeds, sawdust –– almost anything” [41]. Ethyl alcohol, or ethanol, is a two carbon, straight chain alcohol that is found in alcoholic beverages. Ethanol is a renewable, biodegradable, clean burning, alternative fuel that is usually produced by the fermentation of carbohydrates from sugar, corn, or fruits [13]. Ethanol has replaced methyl tert-butyl ether (MTBE)as an emissions reducing additive in gasoline due to concerns of MTBE ground water contamination that arose in late 2005. Ethanol can be used in current automobiles in blends up to 10% (E10) in gasoline without any engine modifications. Higher percentages of ethanol blends (E85 and E100) can be used in Flex Fuel Vehicles (FFVs). Sugarcane-based ethanol edges out gasoline at an oil equivalent economic price of $40 per barrel [42]. In contrast, US corn-based ethanol has an edge over gasoline when oil price is $60 or higher. “Flex-fuel” vehicles are designed to run on ethanol, gasoline, or a mixture of the two. Ethanol is made through the fermentation of sugars, and sugar cane offers particular advantages. The energetic balance in ethanol production shows that for each unit of energy invested, sugar cane based ethanol yields eight times as much energy as corn [43]. Unlike corn-based fuels, sugarcane requires no fossil fuels to process. Cellulosic ethanol, derived from a range of crops, such as switchgrass and crop waste, is more economical than corn ethanol because it requires far less energy to produce. However, the economics of corn or cellulosic ethanol has been discussed widely in many articles. A central argument is that corn-based ethanol is literally a waste of energy. Detractors say that it takes more energy to grow the corn, process it, and convert it to ethanol than would be saved by using it. According to Pimentel and Pazek [44] “Ethanol production using corn grain required 29% more fossil energy than the ethanol fuel produced.” Wang et al. dispute this and statethat it takes 0.74 BTU of fossil fuel to create 1 BTU of ethanol fuel, compared with a ratio of 1.23 BTUs to 1 BTU for gasoline or 66% more than ethanol [45]. The conclusions of Wang et al. have largely been corroborated by Farell et al. [46]. According to them, “current corn ethanol technologies are much less petroleum-intensive than gasoline but have greenhouse gas emissions similar to those of gasoline.” The authors however opined that cellulosic ethanol would be key to large-scale use of ethanol as a fuel. Hammerschlag compared data from ten different studies and used a parameter, rE , defined as the total product energy divided by nonrenewable energy input to its manufacture [47]. Thus, rE > 1 indicates that the ethanol has captured some renewable energy. The corn ethanol studies showed rE in the range 0.84 ≤ rE ≤ 1.65, and three of the cellulosic ethanol studies indicated a range of 4.40 ≤ rE ≤ 6.61. Because ethanol is made from crops that absorb carbon dioxide, it generally helps reduce greenhouse emissions. Although it is carbon neutral and renewable


the GHG impact depends on farming practices, particularly the use of fertilizers. This is specifically true for ethanol made from corn. When ethanol is made from cellulosic sources there is considerable reduction in GHGs [48]. This is because producers of cellulosic ethanol burn lignin to heat the plant sugars whereas most producers of corn ethanol burn fossil fuels to provide the energy for fermentation. Cellulosic ethanol is a renewable, biodegradable, clean burning, alternativefuel. Cellulosic biomass typically contains 40–50% cellulose, 20–30% hemicellulose, and the remainder, 15–30%, is lignin and other components [49]. Cellulose consists of glucose monomers linked by a β-1 bond which forms a linear polymer [50]. Hemicellulose is a highly-branched complex polymer that is composed mainly of xylose and other five-carbon sugars [50]. Lignin is a phenyl propane polymer that acts as a binder, which cannot be converted into useful products. The hemicellulose is randomly acetylated and acts as an interface between the cellulose and lignin. The cellulose and hemicellulose can be broken down into simple sugars that are used to produce ethanol, while the lignin can be burned to produce heat, which helps to increase overall efficiency. What makes cellulosic ethanol promising is the diverse, abundant, low cost feedstock that is readily available. There are two main methods for the production of ethanol from biomass; enzymatic saccharification and fermentation, and fermentation by cellulolytic microorganisms. However, cellulosic ethanol is not without its challenges and drawbacks. Commercial production of cellulosic ethanol currently requires high initial capital costs and involves risk. In 2002, a DOE study determined that for cellulosic ethanol to be competitive, the production cost would need to be $1.07 per gallon or less [51]. One of the most expensive steps in the production of cellulosic ethanol involves the pretreatment of biomass.

4.2 Comparison of Pretreatment and Manufacturing Processes
Pretreatment is required to alter the physical andchemical properties of the biomass to make it easier to process. The methods of pretreatment are similar for either enzymatic or microbial cellulosic ethanol processing. Removing or altering the lignin allows access to carbohydrates in the biomass. Higher lignin sources require chemical treatment to reduce the level to below 12% to enhance digestibility [50]. To gain access to the cellulose fiber, de-crystallization of the hemicellulose that is covalently bound with the lignin via hydrolysis is required [52]. The conversion of all the sugars derived from hemicellulose is highly desired to increase efficiency and minimize by-products. Pretreatment of the biomass is also required to increase the surface area and pore size, thus making it easier to digest. The increase in surface area is from the combination of hemicellulose solubilization, lignin solubilization, and lignin redistribution caused by various methods of pretreatment [53]. There are several methods by which pretreatment is performed: physical, chemical, and biological. Physical methods include ball and compression milling that shear or shed the biomass to de-crystallize the cellulose and increase the surface area and digestibility. However, these processes do very little to degrade hemicellulose




and lignin polymers. Milling also requires long processing times with high capital and operating costs, thus it is not economical and has not been pursued in scale-up operations [50, 54]. Radiation pretreatment utilizes gamma rays, electron beams, or microwaves to react toweaken and break the chemical bonds between hemicellulose and lignin through chemical reactions such as chain scission [55]. However, the high consumption of energy and capital costs makes this process economically unviable. Dilute-acid pretreatment is a chemical process that increases the solubility of hemicellulose to 80–100%, extensively redistributes the lignin, and depolymerizes some of the cellulose [53]. The process soaks the biomass in a dilute solution of sulfuric, hydrochloric, or nitric acid and then raises the temperature by injecting steam to enhance the pretreatment method [50]. Autohydrolysis generates acids by the introduction of saturated steams into the biomass to breakdown the hemicellulose and lignin [50]. The pressure is rapidly released resulting in the breakup of the biomass due to the instant vaporization of the trapped water. This process is known as steam explosion pretreatment and results in 80–100% solubilization into a mixture of monomers and oligomers of hemicellulose. It also redistributes the lignin, and depolymerizes some of the cellulose [53]. Similar to steam explosion, ammonia fiber explosion pretreatment (AFEX) uses high temperature and pressure ammonia to de-crystallize cellulose, and increase the solubility of lignin by 10–20%, and of hemicellulose up to 60% while hydrolyzing about 90% to oligomers [53]. Other chemical pretreatment methods include “hydrothermal” processes using liquid hot water, supercritical carbon dioxide, “organosolv” processes that involve organic solvents in an aqueous medium, concentrated phosphoric orperacetic acid treatment, and strong alkali processes using sodium hydroxide or lime [50, 53]. A biological pretreatment process utilizes fungi, such as white rot, brown rot, and soft rot, to hydrolyze the cellulose component of biomass. Filamentous fungi, typically Trichoderma and Penicillium species, can be used directly for cellulose hydrolysis because of the greater capacity for extracellular protein production than that of cellulolytic bacteria [56]. However, it requires a three-fold reduction in cost for commercialization and the reaction rates for the hydrolysis of cellulose are relatively low in comparison to chemical pretreatment methods [56]. Enzymatic saccharification utilizes enzyme blends for recovering carbohydrates from the hydrolyzate generated after pretreatment [51]. Commonly, cellulase and hemicellulase enzymes are used as a “cocktail” with other enzymes to enhance yields and reduce enzyme costs. The products of enzymatic saccharification – the process of breaking a complex carbohydrate into its monosaccharide components – severely inhibit cellulases and hemicellulases [57]. To overcome this difficulty, Simultaneous Saccharification and Fermentation (SSF) of the pretreated hydrolyzate is preferred. Once the structure of the biomass is disrupted, the cellulose and hemicellulose is enzymatically converted to sugars by the saccharification process. During the fermentation process, yeasts such as Saccharomyces cerevisiae, convert the sugars to ethanol. The advantage of SSF over Separate Hydrolysis and Fermentation (SHF) is higher yields of ethanol but SSF requiresmore than double the fermentation time [58]. However, the hydrolyzate also contains acetic acid



and other toxic compounds. Together with increasing ethanol concentrations, this can inhibit the enzymes and fermentation organisms, thus lowering yields. New developments in enzymatic saccharification and fermentation have been developed by Iogen Energy Corporation and the NREL to develop effective “cocktails” of enzymes along with modified strains of yeast that can break down complex sugar molecules, which conventional fermentation yeast cannot. Recently, Royal Dutch Shell (Shell) announced a partnership with Iogen Energy Corporation to advance cellulosic ethanol from agriculture residues such as cereal straw and corn cobs and stalks. And just recently, Iogen Energy shipped 100,000 L (26,417 gal) to Shell, which is the first installment of the initial order of 180,000 L (47,550 gal) of cellulosic ethanol. Iogen’s demonstration facility located in Ottawa, which first began producing cellulosic ethanol in 2004, is being purchased by Shell for use in upcoming fuel applications [59]. Cellulolytic microorganisms, an alternative to yeast, utilize ethanol fermenting microbes that both hydrolyze and ferment the sugars into ethanol from a milder pretreatment process. Gram-negative bacteria, such as Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis, are being investigated as potential microorganisms for industrial production of ethanol [52]. Using genetic and metabolic engineering, NREL has developed a strain of Z. mobilis (Zymo) that can breakdown complex sugars like xylose, and tolerate higher concentrations of acetic acid [51]. Other studies have shown that the Z. mobilis strain can produce theoretical yields up to 95% and handle a wider range of feedstocks [52]. High technological costs have impeded the widespread production of cellulosic ethanol by microorganisms. Consolidated bio-processing or CBP has been developed to address this problem. This process utilizes cellulolytic microorganisms to perform the hydrolysis of biomass and the fermentation of sugars into ethanol within a single process, which is a large cost reducing strategy [53]. CBP is expected to reduce overall production costs by eight-fold compared to SSF under similar conditions. Mascoma Corporation has dedicated their research team to focus on the commercialization of CBP, which is seen as the lowest cost configuration for cellulosic ethanol. Mascoma Corporation is in the process of developing a cellulosic fuel production facility that will use non-food biomass to convert woodchips into fuel. They are predicting that the new facility will produce 40 million gallons of ethanol and other valuable fuel products per year [60].

4.3 Summary
Cellulosic ethanol is ethyl alcohol produced from wood, grass, or the non-edible parts of plants, and is a sustainable and renewable biofuel that is biodegradable. The promising features of cellulosic ethanol are the diverse and abundant feedstock that can utilize existing waste by-products. Iogen Energy Corporation is currently producing cellulosic ethanol for Shell using enzymatic saccharification andfermentation in a small-scale commercial facility. Another approach to cellulosic ethanol is via the use of cellulolytic microorganisms. As commercialization of cellulosic

ethanol expands, it can be used to increase ethanol production without causing food shortages or demands, and will reduce greenhouse gas emissions and our dependence on fossil fuels.

5 Final Thoughts
Research on renewable and environmentally sustainable fuels has received a lot of impetus in recent years. With oil at high prices, alternative renewable energy has become very attractive. Many of these technologies are eco-friendly. Besides ethanol, other environmentally sustainable fuels include biodiesel and biobutanol. A recent United Nations report urges governments to beware the human and environmental impacts of switching to energy derived from plants. There should a healthy debate about turning food crops or animal feed into fuel and the consequences of the switch to biofuels needs to be carefully thought out. Thus the focus of biofuel production needs to be on non-edible and waste sources. In the case of biodiesel, these include restaurant grease, non-edible sources like Jatropha as well as microalgae. Biobutanol is a renewable, biodegradable, alternative fuel, which can be used neat or blended with gasoline. Properties such as energy density, octane value, and Reid vapor pressure (RVP) are similar to gasoline; hence current vehicles can use biobutanol without any engine modifications. Biobutanol can be produced from biomass by the fermentationof sugars and starches or by thermochemical routes using gasification. Ethanol is made through the fermentation of sugars, and sugar cane offers many advantages. Unlike corn-based fuels, sugarcane requires no fossil fuels to process. Cellulosic ethanol, derived from a range of crops, such as switchgrass and crop waste, is more economical than corn ethanol because it requires far less energy. While there is no single magic bullet that can completely replace our dependence of petroleum, the focus needs to shift on fuels that can not only alleviate our dependence on petroleum but are also renewable and environmentally sustainable.

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