Currently, over a dozen companies have demonstrated strong interest in explor­ing advanced R&D and/or pilot-scale facilities, with a view to building future commercial-scale plants. The following are a few examples, showing the range of locations, technologies, and feedstocks:

Abengoa Bioenergy, Inc. (http://www. abengoabioenergy. com) began to build the world’s first commercial lignocellulosic ethanol plant in Babilafuente (Salamanca), Spain in 2005. With $76 million in funding from the DOE, the company is planning to build a lignocellulosic ethanol plant in Kansas by 2011, which will evaluate the use of corn stover, wheat straw, and other agricultural biomass.

BlueFire Ethanol, Inc. (http://www. bluefireethanol. com) recently received DOE funding of $80 million to build a 19 million gallons per year lignocellulosic ethanol plant in California. They plan to use urban trash (post-sorted MSW), rice straw, wood waste, and other agricultural residues as feedstock, combined with a concentrated acid process.

Coskata, Inc. (http://www. coskata. com) is exploring the integration of ther­mochemical and biochemical conversions: syngas is generated by gasification of lignocellulosic biomass and then converted into ethanol from the gas phase by anaer­obic fermentation [61]. The company claims this technology can produce more than 100 gallons of ethanol per dry metric ton of feedstock with production cost of less than $1/gallon. There is no indication of when such numbers will be achieved in a practical large scale operation.

DuPont Danisco Cellulosic Ethanol LLC. (http://www. ddce. com) is a joint- venture between DuPont and Genencor (a subsidiary of Danisco). The company is cooperated with University of Tennessee to build a pilot lignocellulosic ethanol facility (PDU, 0.25 MG/y) in Tennessee by 2009. The plan is to combine DuPont’s proprietary mild alkaline pretreatment and fermentation technologies with Genencor’s enzymatic hydrolysis methods to convert corn stover and sugarcane bagasse into ethanol.

Etek Etanolteknik AB (http://www. sekab. com/) is located in Sweden and has set-up a pilot lignocellulosic ethanol plant with a capacity of about 400-500 L of ethanol/day (~2 ton dry substance/day). The plant has been functional since 2004, using the two-step dilute-acid hydrolysis process in combination with enzy­matic hydrolysis. Feedstocks include cereal straws, organic waste, wood clippings, or forestry residues.

Iogen Co. (http://www. iogen. ca/) is located in Canada and has more than a decade of experience in ethanol production from lignocellulosic materials. The com­pany currently runs a demonstration lignocellulosic ethanol plant using a modified steam-explosion pretreatment technology (dilute acid) and enzymatic hydrolysis, with an annual capacity of 1 million gallons of ethanol. Feedstock includes wheat straw, barley straw, corn stover, and waste wood [62].

Mascoma Corporation (http://www. mascoma. com) is located in Massachusetts and was founded around the key technology of genetically-engineered bacteria that are capable of fermenting both hexoses and pentoses into ethanol. The com­pany has recently raised $30 million and is building a 1.5-2.0 million gallon/year demonstration level lignocellulosic ethanol plant.

Poet (http://www. poetenergy. com) is one of the largest corn-based ethanol pro­ducers. With the help of an $80 million DOE grant, the company is expanding one of its plants in Iowa to produce 125 million gallons/year, of which 25 million gallons will be from lignocellulose (corn cobs and/or corn kernel fiber). Poet is currently researching possible methods for the collection and storage of corn cobs and the expanded facilities are expected to be operational by 2011.

Ranger Fuels (http://www. rangefuels. com/home) has began construction of a demonstration 20 million gallons/year lignocellulosic ethanol plant in Georgia (to be commissioned in 2009). The plant will use a thermochemical process (gasification and catalyst transformation) to turn wood, grasses, corn stover, and other available agricultural biomass into fuel ethanol.

Verenium (http://www. verenium. com/) was created by the merger of the former Celunol and Diversa companies. With DOE funding of $40 million, the company is in the process of building a 1.4 million gallon/year demonstration plant at Louisiana. The feedstock will include sugarcane bagasse, hard wood, rice hulls, and other agricultural residues.

ZeaChem, Inc. (http://www. zeachem. com/) has a technology that biologically transforms hemicellulose and cellulose into acetic acid. The acetic acid is then hydrogenated in a thermochemical process using hydrogen produced from gasifi­cation of lignin, to produce ethanol. Since no carbon dioxide is released during the biochemical conversion process, this process has a higher ethanol yield (up to 160 gallons/dry ton biomass) compared to the hydrolytic methods [63]. The plan is to build a 1.5 million gallon per year plant in Oregon with operational start-up in late 2009.

Om V. Singh and Steven P. Harvey

Introduction

Even given the seemingly unlikely near-term resolution of issues involving atmo­spheric CO2 levels and their effect on the climate, the adoption of global conser­vation measures, and the stabilization of fossil fuel prices, it is still a certainty that global oil and gas supplies will be largely depleted in a matter of decades. That much is clear from even a cursory comparison of the independent estimates of the world’s oil and natural gas reserves and the respective data on their consumption, as published regularly on the internet by the US Government Energy Information Administration [1]. Nature of course, offers abundant renewable resources that can be used to replace fossil fuels but issues of cost, technology readiness levels, and compatibility with existing distribution networks remain. Cellulosic ethanol and biodiesel are the most immediately obvious target fuels, with hydrogen, methane and butanol as other potentially viable products. Other recent reports have cov­ered various aspects of the current state of biofuels technology [2-4]. Here we continue to bridge the technology gap and focus on critical aspects of lignocellu — losic biomolecules and the respective mechanisms regulating their bioconversion to liquid fuels and value-added products of industrial significance.

The lignocellulosic structure does not readily yield its component five — and six- carbon sugars so the efficient biological conversion of biomass typically requires a pretreatment step to render the polysaccharide molecules accessible to enzymes. Several thermochemical or biochemical approaches are currently in various stages of development, and have the potential for major impact on the economics of biofuel

O. V. Singh (b)

Division of Biological and Health Sciences, University of Pittsburgh, 300 Campus Drive, Bradford, PA 16701 USA

e-mail: ovs11@pitt. edu, ovs11@yahoo. com S. P. Harvey (в)

U. S. Army Edgewood Chemical Biological Center, AMSRD-ECB-RT-BC, Bld E3150, 5183 Blackhawk Rd, Aberdeen Proving Ground, MD 21010-5424, USA e-mail: steve. harvey@us. army. mil

Xiii

production. In order to derive a stable and cost-effective approach, a greater fun­damental understanding is needed of the exact effects of these processes on plant anatomy. These are difficult experiments to conduct and in Chapter 1 “Heat and Mass Transport in Processing of Lignocellulosic Biomass for Fuels and Chemicals”, Viamajala et al. provide an in-depth report on the effects of heat and mass transport on the efficiency of biomass conversion. Further, Wu et al. in Chapter 2 “Biofuels from Lignocellulosic Biomass”, give the matter a more detailed consider­ation by comparing thermochemical and biochemical approaches to the production of biofuel from lignocellulosic biomass.

As compared to gas and oil, relatively greater potential reserves exist for both coal and uranium (probably on the order of a century) but neither is renewable and each is associated with its own environmental conundrum (carbon release and waste storage, respectively). Linus Pauling expressed a particular concern for the destruc­tion of the element uranium, saying “In a thousand or ten thousand years the world may require uranium for a purpose about which we are currently ignorant.” [5]. Looking beyond the immediate temporal horizon, we are unavoidably confronted with the need to develop permanently renewable sources of energy.

Earth’s most plentiful and renewable energy resources typically include sunlight, wind, geothermal heat, water (rivers, tides and waves), and biomass. All of these are suitable for the generation of electricity but biomass is the current main renewable feedstock for the production of “liquid” fuels — typically ethanol, and biodiesel and possibly to include butanol, hydrogen and methane. These liquid fuels, or energy carriers lie at the heart of the solution to the global energy problem, since they are the materials currently most suitable for use in the transportation sector and for the direct replacement of the immediately endangered fossil resources of oil and gas. Vasudevan et al. in Chapter 3 “Environmentally Sustainable Biofuels — The Case for Biodiesel, Biobutanol and Cellulosic Ethanol” provide a detailed dis­cussion of the case for ethanol, butanol and biodiesel. Significantly, a potential technical hurdle confronting the production of biofuels is the efficiency of utiliza­tion of hemicellulose-derived sugars. In Chapter 4 “Biotechnological Applications of Hemicellulosic Derived Sugars: State-of-the-Art”, Chandel et al. examine the challenges associated with the successful utilization of this second most abundant polysaccharide in nature.

Energy-yielding materials are found in various guises, one of which is garbage. Although not always classified as a resource, garbage clearly is renewable (increas­ingly so, in fact), and processes that convert it into energy are obviously dually beneficial. In Chapter 5 “Tactical Garbage to Energy Refinery (TGER)”, Valdes and Warner present a hybrid biological/thermochemical system designed for the conversion of military garbage into ethanol and electricity, with clear potential for applications in the civilian sector.

Agricultural waste (e. g. livestock, manure, crop residues, food wastes etc.) is a high impact feedstock with particular utility in the production of bio­gas. In Chapter 6 “Production of Methane Biogas as Fuel Through Anaerobic Digestion”, Yu and Schanbacher discuss the anaerobic conversion of biomass to methane. Untreated wastewater also contains biodegradable organics that can be used to produce hydrogen or methane. In Chapter 7 “Waste to Renewable Energy: A Sustainable and Green Approach Towards Production of Biohydrogen by Acidogenic Fermentation”, Mohan provides a detailed review of the state of the art with regard to biological hydrogen production using waste and wastewater as substrates with dark fermentation processes.

Many biological processes use mixed cultures operating under non-sterile con­ditions (e. g. biological hydrogen and methane production, as discussed above). Watanabe et al. in Chapter 8 “Bacterial Communities in Various Conditions of the Composting Reactor Revealed by 16S rDNA Clone Analysis and Denaturing Gradient Gel Electrophoresis” demonstrate the utility of 16S rRNA analysis and denaturing gradient gel electrophoresis (DGGE) techniques for tracking microbial communities within a mixed and changing culture. Their work uses a composting process, which offers a typically cost-effective alternative to incineration for the remediation of contaminated soil.

The production of liquid fuel from biomass necessitates the consideration of var­ious issues such as the effects on the food supply, the rainforest, and greenhouse gas production, as well as carbon sustainability certification. Some of these issues may require appropriate regulations and in Chapter 9 “Perspectives on Bioenergy and Biofuels”, Scott et al., examine these issues closely.

In addition to its environmental advantages, the use of renewable energy resources offers the potential for stimulation of the economies of the nations where they are produced. The potential products of these renewable materials extend well beyond liquid fuels alone. Owing partly to the enormous volume of their produc­tion, fuels are sold for relatively low prices, and the successful implementation of renewable fuels depends, at least initially, on their ability to compete in the mar­ketplace. To this end, it is particularly important to maximize the efficiency of their production in biorefineries where secondary products would be derived from the same feedstock as the fuels. As an example, petroleum refineries have been in oper­ation for over 150 years and now produce lubricants, plastics, solvents, detergents, etc., all from the starting crude oil [6]. Similarly, biomass, in addition to being used for the production of fuels, can be used as a starting material for the pro­duction of other value-added products of microbial bioconversion processes such as fermentable sugars, organic acids and enzymes. In Chapter 10 “Perspectives on Chemicals from Renewable Resources”, Scott et al. describe how, with the aid of biotechnology, Protamylase® generated from starch production, can be used as a medium for the production of a cynophycin polymer, which is a major source of arginine and aspartic acid for the production of many industrially useful compounds including 1,4-butanediamine and succinic acid. In Chapter 11 “Microbial Lactic Acid Production from Renewable Resources”, Li and Cui describe the production of lactic acid from renewable resources such as starch biomass, cheese whey etc. Lactic acid has recently gained attention due its application to the manufacture of biodegradable polymers. Among other renewable resources, Chapter 12 “Microbial Production of Potent Phenolic-Antioxidants Through Solid State Fermentation”, Martin et al. describe the role of agroindustrial residues including plant tissues rich in polyphenols for the microbial bioconversion of potent phenolics under solid state fermentation conditions. Hence, combined with the economy of scale derived from large refineries, secondary products could be key to bridging the price gap between fossil fuels and renewables.

One critical advantage of biofuels is their potential to achieve a reduction in greenhouse gas releases, since the plants from which they are produced derive their carbon from the atmosphere. The overall balance of greenhouse gases however, depends in large measure on the particular feedstocks used and the methods by which they are produced. Corn ethanol for instance, while being potentially car­bon neutral, is not likely to achieve an overall reduction in greenhouse gas release due to its requirement for nitrogenous fertilizer and the associated release of nitrous oxide [7]. An interesting approach to the production of biodiesel is the use of algae to synthesize oil from the CO2 they capture for growth. Algae cultivation offers a potential low-cost alternative to physical methods of carbon sequestration such as pumping liquid CO2 underground or underwater or chemical methods such as base-mediated capture of CO2 and subsequent burial of the resulting carbonates. The algae, while using CO2 as their sole source of carbon for growth, can produce up to 50% of their weight in oil suitable for conversion to biodiesel. Algae are one of the best sources of plentiful biomass on earth; their potential for biosynthesis of astaxanthin, a red carotenoid nutraceutical responsible for the color of salmon flesh, was explored in Chapter 13 “Photoautotrophic Production of Astaxanthin by the Microalga Haematococcus pluvialis”, Del Rio et al.

In a biological system, the biosynthesis of industrially useful compounds has long been recommended. Heparin, a low-molecular weight highly sulfated polysac­charide represents a unique class of natural products, that has long been used as an anticoagulant drug. Due to recent outbreaks of contamination and seizure of hep­arin manufacturing facilities [8], an efficient bioconversion process of heparin is required. In Chapter 14 “Enzymatic Synthesis of Heparin”, Liu and Liu describe novel enzymatic approaches for the biosynthesis of heparin sulfate that mimic E. coli heparosan.

Discovering new and sustainable resources can help refuel industrial biotech­nology. Adverse environmental conditions which normal earth microbiota do not tolerate, offer potential sites to explore specific sets of microorganisms desig­nated as “Extremophiles”. The discovery of these microorganisms has enabled the biotechnology industry to innovate unconventional bioproducts i. e. “Extremolytes”

[9] . In Chapter 15 “Extremophiles: Sustainable Resource of Natural Compounds — Extremolytes”, Kumar et al. provide an overview of these extreme habitats. The applications of extremophiles and their products, extremolytes, with their possible implications for human use are also discussed broadly.

This book “Sustainable Biotechnology: Sources of Renewable Energy” is a col­lection of research reports and reviews elucidating several broad-ranging areas of progress and challenges in the utilization of sustainable resources of renewable energy, especially in biofuels. This book comes just at a time when government and industries are accelerating their efforts in the exploration of alternative energy resources, with expectations of the establishment of long-term sustainable alter­natives to petroleum-based liquid fuels. Apart from liquid fuel this book also

emphasizes the use of sustainable resources for value-added products, which may help in revitalizing the biotechnology industry at a broader scale.

We hope readers will find these articles interesting and informative for their research pursuits. It has been our pleasure to put together this book with Springer press. We would like to thank all of the contributing authors for sharing their quality research and ideas with the scientific community through this book.

1.2 Gasification of Lignocellulosic Biomass

1.2.1 Overview

Gasification is a process where carbonaceous feedstocks react with oxygen and steam at elevated temperatures (500-1500°C) and pressures (up to 33 bar or 480 psi) to yield a mixture of gasses. The mixed-gas product is called synthesis gas or “syngas,” consisting primarily of hydrogen (H2) and carbon monoxide (CO), with varying amounts of carbon dioxide (CO2), water (H2O), methane (CH4), and other elements, depending on the feedstock, gasifier type and conditions [21].

Global energy consumption will continue to increase, even as the reserves of eas­ily available fossil fuels decline. Until alternative energy sources are developed for transportation, liquid fuels will remain in high demand. Crude oil production will be unable to meet future demands at affordable prices and fuels from renewable feedstocks will play a key role in contributing to the supply of liquid transport fuels.

Lignocellulose is a natural abundant material created by plants from sunlight, nutrients, and CO2 capture. The potential volume of lignocellulose that can be the­oretically produced and harvested is considerable and sufficient to make a major contribution to liquid transport fuel volume. In practice, there are several major chal­lenges to lignocellulosic biomass production, collection, and storage that were not addressed in this chapter but are the focus of research in many projects. Ultimately, the real cost of feedstock delivered to the conversion facility will be a major factor determining the magnitude of success for lignocellulosic biomass. Potential out­put products could include ethanol, butanol, biogasoline, FT liquids, and a range of chemical intermediates. Reaching this potential in an economically acceptable man­ner is a challenge, and requires an improved ability to convert the lignocellulosic feedstock to a useable fuel.

After more than two decades of intensive R&D, several technologies have been evaluated for biofuel production at the laboratory level. A few are now at the stage of advanced testing and pilot-scale evaluations. Presently, the challenges facing com­mercial conversion are such that no one technology has an absolute advantage over the others. The approach of thermochemical pretreatment and enzymatic hydroly­sis followed by microbial fermentation has been the most extensively studied. The remaining challenges for this approach include further lowering pretreatment cost, improving hydrolysis efficiency and cost of cellulases (and hemicellulases), and improving the performance of fermentation organisms. The approach of thermo­chemical gasification combined with FT catalytic conversion has also been widely explored and may be promising under the appropriate conditions. The gasifica­tion approach would benefit from improved gasification efficiency, easier syngas cleanup, and better FT factors such as catalyst selectivity and longevity.

In some projects, various combinations (thermochemical front + biochemical, biochemical front + thermochemical) have been evaluated. For economic operation in an integrated biorefinery, it may be that such combinations of approaches will be required and that the combination utilized will depend on the feedstock, the location, the desired product stream, the degree of environmental impact, and the level of investment available. It is expected that the best technologies for specific challenges will be selected and implemented over the next 5-10 years and that the definitive answer on the size of the contribution from lignocellulosic biomass will become evident during that time.

Sridhar Viamajala, Bryon S. Donohoe, Stephen R. Decker, Todd B. Vinzant, Michael J. Selig, Michael E. Himmel, and Melvin P. Tucker

Abstract Lignocellulosic biomass, a major feedstock for renewable biofuels and chemicals, is processed by various thermochemical and/or biochemical means. This multi-step processing often involves reactive transformations limited by heat and mass transport. These limitations are dictated by restrictions including (1) plant anatomy, (2) complex ultra-structure and chemical composition of plant cell walls, (3) process engineering requirements or, (4) a combination of these factors. The plant macro — and micro-structural features impose limitations on chemical and enzyme accessibility to carbohydrate containing polymers (cellulose and hemicel — lulose) which can limit conversion rates and extents. Multiphase systems containing insoluble substrates, soluble catalysts and, in some cases, gaseous steam can pose additional heat and mass transfer restrictions leading to non-uniform reactions. In this chapter, some of these transport challenges relevant to biochemical conversion are discussed in order to underscore the importance of a fundamental understanding of these processes for development of robust and cost-effective routes to fuels and products from lignocellulosic biomass.

Keywords Lignocellulose ■ Biomass ■ Biofuels ■ Heat transport ■ Mass transport

1 Introduction

The biochemical conversion of lignocellulosic biomass requires several processing steps designed to convert structural carbohydrates, such as cellulose and hemicellu — lose, to monomeric sugars, which include glucose, xylose, arabinose, and mannose. These sugars can be fermented to ethanol and other products, to varying degrees of effectiveness, by wild type and modified microbial strains. The front end of the process includes feedstock size reduction followed by a thermal chemical treatment, called pretreatment. In practice, this unit operation usually involves the exposure of

S. Viamajala (B)

Department of Chemical and Environmental Engineering, The University of Toledo, Toledo, OH 43606-3390

e-mail: sridhar. viamajala@utoledo. edu

O. V. Singh, S. P. Harvey (eds.), Sustainable Biotechnology,

DOI 10.1007/978-90-481-3295-9_1, © Springer Science+Business Media B. V. 2010 biomass to acid or alkaline catalysts at temperatures ranging from 120 to 200°C. Pretreated slurries (the hydrolysate liquor containing soluble sugars, oligosaccha­rides, and other released solubles plus the residual solids) are then enzymatically digested at 40-60°C to release sugars from the polysaccharides and oligomers remaining after pretreatment [1-9]. In both of these steps, adequate heat, mass, and momentum transfer is required to achieve uniform reactions and desirable kinetics.

Plant cell walls, which make up almost all of the mass in lignocellulosic biomass, are highly variable both across and within plant tissue types. At the macroscopic scale, such as within a stem or leaf, uneven distribution of catalyst (chemical or enzyme) due to the different properties of different tissues results in heterogeneous treatment, with only a fraction of the plant material exposed to optimal conditions [10-13]. Tissues that do not get exposed to sufficient amounts of catalyst during pretreatment are incompletely processed, resulting in decreased overall enzymatic digestibility of pretreated biomass [6]. When pretreatment severity is increased, by increasing temperature, catalyst concentration, or time of reaction, areas of biomass readily exposed to catalyst undergo excessive treatment leading to sugar degra­dation and formation of toxic by-products (furfural, hydroxymethyl furfural, and levulinic acid) that inhibit downstream sugar fermentation and decrease conversion yields [1]. This problem continues at a microscopic scale due to the compositional and structural differences between middle lamella, primary cell wall, and secondary cell wall. At even smaller scales, intermeshed polymers of cellulose, hemicellulose, lignin, and other polysaccharides present another layer of heterogeneity that must be addressed during bioconversion of plant cell walls to sugars.

Milling to fine particle sizes improves some of these mass transfer limitations, but can add significant costs [14, 15]. Size reduction, however, may not overcome heat transfer limitations associated with short time-scale pretreatments that employ hot water/steam and/or dilute acids. When such pretreatments are carried out at high solids loading (>30% w/w) to improve process efficiency and increase product con­centrations, heat cannot penetrate quickly and uniformly into these unsaturated and viscous slurries. It is thought that steam added to high-solids pretreatments can con­dense on particle surfaces impeding convective heat transfer. Depending on particle and slurry properties, the condensed steam can form temperature gradients within biomass aggregates, resulting in non-uniform pretreatment.

Besides limiting heat transfer rates, biomass slurries can pose other process­ing challenges. At high solids concentrations, slurries become thick, paste-like, and unsaturated. Limited mass transfer within these slurries can cause localized accumulation of sugars during enzymatic hydrolysis, decreasing cellulase and hemi — cellulase activity through product inhibition [16-23]. In addition, slurry transport through process unit operations is challenging at full scale. As solid concentrations increase, hydrodynamic interactions between particles and the surrounding fluid as well as interactions among particles increase. At high solids concentrations “dense suspensions” are formed and the resulting multiple-body collisional or frictional interactions and entanglement between particles creates a complex slurry rheology [24-26]. A further complicating aspect is water absorption by biomass, causing the bulk to become unsaturated at fairly low insoluble solids concentrations (~ 30-40%
w/w) and behave as a wet granular material [27]. This material is highly compress­ible and the wet particles easily “stick” to each other and agglomerate. With no free water in the system, the material becomes difficult to shear or uniformly mix.

At the ultrastructural scale of plant cell walls, catalysts must penetrate through the nano-pore structure of the cell wall matrix to access the “buried” and inter — meshed carbohydrate polymers. Based on reported average cell wall pore sizes of 5-25 nm [28-31], small chemical catalysts (<1 nm) may not face as significant a penetration barrier as do enzymes (about 10 nm). The most dominant commer­cial cellulase component enzyme, cellobiohydrolase I or Cel7A, has dimensions of ~5 x 5 x 12 nm [32, 33] which is roughly the same size as smallest of these reported nano-pores, likely restricting accessibility to primarily surface cellulose chains. Once they have penetrated the cell wall matrix, these enzymes must locate suitable substrates. For Cel7A, this implies that a region of cellulose microfibril has been sufficiently unsheathed from lignin and hemicellulose to expose the cellulose core (Fig. 1). This unsheathing process may be accomplished by the pretreatment or as an ablative effect caused by the system of cellulase enzymes which can peel away microfibrils from the surface layers. Lignin is also a major impediment to cellulase action because it is difficult to remove uniformly or modify through pretreatment. Furthermore, it is entirely unclear at this time if lignin can be effectively removed from cell walls using enzymes.

wall lumen

pit/pore

inter-cellular

space

Lignin is believed to impede enzymatic hydrolysis of cellulose by interact­ing with biomass surfaces and either blocking the path of processive hydrolases (e. g. Cel7A), preventing enzymatic access to specific binding sites, or through non­specific binding of cellulolytic enzymes [34-36] to lignin. Several low-temperature pretreatment protocols, such as alkaline peroxide [37, 38] or lime and oxygen [39], address these issues by removing substantial amounts of lignin. Although these pro­cesses are highly relevant to the pulp and paper industry, the fate of lignin and its impact on enzymatic digestibility after high-temperature acidic or neutral pretreat­ments has largely been neglected until recently [40-42]. Recent observations show that lignin undergoes significant structural changes during high temperature pre­treatments. These changes cause it to both mobilize during elevated temperatures and then coalesce upon cooling, both within the cell wall matrix and on the biomass surfaces [40]. This mobilized processed lignin, when redeposited onto cellulose sur­faces, can impede enzymatic digestion presumably due to the occlusion of substrate binding sites [42]. All of these transport limitations during lignocellulosic con­version to ethanol impact the overall process performance and thus warrant more detailed further investigation.

Depending on how heat is generated, gasification technology can be classified as either directly — or indirectly-heated gasification. For directly-heated gasification, pyrolysis and gasification reactions are conducted in a single vessel, with heat arising from feedstock combustion with oxygen. The syngas generated from this method has low heating values (4-6 MJ/m3 or ~100-140 Btu/ft3). For indirectly — heated gasification, the heat-generating process (combustion of char) is separated from the pyrolysis and gasification reactions, which generates high heating value syngas (12-18 MJ/m3 or ~300-400 Btu/ft3). Low heating value syngas is usually used to generate steam or electricity via a boiler or gas turbine, while high heat­ing value syngas can also be used as a feedstock for subsequent conversion to fuels and chemicals [22]. According to the flow direction of the feedstock material and oxidant, gasifiers can basically be classified into five types (Table 2, Fig. 3).

Although a portion of the feedstocks are converted to heat during gasifica­tion, conversion efficiencies of biomass to syngas are relatively high: e. g. 50-75% on weight basis [22]. This gasification efficiency is mainly due to the utilization of lignin and other organic substances, which cannot be used directly in acid or enzymatic hydrolyzing processes.

Palligarnai T. Vasudevan, Michael D. Gagnon, and Michael S. Briggs

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], current estimates 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, © Springer Science+Business Media B. V. 2010

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 past century 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 from petroleum 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 cellulose ethanol, respectively.

In a large-scale process, pre-impregnation of catalyst into large pieces of biomass (>1 cm) is often overlooked; however, milling biomass to reduce this problem can incur large energy and equipment costs [1, 14, 15]. This problem is compounded by the widespread use of process irrelevant biomass sizes for laboratory exper­iments. Most laboratory studies on biomass to ethanol conversion processes use finely milled materials (20-80 mesh is standard) where the effects of macroscopic transport processes are not easily observed or are masked altogether [43-45]. In larger pilot studies using compression screw feeders, these transport effects can be further masked by the high-shear feeder causing biomass size reduction [6, 8]. Often this size reduction occurs after catalyst impregnation, limiting catalyst effectiveness on pretreatment. A further complication is that compression of the feed stock may cause biomass pore structure collapse, leading to uneven heat and mass transfer dur­ing pretreatment [10,13] as well as limitation of catalyst access to the interior of the biomass.

Before larger biomass particles containing intact tissues are used in processing, it is essential to understand the catalyst transport processes and pathways and the limitations associated with them (Fig. 1). In living plants, vascular tissues such as xylem and phloem are the primary routes for transport of water and nutrients along the length of the plant stem and leaves. Additional transport within tissues and between adjacent cells is carried out through (1) the pits, areas of thin primary cell wall devoid of secondary cell wall between adjacent cells and (2) the apoplast, the contiguous intercellular space exterior to the cell membranes [46]. In dry senesced plants, studies with dyes to visualize fluid movement through tissues showed that the apoplastic space is the major catalyst carrier route, with limited fluid movement occurring through the vascular tissue [11]. In untreated biomass, the pits do not appear to support significant transport. It is probable that these pits disintegrate and open up during pretreatment allowing fluid to flow through [40]. Thus, new path­ways for catalyst penetration are formed either during the drying process that creates fractures in plant tissues or after some degree of biomass degradation.

The primary major barrier to fluid transport into native dry plant tissue appears to be air entrained in the cell lumen. Simple exposure of tissues to high tempera­ture fluids is insufficient to achieve catalyst distribution to all parts of the biomass [11]. The primary escape route for the intracellular air is most likely through pits. However, the small pit openings (approx 20 nm) could be blocked due to cell wall drying and water surface tension may prevent movement through these narrow open­ings. Forced air removal by vacuum provides additional driving force for the bulk fluid mobility necessary to enhance liquid and catalyst penetration into tissues as demonstrated by Viamajala and coworkers [11]. Heating dry biomass can minimize the amount of entrained air (due to expansion of air by heat) and assist in drawing liquid into the cells by contraction of the entrained air when cooled by immersion in catalyst-carrying liquid. Thus, bulk transport, rather than diffusive penetration, is the dominant mass transfer mechanism into dry biomass.

Although movement of fluids is associated with catalyst transport, the primary goal of catalyst distribution is to deliver the catalyst to cell wall surfaces con­taining fuel-yielding carbohydrates, rather than to empty cytoplasmic space in dry tissues. In fact, entrainment of fluids in the biomass bulk can be detrimen­tal to small time-scale dilute acid or hot water pretreatments, as the presence of excess water increases the net heat capacity of the material, increasing the heating time needed to achieve desired pretreatment temperatures. Data shown in Fig. 2 support this hypothesis. In this set of experiments, un-milled sections of corn stems

(~ 1 inch long) were saturated to various degrees with dilute sulfuric acid (2% w/w) and pretreated in 15 mL of the same acid solution at 150°C for 20 min. Milled corn stems (-20 mesh) pretreated under identical conditions served as controls. All pretreatments were performed in 22 mL gold coated Swage-Lok (Cleveland, OH) pipe-reactors, heated in an air-fluidized sand bath [42]. After pretreatment, whole stem sections were air-dried, milled and enzymatically digested for 120 h with a 25 mg/g of cellulose loading of a commercial T. reesei cellulase preparation (Spezyme CP, Genencor International, Copenhagen, Denmark) supplemented with an excess loading (90 mg/g of cellulose) of commercial Aspergillus niger cellobiase preparation (Novo 188, Novozymes Ltd., Bagsvaerd, Denmark) using procedures described previously [47]. Milled stover pretreated as controls in this experiment was dried and digested similarly, but without any further comminution.

In Fig. 2a, dry internodes pretreated without pre-impregnation of catalyst were poorly pretreated as evidenced by the high amounts of xylan remaining in the biomass after reaction. Stem sections pre-impregnated to achieve 20% satura­tion showed better reactivity and xylan removal and this trend continued when stem sections pre-impregnated to 50% saturation were pretreated. However, when completely saturated (100%) stem sections were pretreated, xylan conversion was observed to be lower. Milled materials with and without pre-impregnation of catalyst — conditions that would have lowest mass transfer limitations, showed com­parable pretreatment performance with each other as well as with the 50% saturated stem sections. These results confirm that only limited catalyst penetration and pre­treatment is achieved when air remains entrapped in cytoplasmic spaces such as in dry internodes. Enhanced catalyst distribution and transport dramatically enhances pretreatability up to a certain point, after which excess fluid impedes pretreatment. Similar conclusions on the negative impacts of poor bulk transfer on biomass pre­treatability can be inferred from other reported studies also. Tucker and coworkers

[10] observed poor pretreatability of biomass during steam explosion of corn stover when materials were not pre-wetted with dilute acid and ascribed their results to mass transport limitations. In another study Kim and coworkers [13] observed poor pretreatment of biomass when the biomass was pressed prior to pretreatment and hypothesized that the mechanical compression of biomass caused pore structure collapse resulting in formation of material that was relatively impervious to heat and mass transfer.

Enzymatic digestion results corresponding to pretreatments shown in Fig. 2a, are presented in Fig. 2b. As expected, release of monomeric sugars from pretreated whole stem sections was proportional to the degree of pretreatment they experi­enced. Unmilled biomass that was 50% saturated with acid before pretreatment showed better digestibility than the sections that were pre-saturated to lower or higher levels. Milled biomass, however, digested best, demonstrating the importance of enhanced enzyme transport — an outcome of the more thorough and uniform pretreatment of milled materials. With woody feedstocks, milling to fine parti­cle sizes may be impractical and pre-impregnation of biomass with catalyst, as practiced in the pulp and paper industry [48], might need to be utilized to improve conversion efficiencies.

Biomass gasification is basically a two-step process, pyrolysis at lower tempera­ture followed by gasification at a higher temperature. Pyrolysis is an endothermic process during which the biomass is decomposed into volatile materials (majority)

Flue gas

Bottom ash

Circulating fluid-bed gasifier

Fig. 3 Illustrative structures of different types of gasifiers (modified from Dr. R. L. Bain’s 2004 presentation at DOE/NASCUGC Biomass and Solar Energy Workshop)
and char. Volatiles and char from the pyrolysis process are further converted into gases during the gasification process. Although the exact chemical reactions and kinetics are complex and not yet fully-understood, biomass gasification includes the following:

The major components of typical syngas generated from wood are listed in Table 3, and it is evident that output variation occurs, even in the same type of gasi­fier as gasification conditions (temperature, pressure, O2, and steam levels) typically impact the syngas composition.

1.1 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. These vehicles 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, or butanol) 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]