Depending on their origin, the most promising lignocellulosic materials regard­ing their conversion into fuel ethanol can be classified into seven big groups (see Table 3.10):

1. Agricultural residues

2. Agro-industrial residues

3. Hardwood

4. Softwood

5. Herbaceous biomass

6. Cellulosic wastes

7. Municipal solid waste

The agricultural residues comprise those lignocellulosic materials derived from the cropping and harvesting of plant species with economic importance. In particular, the exploitation of cereal plantations implies the generation of a great

Coumaryl alcohol Coniferilyc alcohol

^hydroxyphenyl unit Guaiacyl unit

Cellulose

/

к

FIGURE 3.9 Schematic diagram of the structure of a lignocellulosic complex.

Classification of Some Materials with High Content of Lignocellulosic Biomass

volume of residues, among which the straw should be highlighted. The straw is the dried, crushed or not crushed material coming from plants of the family Gramineae once it is separated from the grain. The straws most evaluated for ethanol production purposes are wheat straw, rice straw, and barley straw. The agro-industrial residues refer to the by-products and wastes generated during the commercial transformation of agricultural crops. The bagasse should be listed among these residues. The most studied agro-industrial residue is the sugarcane bagasse, the fibrous residue obtained after juice extraction during the milling step of sugarcane. Other promising residue is the sweet sorghum bagasse. Other mate­rials belonging to this category are corn stover and olive stone and pulp.

The materials having origin in hardwood constitute a separate group of ligno — cellulosic feedstocks. These materials comprise not only the wood itself, but also its derivatives like sawdust, shavings, and the collected biomass resulting from forestry activities, such as branches, stalks, and trunk pieces. The wood obtained from trees of the angiosperm species—poplar, eucalyptus, aspen, oak, maple, birch, rosewood, and mahogany—belongs to hardwood. The softwood, in turn, comprises the wood of conifers. The wood from trees of the gymnosperm spe­cies, such as pine, fir, spruce, larch, and cedar, can be included in this group. Softwood has higher lignin content than hardwood.

The herbaceous biomass refers to the materials coming from herbaceous plants, i. e., those plants not generating wood. The grasses are plants that present neither woody stems nor woody roots. In general, their stems are green. The most studied herbaceous biomass for ethanol production purposes comprises differ­ent types of grasses like switchgrass, used in North America for hay production, alfalfa hay, reed canary grass, coastal Bermuda grass, and timothy grass that covers almost two-thirds of the livestock meadows in the United States. These herbaceous plants have a great importance because they grow very fast and have reduced nutritional requirements. Thus, these plants are excellent candidates for their exploitation as crops dedicated to bioenergy production.

Among the cellulosic wastes are residues resulting from industrial activities, mostly related to paper processing, which have an elevated content of cellulose compared to other types of lignocellulosic biomass. As examples of this group, newspaper, waste office paper, and paper sludge, one of the effluents of plants for paper recycling, should be highlighted. Finally, the organic fraction of munici­pal solid wastes are composed of materials with high lignocellulosic content like wasted paper, cardboard, fruit and vegetable peels, garden residues, and wood items, among others.

The composition of lignocellulosic biomass, expressed as the proportion of cellulose, hemicellulose, and lignin, depends on its origin, though some simi­larities can be observed independent on the group to which all material belongs. The composition of several representative lignocellulosic materials is presented in Table 3.11. As can be observed, hardwood presents high cellulose content mak­ing it very promising regarding the production of second generation bioethanol. In this sense, the hydrolysis of cellulose allows the formation of glucose, which in turn can be transformed into ethanol by fermentation. Likewise, the cellulosic

Percentage Composition (Dry Basis) of Some Lignocellulosic Materials

wastes have high cellulose contents. On the other hand and due to its high lignin content, softwood has a more difficult transformation process increasing the com­plexity of fuel ethanol production.

From the large variety of lignocellulosic materials that have been proved for fuel ethanol production, those with higher availability and production volume have been defined as the most promising. Logically, this selection is made based on the context of each region and country. Thus, the corn stover along with pop­lar wood has become the most potential feedstocks for bioethanol production in the United States. In Europe, the straw of different cereals is the most promising material. In fact, wheat straw and barley straw are the feedstocks defined for the first commercial plant for production of cellulosic ethanol whose construction started in 2005 and will open soon in Salamanca (Spain; Abengoa, 2008). In the case of South American countries like Brazil and Colombia, the most promising feedstock for second generation (lignocellulosic) ethanol is cane bagasse because it has great availability and production volumes. In addition, the participation of the sugar sector could provide the necessary financial resources needed for the implementation of these technologies at industrial level.

In the case of ethanol production from lignocellulosic biomass, the main obstacle to be overcome is the fact that fermenting microorganisms are not able to assimi­late all the sugars released during pretreatment and hydrolysis of biomass in an effective way. Genetic engineering has been contributing to the development of microorganisms exhibiting this feature. To face this challenge, there exist two approaches (Aristodou and Penttila, 2000; Chotani et al., 2000; Zaldivar et al., 2001). The first one consists of modifying microorganisms in such a way that they can assimilate a wide spectrum of substrates, e. g., by introducing the metabolic pathways required for the utilization of xylose or arabinose to good ethanologenic microorganisms such as yeasts and Z. mobilis. The second approach is based on the modification of microorganisms that assimilate a great variety of substrates; in this case, genes encoding the conversion of pyruvate into ethanol are introduced to microorganisms, like Escherichia coli, capable of assimilating hexoses and pen­toses. Some examples illustrating these two approaches are shown in Table 6.3.

Through clonation of genes encoding xylose reductase and xylitol dehydro­genase in S. cerevisiae, the conversion of xylose into xylulose via xylitol can be achieved. The xylulose is a pentose, which can be assimilated by the yeasts. Unfortunately, the productivity is low and xylitol is formed as a by-product, which deviates part of the substrate that could be utilized for ethanol synthe­sis (Claassen et al., 1999). Ingram and Doran (1995) report the development of recombinant strains of gram-negative bacteria (E. coli, K. oxytoca, or Erwinia sp.) in whose chromosomes have been inserted into the genes of Z. mobilis encod­ing the metabolic pathway for ethanol production. In this way, these strains can efficiently convert all the sugars released during the hydrolysis of cellulose and hemicellulose. The saccharification of cellulose is more complicated, although one of the obtained recombinant strains of K. oxytoca has the natural ability for degrading cellobiose that employs a lower dosage of added cellulases for the effective conversion of purified cellulose into ethanol by a simultaneous hydro­lysis and fermentation process. In their documented review on the fermentation

of lignocellulosic hydrolyzates, Olsson and Hahn-Hagerdal (1996) discuss the performance of different pentose-utilizing microorganisms, among them several recombinant strains. Fermentation indexes for lignocellulosic hydrolyzates pre­treated and detoxified by different methods are also provided.

Lynd et al. (2002) point out that the feasibility of a CBP (see Chapter 9, Section 9.2.4) for lignocellulosic ethanol process will be established when a microorgan­ism or microbial consortium can be developed according to one of two strategies. The first of them, called native cellulolytic strategy (A in Figure 6.5), is oriented to engineer microorganisms having a high native cellulolytic activity in order to improve the ethanol production through the increase of their yield or tolerance, i. e., by the improvement of the fermentative properties of a good producer of cel — lulases. In this case, the modifications in the process microorganism should be aimed at reducing or eliminating the production of by-products, such as acetic acid or lactate, and at increasing the ethanol tolerance and titres (Cardona and Sanchez, 2007). Recent studies have demonstrated the possibility of obtaining ethanol-tolerant strains of C. thermocellum growing at ethanol concentrations exceeding 60 g/L, a titer not sufficient to put thermophiles at a disadvantage rela­tive to more conventional ethanol producers (Lynd et al., 2005).

The recombinant cellulolytic strategy (B in Figure 6.5) contemplates the genetic modification of microorganisms that present high ethanol yields and tol­erances in such a way that they are capable of utilizing cellulose within a CBP configuration, i. e., making a microorganism with good fermentative properties to produce cellulases (Cardona and Sanchez, 2007). The second strategy is con­sidered more difficult due to the complexity of cellulases system (Begum and Aubert, 1994). Currently, the production of cellulases by bacterial hosts produc­ing ethanol at high yield such as engineered E. coli, K. oxytoca, and Z. mobilis and by the yeast S. cerevisiae has been studied. For instance, the expression of cellulases in K. oxytoca has allowed an increased hydrolysis yield for microcrys­talline cellulose and an anaerobic growth on amorphous cellulose. Similarly, sev­eral cellobiohydrolases have been functionally expressed in S. cerevisiae (Lynd et al., 2005). Undoubtedly, ongoing research on genetic and metabolic engineering will make possible the development of effective and stable strains of microorgan­isms for converting cellulosic biomass into ethanol. This fact will surely lead to a qualitative improvement in the industrial production of fuel ethanol in the future (Cardona and Sanchez, 2007).

One of the bottlenecks of ethanol production from biomass is the high cost of enzymes, as noted above. The current cost of producing lignocellulolytic enzymes by submerged fermentation mainly using T. reesei, is about US$0.40 to 0.60/gal ethanol, but an increase in the specific activity of the enzymes or in the efficiency of their production through genetic engineering can be expected. This would allow an eventual cost reduction of the enzymes to US$0.07/gal ethanol, as suggested by (suggested by Tengerdy and Szakacs, 2003). However, these authors consider that the genetic improvement of fungi producing these enzymes by solid — state fermentation could have a greater potential than the genetic improvement of fungi synthesizing the same enzymes by submerged fermentation, considering the fact that fungi growing on the surface of biomass have enzymatic complexes with optimal characteristics and proportions for the hydrolysis of lignocellulosic materials. On the other hand, the U. S. Department of Energy has contracted with the world enzyme-producing leaders, Novozymes (Denmark) and Genencor (USA), with the aim of developing research for improving cellulase systems and reducing their costs. The research must be oriented not only to the enhancement of yield, stability, and specific activity of cellulases, but also to the development of an enzyme mixture that will remain active under hard conditions related to such steps as acid pretreatment (Mielenz, 2001).

Undoubtedly, if the worldwide use of fuel ethanol uses the development of the technology of lignocellulosic biomass utilization, genetic engineering will be called to supply the “tailored” microorganisms needed to meet the exigen­cies of this new technology. From this, the importance of the metabolic pathway engineering is inferred. Metabolic pathway engineering is aimed at establishing metabolic pathways and production hosts, which are capable of delivering opti­mal flow of carbon from substrate to final product at high yields and volumetric productivities. In particular, pathway engineering can achieve the integration of the process at the molecular level through the optimization of the primary meta­bolic pathways for the synthesis of the targeted product (Chotani et al., 2000).

In Chapter 7, Section 7.1.4.3, the co-fermentation was presented as a way for a more complete utilization of all the sugars present in the hydrolyzates of lignocellulosic biomass. This process can be considered an example of reaction-reaction integra­tion since two biochemical processes (fermentation of glucose and fermentation of xylose) are combined and simultaneously accomplished in the same single vessel. For this, mixed cultures can be used as shown in Table 7.3. In addition, an enzyme transforming the xylose into another compound more assimilable by conventional yeasts can be added to the culture medium in order to allow the utilization not only of glucose, but also of xylose. The other approach consists of using recombinant microorganisms able to assimilate these two sugars as presented in Chapter 6, Table 6.3. From the viewpoint of process systems engineering, the modeling of this process plays a crucial role for simulation procedures intended to assess different technological configurations of biomass-to-ethanol conversion in the framework of process synthesis. The aspects concerning this topic were discussed in Chapter 7, Section 7.2.2. In general, through co-fermentation, it is possible to implement simultaneous fermentation processes with higher compactness and lower pro­duction costs since a unit for pentose fermentation is not required (Figure 9.3).

Nevertheless, the process itself corresponds to an SHF scheme because the cel­lulose has to be hydrolyzed in a previous bioreactor using cellulases.

An integral study was carried out in Brazil regarding the ecological, economic, and social aspects of fuel ethanol production including the agricultural and indus­trial steps. Three ethanol production plants were analyzed and the results were quantified through the environmental efficiency of each process. According to the analysis performed, none of the three plants achieved the highest level estab­lished for environmental efficiency (Borrero et al., 2003). Prakash et al. (1998), in turn, proposed an indicator called figure of merit, which is expressed as the ratio between the net energy yield of a fuel (the difference between the gross energy produced during its combustion and the energy needed to produce it) and the CO2 emissions produced by that fuel. Using this figure, it was concluded that for anhy­drous ethanol production from cane molasses in India the net energy yield is about 2% the potential of ethanol as a gasoline substitute in road transport has been estimated to be as high as 28%. A similar indicator based on the figure of merit has been proposed by Hu et al. (2004a) as well. Lynd and Wang (2004) developed a methodology for evaluating fossil fuel displacement for biological processing of biomass in the absence of product-specific information other than the product yield and whether fermentation is aerobic or anaerobic. With the help of this meth­odology, the authors answer affirmatively the question: Are there biomass-based processes and products with fossil fuel displacement sufficiently large that they could play a substantial role in a society supported by sustainable resources?

Another methodology that has been used to measure the environmental fea­sibility and sustainability of bioethanol production processes over a long term is the so-called emergy analysis (see Chapter 2, Section 2.2.5). Emergy is the abbreviation of embedded energy. This methodology takes into account the dif­ferent system inputs, such as the renewable and nonrenewable energy sources, goods, labor, and all the materials involved in a process. Each input is assessed under the same physical condition, the equivalent solar energy (emergy) concen­trated to supply the given input. In this regard, the emergy is a measure of the global convergence of energy, time, and space needed to make available a given resource. Bastanioni and Marchettini (1996) analyzed four systems for bioethanol production and concluded that mankind is far from a sustainable production of biofuels because it appears that the biomass energy cannot be the basic energy source in countries having a high level of energy consumption, with arable land being the major constraint. Other methodologies for environmental assessment of fuel ethanol production from lignocellulosic biomass have been reported as the application of the sustainable process index (SPI), a highly aggregated indicator of the environmental pressure of a process, to the bioenergy assessment model (BEAM), which have allowed showing that the bioenergy integrated systems are superior to the fossil fuel systems in terms of their environmental compatibility (Krotscheck et al., 2000).

Berthiaume et al. (2001) propose a method to quantify the renewability of a biofuel taking as an example the ethanol production from corn. This method con­siders the exergy (useful or available energy) as the quantitative measure of the maximum amount of work that can be obtained from an imbalance between the physical system and the environment surrounding it. The exergy was accounted for evaluating the departure from ideal behavior of a system caused by a con­sumption of nonrenewable resources through the so-called restoration work. These authors point out that ethanol production is not renewable, though they emphasize that this evaluation was performed employing many simplifications. Thus, further research is needed to improve the accurateness of the results and the validity of the conclusions.

Undoubtedly, the unification of the environmental assessment criteria is required in such a way that the main aspects of fuel production and utilization, including both fossil fuels and biofuels, are taken into account. The large number of consideration, supposition, assumption, approximation, and methodological approaches limit the use and comparison of the environmental indicators that have been applied to fuel ethanol production. An example is the contradictory results about the environmental and energy performance of corn ethanol that have been published (Patzek et al., 2005; Pimentel, 2003; Shapouri et al., 2003; Wang et al., 1999). This difficulty imposes some constraints when different pro­cess alternatives for bioethanol production are being evaluated, especially if the optimization of an objective function considering not only the technoeconomic indicators but also the environmental performance indexes of the different tech­nological configurations proposed is performed.

Demand for energy, enough food, and a good environment is the most important concern in the entire world today. Then, the possibility of obtaining a renewable, available, safe, and effective source of energy is one of the challenges that human­ity should address. The biofuels, particularly bioethanol, are an environmentally clean source of energy. However, production costs of fuel ethanol are higher than production costs of gasoline. Nevertheless, many groups and research centers in different countries are continuously carrying out studies aimed at reducing etha­nol production costs for a profitable industrial operation. Diverse research trends and process improvements could be successful when trying to lower ethanol costs. These research tendencies are related to the nature of utilized feedstocks (looking for the most productive and cheap raw materials), tools of process engi­neering (mainly process synthesis, integration, and optimization), food security, and environmental impacts.

13.1 FEEDSTOCKS

The three kinds of feedstocks used for fuel ethanol production correspond to

resources that are present in almost all the countries. In particular, all populated regions in the world account for vast amounts of lignocellulosic waste materials that eventually can be converted into ethanol. Tropical countries exhibit compara­tive advantages in the availability of sucrose — and starch-containing feedstocks for ethanol production in comparison to European or North American countries. In fact, the dynamics of the global ethanol market could require these countries to supply the growing demand of those countries that have implemented or will implement ambitious programs for the partial substitution of fossil fuels with renewable liquid fuels. These programs may have dissimilar motivations other than environmental concerns, but humankind and global climate will be ben­efited in any case.

For the three main types of feedstocks, the development of effective, continu­ous fermentation technologies with near 100% yields and elevated volumetric productivities is one of the main research subjects in the ethanol industry. To this end, many of newly proposed technologies for reducing the product inhibi­tion effect on the cell growth rate should be scaled up at the industrial level. Additionally, past research tendencies for cell-free ethanol production, using
only the enzymes involved in the conversion of glucose to ethanol, may offer a practical and beneficial alternative. This progress should complement the intense efforts oriented to the selection and development of microbial strains with par­ticular traits, such as specific flocculating properties or increased tolerance to ethanol, inhibitors, and salts.

Consequently, an important part of the research trends on fuel ethanol produc­tion is geared to the reduction of feedstock costs, especially through the utiliza­tion of less expensive lignocellulosic biomass. In general, most of the research efforts are oriented to the conversion of lignocellulosics into fermentable sugars and then to ethanol. One of the key factors for enhancing the competitiveness of the biomass-to-ethanol process is the economic and concentrated access to large quantities of biomass distributed in rural areas of the world. Another important issue in transformation technologies for lignocellulosic feedstocks is the design of low-cost methods for its delignification. After that, the increase in the specific activity of cellulases and the decrease in their production costs play a crucial role in the process costs. The technology of recombinant DNA will provide impor­tant advances for the development of the fuel ethanol industry. The development of genetically modified microorganisms capable of converting starch or biomass directly into ethanol and with a proven stability under industrial conditions will allow the implementation of the consolidated bioprocessing of the feedstocks.

The massive utilization of fuel ethanol in the world requires that its production technology must be cost effective and environmentally sustainable. In particular, ethanol production costs should be lowered. For current technologies employed at the commercial level, the main share in the cost structure corresponds to the feed­stocks (above 60%) followed by the processing expenditures. In general, the use of sucrose-containing materials such as cane molasses allows producing ethanol with the lowest costs compared to using the starchy materials (mostly grains).

Although the ethanol yield from corn is higher than that from sugarcane, the lower annual yield of corn per cultivated hectare makes it necessary to use larger cropping areas. On the other hand, the lignocellulosic biomass represents the most promising feedstock for ethanol production. The availability and low cost of a wide range of lignocellulosic materials offer many possibilities for the develop­ment of bioindustries that could support the growth of the international biofuel market and contribute to the reduction of greenhouse gas emissions worldwide. A summary about research perspectives of feedstocks for ethanol production is presented in Table 13.1.

Optimization-based process synthesis makes use of optimization tools to identify the best configuration of a process flowsheet. For this, the definition of a super­structure that considers a significant amount of variations in the topology of the technological configurations for a given process is required. In general, the evalu­ation and definition of the best process flowsheet is carried out solving a mixed — integer nonlinear programming (MINLP) problem. In this way, process synthesis is accomplished in an automatic way excluding so far as possible the formulation of heuristic rules.

The great advantage of this approach lies in the generation of a generic frame­work to solve a large variety of process synthesis problems carrying out a very rigorous analysis of the global process structure. In particular, all the equations corresponding to the models of each process unit should be specified. This allows the definition of the accuracy level during the description of unit processes and the operations involved. This implies solving an optimization problem while simultaneously taking into consideration all the models of the units (equation- oriented approach). In contrast, most of the commercial process simulators some­times used for knowledge-based strategy are based on the modular-sequential approach in which the calculation scheme involves the models for each unit to which the user does not have direct access. Thus, the simulation is solved taking into account the strict order of the units (from feedstocks to end products) mak­ing up the process flowsheet. Main drawbacks of the optimization-based strategy are related to the fact that the optimal configuration can only be found within the alternatives considered in the formulated superstructure (Li and Kraslawski, 2004). Furthermore, this approach has the additional disadvantages of having a significant mathematical complexity as well as the difficulties arising during the definition of the superstructure of technological configurations, i. e., the difficulty to ensure that the initial superstructure contains the “best” solution (Barnicki and Siirola, 2004). In this sense only, it is possible to formulate the design problem as a mathematical programming (optimization) problem when all the alternatives to be considered can be enumerated and evaluated quantitatively (Westerberg, 2004). In addition, this approach presents a number of difficulties when deal­ing with the optimization of under-defined design problems and uncertainties that result from the multi-objective requirements of the design problem (Li and Kraslawski. 2004).

Biological pretreatment has low energy requirements and mild environmental conditions (Table 4.5). However, most of these processes are too slow, which lim­its their application at an industrial level for ethanol production process. Fungi are enzyme producers when they grow on the surface of wood and other lignocel — lulosic materials. Brown-rot fungi mostly attack the cellulose, while white — and soft-rot fungi attack both cellulose and lignin. As many white-rot fungi degrade the lignin, they have been employed for production of ligninases and degradation of lignocellulosics. Evans et al. (1994) describe how enzymes released by these fungi attack lignocellulosic materials and emphasized the role of small molecular agents involved in this process. These authors point out that the most important fungi of this class are Phanerochaete chrysosporium and Phlebia radiate, which synthesize significant amounts of extracellular peroxidases. Lee (1997) reports on the main microorganisms producing lignin-degrading enzymes and suggests the fermentation processes for producing them through both submerged culture and solid-state fermentation. In fact, the fungus P. chrysosporium has been proposed in the patent of Zhang (2006) for degrading lignin in a biomass-to-ethanol process scheme involving the separate fermentation of pentoses and hexoses (Sanchez and Cardona, 2008). The cellulases required by the process for bioethanol production can be obtained from lignocellulosic materials in which the fungi grow. Tengerdy and Szakacs (2003) and Kang et al. (2004) highlight the viability of producing cellulases and hemicellulases by solid-state fermentation compared to conven­tional submerged fermentation (Warzywoda et al., 1992).

When a technological flowsheet involving an SHF process is employed, the detoxified hemicellulose hydrolyzate can be unified with the cellulose hydrolyz — ate coming from the enzymatic reactor. The resulting stream contains mostly glucose, but also xylose and other hexoses released during biomass pretreat­ment. The simplest scheme includes the cultivation of S. cerevisiae that converts the glucose present in the medium into ethanol remaining in the xylose and the other hexoses. This implies a reduction in the amount of ethanol that could be obtained if the xylose and remaining sugars were utilized. To increase the amount of sugars converted into ethanol, yeast assimilating the xylose besides glucose can be used, but in this case the biomass utilization rates are lower relative to microorganisms that assimilate only hexoses. This is explained by the diauxic growth of this type of yeast (see Chapter 6). To offset this effect, sequential fermentations are employed in which S. cerevisiae utilizes the hexo — ses during the first days of cultivation and later xylose-utilizing yeast is added in order to complete the conversion to ethanol (Chandrakant and Bisaria, 1998), but achieved ethanol yields are not very high. Therefore, the most suitable con­figuration corresponds to the scheme of Figure 7.6 where both fermentations are performed independently.

One of the main challenges in pentose fermentation lies in the fact that the productivities of pentose-utilizing microorganisms are less than those of hexose — fermenting ones. Comparisons are conclusive: ethanol productivity for S. cer- evisiae can attain values of 170 g/(L/h) in the case of continuous systems with cell recycling, whereas the productivity for C. shehatae at high cell concentra­tions reaches values of only 4.4 g/(L/h) (Olsson and Hahn-Hagerdal, 1996). On the other hand, there are a few cases where the immobilization of these yeasts increases the ethanol productivity (Chandrakant and Bisaria, 1998), unlike the case of hexose-fermenting yeasts or Z. mobilis. In spite of these drawbacks, pen­tose-utilizing microorganisms are important for the design of processes involving separate fermentation of hexoses and pentoses during the processing of resulting streams from biomass pretreatment. As occurs with S. cerevisiae or Z. mobilis, most pentose-fermenting yeasts are mesophiles (Sanchez and Cardona, 2008). On the other hand, although thermotolerant yeasts such as Kluyveromyces marxi — anus have demonstrated their capability to ferment glucose at 45°C (Singh et al., 1998), there are no data about the assimilation of xylose by this yeast, according to Ryabova et al. (2003). These authors described the separate fermentation of glucose and xylose by native strains of methylotrophic yeast Hansenula polymor — pha at 37°C in flask cultures during 60 h achieving ethanol concentrations of 13.2 g/L and 2.98 g/L from glucose and xylose, respectively.

Lynd et al. (2001) report that for Thermoanaerobacterium thermosaccharolyt — icum cultivated in xylose-based media during batch and continuous cultures, the ethanol concentrations obtained are low (in the order of 25 g/L). These authors studied the influence of different factors limiting the substrate utilization for con­tinuous cultures at progressively higher feed xylose concentrations. Their results indicate that the salt accumulation due to the utilization of bases for pH-control of fermentation limits the growth of this bacterium at elevated values of xylose con­tent in the feed. These outcomes can explain the differences between the tolerance to added ethanol and the maximum concentration of produced ethanol for these microorganisms. Xylose-fermenting termophilic bacteria are prospective organ­isms to be co-cultured with cellulose hydrolyzing bacteria such as Clostridium thermocellum in order to directly convert pretreated lignocellulosic biomass into ethanol, a process called consolidated bioprocessing (CBP). Another promising microorganism capable of fermenting a great variety of sugars (including hexoses and pentoses) is the fungus Mucor indicus reaching ethanol yields of 0.46 g/g glucose when it is cultivated under anaerobic conditions. In addition, this fungus assimilates the inhibitors present in dilute-acid hydrolyzates (Sues et al., 2005). Other pentose-fermenting zygomycetes were also evaluated (Millati et al., 2005). The use of the fungus Chalara parvispora for ethanol production from pentose — containing materials has been patented (Holmgren and Sellstedt, 2006). Ogier et al. (1999) have compiled information about the main fermentative indexes for the pentose-assimilating yeasts Candida shehatae, Pichia stipitis, and Pachysolen tannophilus, and for the xylose-assimilating thermophilic bacteria T. thermosac — charolyticum, T. ethanolicus, and Bacillus stearothermophilus.

Ethanol can be removed from the culture broth through absorption employing a stripping gas. This makes possible the increase of sugar concentration in the feed stream entering the fermenter. This process has been studied in the case of corn mashes obtained by the dry-milling process. Taylor et al. (1998) stud­ied this integrated process in the case of the dry-milling ethanol process in a pilot plant integrating a 30 L fermenter with a 10 cm packed column for ethanol removal by the CO2 (stripping gas) released during the fermentation. A simpli­fied scheme of this process is presented in Figure 9.9 where two circulation loops are employed. In this scheme, concentrated solutions of the product are obtained

from the condensation of ethanol. The model proposed by these authors showed that ethanol inhibition influences especially the cell yield reaching a value of 60 g/L of ethanol in the broth above which the inhibition is very strong. The authors point out that the values of kinetic parameters depend in a high degree on the type of fermentation: batch or continuous. Later, Taylor et al. (2000) employed a saccharified corn mash containing high levels of suspended solids as a feed and compared the results obtained with a state-of-the-art dry-milling process using Aspen Plus. Savings of US0.8 cents/L of ethanol can be attained in comparison with the state-of-the-art process for which saccharification and fermentation are carried out separately (Cardona and Sanchez, 2007).

Other variants of this type of integrated configuration have been proposed as evidenced in Table 9.7. Gong et al. (1999) report the simultaneous variant of the fermentation-stripping process using an air-lift reactor with a side arm (external loop) that improves liquid circulation and mass transfer. A more complex configu­ration integrating the fermentation and stripping was developed by Bio-Process Innovation, Inc. (West Lafayette, IN, USA). A pilot plant was designed and built for ethanol production from lignocellulosic biomass using a 130 L multistage, con — tinuous-stirred, reactor separator (MSCRS) for the SSF of cellulose and hemicel — lulose (Dale and Moelhman, 2001). The MSCRS consists of a series of six stirred

TABLE 9.6 Reaction-Separation Integration for alcoholic Fermentation Processes through ethanol removal by Bioagent/unit Vacuum Technology operation feedstock/Medium remarks references Continuous vacuum fermentation Saccharomyces cerevisiae/vacuum system Glucose-containing medium 50 mm Hg; with and without cell recycling; sparging of oxygen; 33.4 % glucose feed; productivity 40-82 g/(L x h) Cysewski and Wilke (1977) Continuous fermentation coupled with vacuum flashing S. cerevisiae/extractive vacuum flash chamber Sugarcane molasses Modeling based on kinetic approach; 4-5.33 kPa; recycling of liquid stream from flash; cell recycling; 98% conversion; 23-26.7 g/(L x h) productivity Costa et al. (2001) da Silva et al. (1999) Source: Modified from Cardona, C. A., and O. J. Sanchez. 2007. Bioresource Technology 98:2415-2457. Elsevier Ltd.

stages in which the SSF of biomass is carried out. Each stage has a stirred tank for the reaction and a gas-liquid separation contactor. In the three upper stages, SSF of cellulose is carried out at 42°C using a thermotolerant K. marxianus, while in three lower stages, the fermentation of xylose is achieved using the yeast P. stipi — tis at 30°C. In addition, part of the broth containing enzymes is recirculated from the last stage to the first upper stage in order to favor the reaction with the fresh pretreated biomass. Reaction-reaction integration is implemented because com­mercial cellulases were added for the saccharification of pretreated biomass. This defines the process temperature helping the generation of ethanol vapors. The broth overflowing from one stage into the next stage is contacted with a stripping stream of CO2 that entraps the ethanol. A gas stream passes across the reactor and then through an absorption tower where water is used for removing ethanol vapors. CO2 is again recirculated to the reactor. In this way, reaction-separation integration is verified through the in situ removal of ethanol produced in each stage. The same company has installed a pilot plant reactor using MSCRS tech­nology (Dale, 1992) in the plant of Permeate Refining, Inc., located in Hopkinton, IA (USA), that produces 11.36 million liters per year of ethanol from starch. This unit has been in operation since September 1995 and employs starch dextrins, thought its use for lignocellulosic biomass has been proposed. Unfortunately, no reports are available describing the modeling and performance of this type of configuration (Cardona and Sanchez, 2007).

Unlike the design of fuel ethanol production processes from sucrose — or starch — containing feedstocks, the use of lignocellulosic materials entails the analysis of multiple alternative variants for accomplishing the conversion of biomass into eth­anol. This means that process synthesis procedures play a significant role during the design of processes based on biomass. In fact, there exists a significant variety of biomass conversion technologies proposed worldwide to produce fuel ethanol. This variety should be thoroughly assessed in order to select the most suitable technological configuration considering the local conditions and the process per­formance in terms of its technical, economic, and environmental effectiveness.

Many efforts have been put forth worldwide to improve the efficiency of bio- mass-to-ethanol conversion. In the United States, the production of ethanol from lignocellulosic biomass is being studied intensively. Ingram et al. (1999) have car­ried out significant research on the development of recombinant strains of enteric bacteria to use for cellulosic ethanol production. Current technology allows the use of a genetically engineered Escherichia coli strain with the natural ability of assimilating both pentoses and hexoses found in the liquid fraction resulting from the dilute-acid pretreatment of lignocellulosic biomass. The main Zymomonas mobilis genes encoding the ability for homofermentative production of ethanol have been integrated into the bacterial chromosome in this strain. The solid frac­tion from this pretreatment that contains cellulose and lignin undergoes SSF using a recombinant strain of Klebsiella oxytoca with the ability to ferment cellobiose and cellotriose, eliminating the need for supplementing Trichoderma reesei cel — lulases with P-glucosidase. This strain also has the genes to encode the produc­tion of ethanol. The proposed overall process can be observed in Figure 11.11a (Cardona and Sanchez, 2007). At present, research efforts are oriented toward the development of a single microorganism capable of efficiently fermenting both hemicellulosic and cellulosic substrates that will make possible the development of the direct conversion of biomass into ethanol.

The model process designed by the NREL comprises a previous hydrolysis of wood with dilute acid followed by a simultaneous saccharification co-fermenta­tion (SSCF) process using cellulases produced in situ by genetically engineered Z. mobilis with the ability to transform both glucose and xylose into ethanol (Figure 11.11b; Cardona and Sanchez, 2007). The process is energetically inte­grated using the heat generated during the combustion of methane formed in the anaerobic treatment of wastewater from pretreatment and distillation steps (Wooley et al., 1999). In addition, the burning of lignin allows the production of energy for the process and a surplus in the form of electricity. The production of one liter of ethanol by this process is calculated at US$0.396, whereas the ethanol production cost from corn is US$0.232 (McAloon et al., 2000). A pilot plant designed for conversion of lignocellulosic biomass into ethanol was built and operated with the aim of supporting industrial partners for the research and development of biomass ethanol technology (Nguyen et al., 1996). In this plant, tests in continuous regime

(a)

Hydrous EtOH

(b)

(c) Steam, power

Biomass

Cogeneration

(d) (e)

FIGURE 11.11 Some proposed flowsheet configurations for ethanol production from lignocellulosic biomass. (a) Process based on utilization of enteric bacteria (Ingram et al., 1999). (b) NREL model process (Wooley et al., 1999). (c) Iogen’s process (Tolan, 2002). (d) Process proposed by Reith et al., (2002). (e) IIT Delhi process (Ghosh and Ghose, 2003). Main stream components: C = cellulose, L = lignin, G = glucose, P = pentoses, I = inhibi­tors, Cel = cellulases, EtOH = ethanol, Sol = solvent, SCP = single cell protein. LF = liquid fraction, SF — solid fraction, HHZ = hemicellulose hydrolyzate, Rec = recombinant, IE = ion exchange. (From Cardona, C. A., and O. J. Sanchez. 2007. Bioresource Technology 98:2415-2457. Elsevier Ltd. With permission.)

for the utilization of lignocellulosic residues of low cost and great availability, such as corn fiber, were carried out (Schell et al., 2004). The objective of these tests was the assessment of the operation of the integrated equipments and the generation of data concerning the process performance. This type of plant allows the acquisition of valuable experience considering the future implementation of the industrial process, the same as the feedback of the models utilized during the design step. In addition, feasibility studies carried out by NREL help industrial partners make decisions about the potential implementation of these technologies for fuel ethanol production (Kadam et al., 2000; Mielenz, 1997). Future trends for costs reduction in the case of the NREL process include more efficient pretreat­ment of biomass, improvement of specific activity and productivity of cellulases, the possibility of carrying out the SSCF process at higher temperatures, improve­ment of recombinant microorganisms for a greater assimilation of all the sugars released during the pretreatment and hydrolysis processes, and further develop­ment of co-generation system (Cardona and Sanchez, 2007). Nagle et al. (1999) proposed an alternative configuration involving a total hydrolysis of yellow poplar using a three-stage countercurrent dilute-acid process validated at experimental level. The obtained hydrolyzate is co-fermented by the recombinant strain of Z. mobilis. In this case, the lignin is recovered prior to the fermentation. Aspen Plus was utilized for generating the needed information to evaluate the economic performance of the whole flowsheet configuration through a spreadsheet model. Optimized values of the key process variables obtained from the simulation are utilized as target values for bench-scale research to design an advanced two-stage engineering-scale reactor for a dilute-acid hydrolysis process.

Some commercial firms have also invested funds in the development of an etha­nol production process employing the lignocellulosic biomass. Iogen Corporation (Ottawa, Canada) developed an SHF process comprising a dilute-acid-catalyzed steam explosion and the removal of the major part of the acetic acid released during the pretreatment, the use of S. cerevisiae as a fermenting organism, distil­lation of broth, ethanol dehydration, and disposal of stillage in landfill (Tolan, 2002). Later modifications involve the co-fermentation of both hexoses and pen­toses using genetically modified strains of microorganisms, such as yeasts or bac­teria (Figure 11.11c). Using the recombinant Z. mobilis strain patented by NREL, Lawford and Rousseau (2003) tested two configurations for ethanol production using the conceptual design based on SHF developed by Iogen. These authors demonstrated that a configuration involving the continuous pentose fermentation using the recombinant Z. mobilis strain, and the separate enzymatic hydrolysis followed by continuous glucose fermentation using a wild-type strain of Z. mobi­lis is the most appropriate in comparison to the use of the co-fermentation process after the enzymatic hydrolysis or the use of an industrial yeast strain during the glucose fermentation (Cardona and Sanchez, 2007).

Reith et al. (2002) have reviewed different processes for production of biomass ethanol and concluded that verge grass, willow tops, and wheat milling residues could be potential feedstock for fuel ethanol production under the regulations of the Netherlands. These authors constructed a model using Microsoft™ Excel™

for the system description of generic biomass-to-ethanol process. This process involves the evaporation of the stream from the saccharification step in such a way that the sugar concentration allows a final ethanol concentration of at least

8.5 vol.% in the fermentation broth. In addition, pretreatment using Ca(OH)2 was included in the analysis (Figure П. Ш). The advantage of using this type of pre­treatment is that inhibitors are not formed making the detoxification step unneces­sary. The evaluation showed that currently available industrial cellulases account for 36 to 45% of ethanol production costs, and therefore, a 10-fold reduction in the cellulase costs and a 30% reduction in capital costs are required in order to reach ethanol production costs competitive with starch ethanol. These evaluation approaches indicate the need for developing processes that contribute to improv­ing one or all of the four critical areas related to cellulase research mentioned in Chapter 5, Section 5.2.3.1.

Ghosh and Ghose (2003) report on the model process for bioethanol production proposed by the Indian Institute of Technology (IIT) in Delhi (India). This process involves two pretreatment steps: steam explosion for xylose production followed by solvent pretreatment for delignification of biomass. The released pentoses are utilized for single cell protein production, while the cellulose undergoes simulta­neous saccharification and fermentation. The SSF reactor is coupled with vacuum cycling and has a stepwise feeding of cellulose (Figure 11.11e). The process has been tested in a pilot plant using rice straw as a feedstock. However, the obtained product is hydrous ethanol (95% v/v) and the production costs (US$0.544/L) are higher than those expected for the production of dehydrated ethanol through the NREL model process (US$0.395). The consideration of adsorption separation stage (instead of distillation) increases the cost of ethanol by about 50%. The possibility of using alkali pretreatment was also assessed, but the costs increased due to lower by-product credits (low quality of obtained lignin as a fuel; Cardona and Sanchez, 2007).

Pan et al. (2005) report the preliminary evaluation of the so-called Lignol pro­cess for processing softwoods into ethanol and co-products. This configuration makes use of the organosolv process for obtaining high quality lignin allowing the fractionation of the biomass prior to the main fermentation. For this, the pro­cess utilizes a blend of ethanol and water at about 200°C and 400 psi. For etha­nol production, SHF and SSF have been tested. Streams containing hemicellulose sugars, acetic acid, furfural, and low molecular weight lignin are also considered as a source of valuable co-products. Until now, the Lignol process has been oper­ated only in a three-stage batch mode, but simulation studies indicate an improved process economics by operating the plant in continuous mode (Arato et al., 2005). Gong et al. (1999) report a fractionation process employing corncob and aspen wood chips as feedstocks and utilizing alkaline pretreatment with ammonia that favors the separation of lignin and extractives. After this step, the hemicellulose is hydrolyzed with dilute acid and released sugars are fermented by xylose-assim­ilating yeast. Finally the cellulose is converted into ethanol by batch SSF using a thermotolerant yeast strain. So and Brown (1999) performed the economic analy­sis of the Waterloo Fast Pyrolysis process comprising a 5% acid pretreatment, fast pyrolysis, levoglucosan hydrolysis, and the use of two cultures, S. cerevisiae and P. stipitis, to ferment hexoses and pentoses, respectively. These authors also analyzed the SSF process of dilute-acid pretreated feedstock that comprises the pentose fer­mentation by recombinant E. coli for xylose fermentation, and the SHF process of dilute-acid pretreated feedstock using a strain of C. shehatae for hexose and pentose fermentation. The evaluation indicates that the cost of the fast pyrolysis process is comparable to the other two processes in terms of capital costs, operat­ing costs, and overall ethanol production costs (Cardona and Sanchez, 2007).

Due to the high costs of the feedstocks accounting for more than 20% in the case of the lignocellulosic biomass (Kaylen et al., 2000), the optimization of cel­lulose conversion is of great importance, especially if it is accompanied with the appropriate handling and utilization of all process streams. Although many related works can be found, the tendency is the optimization of separate process units. This implies that the integration of such separately studied units optimized at different scales does not always provide the correct information on the global process. This situation is particularly important in the case of the integration of the pretreatment step with the biological transformations. De Bari et al. (2002) undertook this prob­lem emphasizing the scale-up features and the potential of produced by-products in the case of steam-exploded aspen wood chips. The pretreatment step was carried out in a continuous steam explosion pilot plant fed with 0.15 ton/h of dry matter that was coupled with the extraction step in order to separate the lignin and the hemicel — lulose and carry out the detoxification. The subsequent conversion to ethanol was made by SSF. The process was completed with a packed distillation column with a maximum reboiler capacity of 150 L working batch-wise. The experimentation allowed the definition of the best combinations of operation parameters, the selec­tion of the best detoxification procedure, the determination of yields and operation conditions of SSF, the analysis of the distillation for its conversion to hydrogen or ethanol, and the determination of the chemical oxygen demand (COD) of the liquid stream from the distillation step. However, this work does not report if any analysis of the process was carried out from the viewpoint of thermodynamic and kinetics fundamentals of the studied system, or if process synthesis procedures were used for the definition of the selected configuration of the process. These tools may help predict the behavior of experimental systems. Similarly, pilot-plant data can pro­vide feedback to the mathematical models used for the analysis of the system, the same as for the study of its stability and operability. For this point, the complemen­tation with simulation tools is invaluable (Cardona and Sanchez, 2007).

In general, it is thought that reductions in processing or conversion costs of lignocellulosic biomass offer the greatest potential for making biobased products like ethanol competitive in the market place in comparison to oil-based products for which high raw material costs are characteristic (Dale, 1999). Therefore, the fundamental research on the development of cost effective processes for biomass processing represents the key for attaining the mentioned competitiveness. Lynd et al. (1999) argue that oil refineries are unlikely to have significant economies of scale advantages in comparison with the expected mature biomass refineries. In this way, the challenges associated with the biomass conversion are related to the recalcitrance of cellulosic biomass (conversion into reactive products like fer­mentable sugars) and to the product diversification (conversion of reactive inter­mediates into valuable products; Cardona and Sanchez, 2007).

In general, process synthesis procedures can be significantly enhanced using process simulation packages. These simulators have allowed the analyzing of sev­eral technological options and the gaining of insight about the process improve­ments (Cardona and Sanchez, 2007). Almost all the approaches for carrying out process synthesis can rely on simulation tools to evaluate different process alter­natives. This is illustrated in the following case study.