The number of studies on economic aspects of ethanol production from biomass is rather limited. This depends to a large extent on the fact that the ethanol production from biomass has not yet been demonstrated on commercial scale. The ethanol production cost varies between the studies performed from about 0.13 to 0.81 US$ per liter ethanol, see Table 1. Dur­ing 2006 the selling price of bioethanol, produced from starch or sugar-based materials, has fluctuated around 0.65 US$ per liter of ethanol with a peak at 1.12 US$/liter [21]. The future selling price will be dependent on demand and availability, which is influenced by political decisions, such as the EU direc­tive mentioned before. Also tax exemptions, e. g. exemption of CO2 tax and energy tax adopted in Sweden, and protective duty, as that applied in the EU, will impact the pricing for customers.

The large differences in ethanol production costs in Table 1 can be ex­plained by variations in the process design and in the assumptions underly­ing the techno-economic evaluations. Process variations are due to different conversion technologies, e. g. an enzymatic process with SHF, SSF or SSCF or the use of various types of raw materials. Thus, for meaningful compari­son the actual differences have to be identified. The discrepancies that arise due to various assumptions, in many cases, overshadow the actual differences.

Typical examples are the raw material cost (even if the same raw material is used), plant capacity and investment parameters, e. g. pay-off time and in­terest on capital. Also, the country in which the proposed plant is assumed to be located is of importance. One of the main influences on the produc­tion cost originates from the assumed annual capacities of the ethanol plants, which varied from 196 000 to 3 110 000 tons of raw material. This has a con­siderable influence on the total production cost (Fig. 4). Another difference is found in the overall ethanol yields assumed, e. g. if pentoses are converted to ethanol or not. Also, changes in process configurations, or a change in the equipment included in the ethanol production process also influences the overall cost, e. g. whether utilities such as process steam and electricity are in­cluded. Therefore, care must be taken when comparing ethanol production costs from different studies. However, this does not mean that the economic studies are without value. They give important information about which parts of the process are most costly, and where bottlenecks, which need to be ad­dressed by further research, can be expected.

Most economic studies performed on the enzymatic bioethanol process during the past ten years have been for a configuration using some kind of

steam pretreatment employing an acid catalyst. NREL has for many years been conducting detailed techno-economic evaluations of ethanol produc­tion from lignocellulosic materials. In the 1999 report [16] hardwood (poplar) was considered as the raw material and the proposed annual capacity was 700 000 dry metric tons. The following process configuration was assumed. The raw material is pretreated with dilute sulfuric acid at 190 ° C for 10 min­utes. The liquid hydrolyzate is detoxified by ion exchange and overlimed, after which an SSF step is employed. Some of the slurry following pretreatment is used for enzyme production. In the SSF step the remaining cellulose is con­verted to glucose and both hexoses and pentoses are considered fermentable. The ethanol is removed from the mash through stripping and the stillage is dewatered by means of centrifugation. The solids, together with the con­centrated liquid from the evaporation step, are transferred to a boiler for steam and electricity production. The estimated ethanol production cost was 0.38 US$ L-1. Especially the database of physical properties [22], but also sev­eral of the unit operation models from this study, has been used by many other investigators.

Lynd et al. [23] evaluated a process based on dilute acid hydrolysis, pentose fermentation and SSF using hardwood as the raw material, assuming the fol­lowing procedure. Distillation bottoms are centrifuged and the solid residue, together with methane and sludge from the anaerobic digester, is sent to the boiler where process steam and electricity are generated. The plant capacity was assumed to be 658 000 dry tons per year. The ethanol production cost was estimated to be 0.31 US$ L-1. A techno-economic evaluation based on the same process concept and the same raw material as the above, with an annual capacity of 640 000 tons, was conducted by Stone et al. [24]. This re­sulted in an estimated production cost of 0.34 US$ L-1. In the study presented by Lynd et al. [23] a more advanced scenario was also evaluated where fu­ture improvements in conversion technology were included. These include, but are not limited to, a higher overall ethanol yield, the use of a microorgan­ism capable of not only fermenting sugars to ethanol but also of hydrolyzing the cellulose (direct microbial conversion), and shorter residence times in the process steps. With these improvements, together with an increased cap­acity (2 738 000 dry tons per year) the projected ethanol production cost was 0.13 US$ L-1.

A comparative study of an SSF-based process and a process using dilute acid hydrolysis was performed by So and Brown [25]. The SSF process used the same conversion technology as the process evaluated by Lynd et al. [23], while the dilute sulfuric acid step was assumed to be carried out at 180 °C with an acid concentration of 5 gL-1. Estimated production costs at a plant capacity of 25 million gallons of ethanol per year (equivalent to around 260 000 tons of dry raw material per year) were 0.34 US$ L-1 for the SSF-based plant and 0.36 US$ L-1 for the process employing a dilute sulfuric acid process.

In the NREL report of 2002 [26] the raw material was changed to corn stover. Several changes were also made to the model from 1999. Instead of running SSF, an SHF configuration (including pentose fermentation) was employed, where the saccharification was carried out separately prior to fer­mentation. The reason for this was to be able to carry out saccharification at a higher temperature than in the fermentation step. The enzyme produc­tion step was also removed and it was assumed that the enzymes had to be purchased from an enzyme-producing company at an estimated cost of 0.10 US$ per gallon of ethanol. This represents a projected future cost rather than the present cost. Changes were also made to the separation of solids from the stillage stream. Instead of centrifuges, as suggested in the 1999 report, a horizontal belt filter (of the type manufactured by Pneumapress Filter Corporation, CA, USA) was employed and the concentration of wa­ter insoluble solids (WIS) in the filter cake was assumed to be about 50%. The overall ethanol yield increased significantly compared to the 1999 report (from 73.6% to 85.5% concerning cellulose, while 85% yield was assumed from all hemicellulose sugars). The estimated production cost was reduced to 0.28 US$ L-1.

According to a techno-economic evaluation of ethanol production from biomass performed by “SRI Consulting’s Process Economic Program” (PEP) [27], the capital investment required for a plant producing ethanol from 2000 metric tons of straw per day would be around 260 million US$. The plant is assumed to produce 190 million liters of ethanol per year, which gives an investment cost of around 1.37 US$ per liter ethanol and 370 US$ per ton raw material. This is about 2.5 times higher than the investment cost of a corn-based ethanol plant with the same feed capacity. On the basis of the ethanol produced, the ratio would increase to above 3 as the yield of ethanol per ton raw material is higher for corn than for lignocellulosic materials.

The highest contribution to the capital cost, 45% of the total, was equip­ment for the production of heat and electricity for the process and for sale to the grid, wastewater treatment and other utilities. This is not a direct cost of the ethanol production equipment, and in our opinion is often underesti­mated in most studies on ethanol production cost. Another cost that differs widely between studies is the cost of raw material. This depends on both dif­ferences in the type of raw material (agricultural residues, forest residues or energy crops) and on the location of the raw material. According to the Road Map for Agricultural Biomass Feedstock Supply in the US presented by the DOE [28] the goal is to reach a feedstock cost of 30 US$ per dry ton. On the basis of this figure the net raw material cost, i. e. after by-product credit, in the PEP study would be about 0.07 US$ per liter of ethanol, which corres­ponds to 20% of the total production cost of 0.34 US$ L-1. This production cost is, however, without any profit. The cost of biomass in Sweden, and other European countries is much higher, exceeding 90 US$ per metric ton of dry matter [29], which results in a net raw material cost of about 60 US$ per met­ric ton. This would have increased the total production cost in the PEP study to about 0.44 US$ L-1. However, according to the PEP study, the main reason for the higher investment cost for biomass-produced ethanol is due to the cost of conditioning and pretreating the biomass to make the cellulose accessible to enzymatic hydrolysis, which was estimated to represent 27% of the total fixed capital.

Eggeman et al. [30] investigated the pretreatment cost in ethanol produc­tion from corn stover for five different pretreatment methods: dilute acid, hot water, ammonia fiber explosion (AFEX), ammonia recycle percolation (ARP) and lime. The pretreatment design was based on experimental data from various research groups [31] and was implemented in the Aspen Plus model for a full-scale bioethanol plant previously developed by NREL [26]. The model was based on a corn stover feed rate of 2000 dry metric tons per day. The process configuration was based on pretreatment, SSF, ethanol re­covery and internal production of heat and electricity from the syrup and solid residue from the process. The process configuration was identical for all processes except for the pretreatment step. The dilute acid pretreatment process resulted in the lowest ethanol production cost, 0.26 US$ L-1 for the base case alternative where oligomers released in the pretreatment and hydro­lysis steps were not considered for ethanol production. The production cost includes depreciation, but no income tax or return on capital, to make it com­parable to the other costs presented in this review. The total investment cost was estimated to be 185.8 million US$, of which the pretreatment step consti­tuted 25 million US$, i. e. 13.5% of the total. The largest investment cost was for steam and power, 41.8 million US$, which represents 22.5% of the total investment cost.

Two-step pretreatment has been suggested to improve the overall sugar yield in several studies [32-34]. The first step is performed at low severity to release hemicellulose sugars, which are then removed, followed by the sec­ond step at more severe conditions to make the cellulose more accessible to enzymatic attack. Wingren et al. [35] compared the ethanol production cost of two-step steam pretreatment of SO2-impregnated spruce with that for one — step pretreatment, based on experimental data for pretreatment at optimal conditions. The production plants considered were designed for a yearly cap­acity of 200 000 tons raw material, and the only difference between the two plants was the pretreatment step. The process was based on SSF of the pre­treated material. The two-step process resulted in a higher ethanol yield (see Table 2) and a lower requirement for enzymes. However, due to the higher energy demand and higher capital cost the estimated ethanol production cost was the same, 0.55 US$ L-1. In the most optimistic scenario, where the material from the first step was dewatered to 50% dry matter (DM) with­out reducing the pressure, and the overall ethanol yield was assumed to be the highest achieved in the experimental work, 77%, the production cost de­creased by 5.6% to 0.52 US$ L-1. This shows the potential of the two-step pretreatment process, which, however, remains to be verified in pilot trials.

Hamelinck et al. [36] investigated the effect of expected future improve­ments in the conversion of biomass to ethanol, with poplar as a model raw material. The paper contains detailed information on the technical and eco­nomic data used in the analysis. Three scenarios were investigated: short­term (5 y), middle-term (10-15 y) and long-term (> 20 y). Improvements were mainly expected to be in enhanced pretreatment and bioconversion steps, changing from SSF to SSCF of hexose and pentose sugars and finally Consolidated BioProcessing (CBP), resulting in higher ethanol yield and re­duced capital costs. This is based on improvements of both enzyme efficiency and fermentation microorganisms. For CBP a completely new microorgan-

ism, not yet known, was assumed. The production capacity is also assumed to increase, from a biomass input (in metric tons per year) of 620 000 to 1550 000 and 3 110 000 for the short, medium and long term, respectively. Also, the cost of the raw material was assumed to fall from about 58 US$ per metric ton for the short term to 48 and 39 US$ for the middle and long term.

The total investment cost for the short-term scenario was about 395 mil­lion US$, i. e. 475 US$ per ton raw material yearly capacity. The ethanol production cost was determined to be 0.51 US$ L-1. Forty-five percent was due to capital cost and about 35% to the net raw material cost, i. e. after credit for co-produced electricity. For the middle-term scenario the total invest­ment cost increased to around 465 million US$, corresponding to 300 US$ per ton raw material yearly capacity. The ethanol production cost decreased to 0.31 US$ L-1, of which about 44% arose from capital cost and about 31% due to net raw material cost. For the long-term scenario the total invest­ment cost increased to around 820 million US$, corresponding to 263 US$ per ton raw material yearly capacity. The ethanol production cost decreased to 0.20 US$ L-1, of which about 50% was due to capital cost and about 42% to the net raw material cost. The long-term ethanol production cost was still considerably higher than that predicted by Lynd et al. [23] who estimated the future ethanol production cost for a plant based on CBP to be between 0.1 and 0.16 US$ L-1. The main reasons for their lower cost are a higher conversion yield and lower capital costs. It must be pointed out that these are scenarios based on future projected improvements, which are very uncertain.

The number of studies on softwood is more limited. Fransson et al. [27] studied the potential for a two-stage dilute acid process using softwood as raw material. The plant is assumed to be co-located with a heat and power plant from which steam is purchased and co-products (solid residue) are sold. The first hydrolysis step is assumed to be run in co-current fashion, whereas the second reactor is working in counter-current mode to maximize sugar yield and reduce sugar degradation. A sulfuric-acid concentration of 5 g/L is used in both steps. The sugar stream is detoxified in an overliming step and then fermented to ethanol. The dilute ethanol is concentrated in a distillation step and the stillage is then evaporated. The plant is designed to process around 300 000 tons of raw material annually. The estimated ethanol production cost was 0.53 US$ L-1.

In another study by Wingren et al. [38], the production of fuel ethanol from spruce using the enzymatic process was investigated. The softwood was steam pretreated after impregnation with SO2. Two configurations, one based on SSF and the other on SHF, were evaluated and compared. The process conditions selected were based mainly on laboratory data and the processes were simulated using Aspen Plus, while the capital costs were estimated using Icarus Process Evaluator. The ethanol production cost was estimated to be 0.69 and 0.76 US$ L-1 for the SSF and SHF cases, respectively, based on a raw material cost of 63 US$ per dry ton. The main reason for SSF being less ex­pensive was due to the capital cost being lower and the overall ethanol yield higher. Improvements in the SSF process, by running SSF at 8% WIS rather than at 5%, and by recycling of process streams, were shown to result in a de­crease in production cost to 0.51 US$ L-1.

3.1

A number of components in lignocellulosic hydrolysates can inhibit the growth and ethanol production of bacteria and yeasts, and acetic acid has been identified as a major potential inhibitor of Z. mobilis in such acid — produced hydrolysates [61-65]. Lawford and Rousseau [32] examined the role of glucose feeding as a means of improving fermentation perform­ance in acetate-containing media. Another approach to solving this problem has been to use a hydrolysate-fed chemostat to produce adapted or mutant strains [33,34]. Following chemical mutagenesis, Joachimsthal et al. [66] iso­lated a mutant strain, designated ZM4/AcR with a higher acetate resistance than the parent strain. This strain was then transformed by Jeon et al. [67] to the mutant recombinant ZM4/AcR (pZB5). Compared to ZM4 (pZB5), this strain showed enhanced kinetics in batch culture in the presence of 12 gL-1 sodium acetate (8.8 g L-1 acetic acid) at pH = 5.0 in batch culture on 40 g L-1 glucose, 40 g L-1 xylose medium. In continuous culture there was evidence of increased maintenance energy requirements/uncoupling of metabolism in the presence of acetate.

In more recent studies Saez-Miranda et al. [68] have determined ATP levels for growth on glucose/xylose media in the presence of different concentra­tions of acetic acid. From their results they have found that ATP production and accumulation rates are most sensitive to acetic acid at lower pH values— a result consistent with the earlier NMR studies by Kim et al. [57] which demonstrated increasing de-energization of the cells as the inhibitory effects of acetic acid increased. The greater toxicity of acetic acid at lower pH is related to its pKa value as only unprotonated acid can be transported into the cells.

The effects of a range of inhibitory compounds at levels reported previ­ously for a pre-treated hardwood hydrolysate [65] on specific rates of xylose utilization and ethanol production for ZM4 (pZB5) have been analyzed by Kim et al. [57]. From the results, sodium acetate was found to have the greatest inhibitory effect at the concentration tested (10.9 gL-1 at pH = 6.0), followed by vanillin (0.04 gL-1), syringaldehyde (0.13 gL-1) hydroxymethyl- furfural (0.9gL-1) and furfural (0.3 gL-1). Vanillic acid (0.08 gL-1) did not show any inhibitory effects at this experimental concentration. At the levels tested, these inhibitory compounds did not affect ethanol yields on xylose. Volumetric rates of xylose utilization and ethanol produc­tion were reduced by up to 20% by addition of the individual inhibitory components.

2.7

The ability of biofuels to contribute positively to the environmental and eco­nomic performance of a country, and to improve energy security in the long term, makes the nascent industry a tool that policymakers can employ to meet national priorities in these areas. A review of the priorities that gov­ernments are pursuing when designing biofuel-related policy illustrates some issues that the emerging bioethanol industry might consider. These issues may have particular relevance to the commercialization of the lignocellulosic — based component of the industry.

In the USA, the primary political drivers that support research and de­velopment into bioethanol for fuel are related to the economy and to energy security. Two agencies have become the primary implementing bodies for US policies related to bioethanol. The Department of Agriculture (USDA) has a mandate to increase rural employment, diversify agricultural economies, and stimulate rural development by harnessing crops and crop residues and identifying new uses for this material. The Department of Energy (DOE) has a mandate to diversify the energy supply, expand the availability of renew­able energy sources, and develop new technologies to exploit renewables in all forms.

From an economic perspective, bioethanol policy in the USA has been highly successful. Since 1976, bioethanol production capacity has grown sig­nificantly. Almost a decade ago, the US industry passed 5 billion L in an­nual production and was credited with the creation of an estimated 200 000 new jobs and US$ 500 million in annual tax receipts [4]. Today, there are 94 bioethanol plants in the USA, producing about 18.5 billion L year-1, with an additional 16 plants and 2.5 billion L of capacity under construc­tion [13]. Urbanchuk [85] estimated that expansion to this level would require US $ 5.3 billion investment in new facilities and would increase demand for crops by 1.6 billion bushels per year. In that report, the author anticipates that a bioethanol industry of this size could reduce the US trade deficit by US$ 34 billion year-1, create 214 000 new jobs within the USA, and gener­ate US$ 51.7 billion in new US household income. It should be noted that the success of the US industry is in part due to the presence of import tar­iffs on bioethanol (duty of 2.5% market value, plus US$ 0.143 L-1) [23]. While some regions (notably the Caribbean) may export duty-free bioethanol within a quota, the maximum amount of duty-free bioethanol entering the USA is currently 7% per year. This means that it is not cost-effective to import large supplies of bioethanol from other producers, such as Brazil.

From a security perspective, bioethanol policy has been less success­ful. American demand for petroleum continues to outpace domestic sup­ply, resulting in growing petroleum imports, anticipated to be nearly 70% by 2020 [18]. Only about 3% of US energy requirements are supplied by biomass [56], and only about 2.6% of American total transportation fuel consumption is derived from biofuels [18]. Five individual US states (South Dakota, Nebraska, Minnesota, Iowa, and Illinois) now produce enough bioethanol to provide an E10 option to their entire local population. From the perspective of energy security, the USA could benefit from continued ex­pansion of the bioethanol industry and increased utilization of the industry’s potential.

Globally, Germany has the best capacity to substitute biofuels for fossil — based fuels, with current capacity of about 3.75% total demand, followed by the USA (2.6%), Sweden (2.2%), France (1.2%), Austria (1.1%), and Spain (0.44%) [9,35,36,43,48,60].

The issue of climate change has become a major, global concern, but the sectors most closely linked to bioethanol production — including energy pro­ducers, farmers, and foresters — will feel the impact of this issue more closely. Climate change is the driver behind many new policies that influence the ac­tions taken by these sectors. Perhaps the best-known of these is the Kyoto Protocol, which has been ratified by Russia, by the members of the EU, and by Canada in North America. The Clean Skies Initiative in the USA is another example of these policies. Because the use of bioethanol has the potential to significantly reduce net greenhouse gas emissions compared to petroleum products, an expansion of bioethanol production may become a significant part of national climate change strategies. It must be noted, however, that sig­nificant amounts of bioethanol must be substituted for petroleum products in order for these reductions to make a significant impact on total greenhouse gas emissions.

6

Conclusions

Successful policy options to support biofuel production may take a number of forms, including targets and mandates, exemption of biofuels from national excise taxation schemes, direct government funding of capital projects to in­crease capacity or upgrade distribution networks, or consumption mandates for government or corporate vehicle fleets. As discussed in this review, these policies can be differentiated by their relative emphasis on government, in­dustry, or consumer actions. In most biofuel-producing countries examined here, a number of policies have been enacted in order to develop industrial capacity and encourage consumption. It is very difficult to measure the indi­vidual success of these policies because of the synergistic effects that multiple policies may have.

In the USA, an analysis of state-level excise tax exemptions shows no correlation with bioethanol industry capacity, which suggests that these ex­emptions are not a crucial factor in the creation of industrial facilities. Direct funding and support was found to play a much more positive role in the creation of production capacity. It was noted that strong funding for es­tablishment of facilities, including all aspects of research, development, and deployment, was present in each of the states where significant bioethanol production was present. In a comparison of production capacity between 2003 and 2005, it was observed that the correlation between direct funding opportunities and bioethanol production capacity has dropped somewhat. This indicates that other factors, including feedstock supply, the presence or absence of interested industrial players, and other market forces play a signifi­cant role in the establishment of the industry.

In advising governments on the creation of bioethanol-friendly policy, the US experience offers some valuable lessons to consider. The US goals behind policies supporting the bioethanol industry are dominated by (1) economic and social issues, and (2) security-based concerns. Of these priorities, the bioethanol industry has been more successful in meeting social criteria such as rural employment. The starch-based segment of the bioethanol industry has enjoyed particular success in the USA, particularly in Minnesota, Illi­nois, and Iowa. In the past, these jurisdictions have utilized a number of schemes, including direct payments, grants, corporate tax breaks, and ex­cise tax exemptions, as incentives to lure the industry and build bioethanol capacity.

The ability of the industry to increase energy security in the USA, on the other hand, has been limited by the relatively small capacity of their production facilities at the current time. This should serve as a cautionary measure for governments in both Canada and the EU, who have invested biofuel-related policy with more emphasis on the environment and on energy security than they have upon social or economic concerns. Improved en­ergy security through biofuel production can only be achieved when enough capacity is brought on-line. Thus, security-related policy geared to the short­term cannot succeed to any great extent. Policymakers must realize that, in the immediate future, the goals of most successful policies will be related to the economy, and perhaps to the environment. The implication here is that security-related policy, such as mandated renewable fuel use, is likely to take the form of long-term programs that have very little immediate reward.

One important finding was that a balance between research funding and funding for the creation of facilities might be more conducive to support­ing the industry. It was noted that the USA has devoted a significant amount of funds to research as well as to supporting facility creation. A commit­ment to advancing the technology and improving efficiencies may serve to increase the industry’s comfort level in committing resources to this sec­tor. The US example may have important lessons for other countries, where an effective balance between research and commercialization has not been reached. For instance, the total French commitment to biofuels in 2002 was just under US$ 200 million, of which about US$ 180 million is devoted to investment subsidies for biofuels, and a further US $ 11 million was put to­wards wood energy programs. Only about US $ 9 million was earmarked for research and development into renewables, including biofuels research [32]. Although the incentives that the French government offer are dramatic, the research focus of this country has been in other areas, notably nuclear power. This may in part explain the relatively low level of bioethanol production in France, which is currently at about 140 million L year-1 (or 629 million L when bio-ETBE production is considered) [35]. In Spain, the total investment is much lower at approximately US $ 30 million per year, but over half of this amount (US $ 17 million) is available for research and development into var­ious renewables, while the other half may be used for commercial facilities or demonstration plants [32]. Perhaps because of this, Spanish production of bioethanol is at about 521 million Lyear-1 [43]. The balance between research and production incentives that is present in both Spain and the USA, and the resultant human capital, may in part account for the success that these nations have had in nurturing the bioethanol industry.

The experiences gained in developing bioethanol capacity, using both sugar — and starch-based processes, contain many lessons for other biofuels, including biodiesel and the lignocellulose-based bioethanol industry. These fuels can be seen as a response to a variety of domestic issues, including the need to diversify local economies, increased concerns over environmen­tal damage associated with fossil fuel use, and a growing security rationale for a shift to domestic fuel sources. The emerging industry, including the lignocellulosic-based sector, may in turn find opportunities for strategic link­ages and partnerships that capitalize upon these political issues.

Our findings indicate that successful policy interventions can take many forms, but that success measured as biofuel production capacity is equally dependent upon external factors, which include feedstock availability, an ac­tive industry, and competitive energy prices. It is important that policies be crafted that reflect “realistic” use scenarios for bioethanol and other biofuels over future time-frames.

Acknowledgements The authors would like to thank the International Energy Agency (IEA) Bioenergy Task 39 for providing some of the funding required to support this work. The authors also recognize the assistance of Jack Saddler, John Neeft, and other colleagues within Task 39, as well as the Forest Products Biotechnology Group at the University of British Columbia.

[1] The unit bbl is an abbreviation for barrel, a common unit of measurement for petroleum, equiva­lent to 42 US gallons or approximately 159 L.

We have combined biological production of ethanol, hydrogen, and methane in the Maxifuel concept (Fig. 2). The concept is designed to address the major barriers for bioethanol production from lignocellulosic materials. The overall process outline has been defined to yield the maximum amount of biofuels per unit of raw material and to increase the process benefit by utilization of the residues for further energy conversion and by-product refining. The main product is bioethanol for use as transportation fuel and emphasis has been on optimizing ethanol production. The supply and efficient conversion of the raw material is the major economic burden of bioethanol production and full and optimized use of the raw material is a key to success. Production of other bio­fuels such as methane and hydrogen, and other valuable by-products such as
a solid fuel from the parts of the biomass not suitable for ethanol production, adds full value to the overall process. The concept exploits an environmen­tally friendly way of producing bioethanol where recirculation and reuse of all streams produced in the process are integrated into the process. This is in contrast to most other bioethanol process schemes where water has to be added continuously and toxic waste water is left after the process. The basic ideas of producing biogas along with bioethanol and then to recycle the pro­cess water, or part of the process water, within the process are patented [14]. A combination of these innovative ideas along with the best available tech­nologies has ensured an economic feasibility with a competitive advantage over other concepts. The development of the optimized process of bioethanol production from lignocellulosic biomass can be further integrated into a con­ventional bioethanol production where corn or grain fibers will be a residue of low value. Conversion of this fraction into ethanol can increase the produc­tivity by up to 20% along with an improvement of the protein feed produced in the process (Fig. 3).

The Maxifuel concept is patented and consists of the following process steps (Fig. 2):

• Pretreatment

• Hydrolysis

• Fermentation of C6 sugars

• Separation

• Fermentation of C5 sugars

• Anaerobic digestion of process water and recirculation

All fermentable carbohydrates in the raw material are converted into ethanol and hydrogen, while much of the unused fraction such as residues from

tion can be recirculated to the pretreatment unit and used together with fresh raw material.

4.1

Xylitol has recently been recognized as one of the top 12 value-added chem­icals from biomass by the DOE [139]. This pentahydroxy sugar alcohol is commonly used to replace sucrose in food products and in toothpastes as a natural, non-nutritive sweetener that inhibits dental caries [140]. In add­ition, xylitol can serve as a valuable synthetic building block for deriva­tives intended for new polymer opportunities [139]. Production of xylitol, which typically involves hydrogenation of xylose derived from hemicellulose — xylan hydrolysates with an active catalyst such as nickel, ruthenium, or rhodium [139], is currently very limited. Numerous yeast strains have been developed that are capable of producing xylitol in complex medium [141 — 144]. Xylitol production (up to 237 gL-1) by Candidata tropicalis has been optimized by growth in complex media containing urea and numerous ex­pensive vitamin supplements [145]. More recently, strain PC09 was derived from E. coli W3110, which is capable of fermenting a broad range of sug­ars in mineral medium. PC09 can process glucose and xylose blends into xylitol by using an NAD(P)H-dependent xylose reductase from Candida boi — dinii (CbXR) to reduce xylose to xylitol, whereas glucose serves as the cell growth substrate and to regenerate the reducing equivalents [146]. Resting cells and controlled fermentations of PC09 produced 71 and 250 mM xylitol while consuming 15 and 150 mM glucose, respectively. In the controlled fer­mentations, approximately 25 mM xylulose was formed as co-product [146].

Because glucose was used to regenerate reducing equivalents and was not converted to xylitol, the xylitol yield was quantified in terms of a molar yield of reduced product formed per glucose consumed. In the case of zero growth, a maximum molar yield of 10-12 is expected; resting cells and controlled fermentations of PC09 had molar yields of 4.7 and 1.7, respectively. While the molar yield is relatively low compared to the theoretical maximum, this process could prove to be more economical after further optimization and metabolic engineering.

5.4

Process simulation of ethanol production from spruce using a process con­cept based on SO2-catalyzed steam pretreatment followed by SSF, as shown in Fig. 3 ([20], Wingren et al. 2007 (submitted)), has been used to illustrate the effect of various process parameters on the energy demand and on the ethanol production cost. The general conclusions are, however, also valid for most of the process configurations described in Table 1. The model input was based on experimental data obtained from a process development unit. SSF was performed at 10% WIS with 2 gL-1 yeast. In the model, the overall ethanol yield was 296 liters per metric dry ton, corresponding to 69.4% of the theoretical based on the hexosan content in the raw material. Pentose fer­mentation was not included. Regarding production cost data, the proposed ethanol plant is assumed to be located in Sweden, with a capacity of 200 000 dry tons of raw material annually.

The ethanol yield affects both the raw material and capital costs and is the single most important parameter in reducing the cost of ethanol pro­duction, as was already stated in 1988 [39]. High energy efficiency is also of great importance for the process to be economically feasible. In most techno­economic evaluations, live steam for the process is generated in a steam boiler by burning part of the solid residue. From the excess solids it is possible to generate heat and electricity or pellets that can be sold to improve the pro­cess economics. Thus, the energy demand of the process affects the amount of solid residue that may add to the income as a solid fuel co-product and, therefore, it is very important for the process to be energy-efficient.

The heat duty of the process depends to a large extent on the process con­figuration. For the process alternative described above, the heat duty of the energy-demanding process steps is shown in Fig. 5. The white bars represent the primary steam demand while the gray bars represent the amount of sec­ondary steam that is generated in each process step. The overall process heat duty, i. e. the total energy demand in the form of boiler-generated steam, is the sum of the black bars. Distillation (including preheating of the SSF broth) and evaporation account for the major part of the process energy demand. The contributions from pretreatment and drying, with the latter assumed to work as a steam dryer, are comparatively small, due to the generation of secondary steam in these process steps.

The energy demand of the distillation step, in which the ethanol in the mash from fermentation is concentrated, is highly dependent on the ethanol

Ethanol feed concentration (% [w/w])

Fig. 6 Energy demand in the distillation step, where ethanol is concentrated to 94 wt %, as a function of the ethanol feed concentration. The step was assumed to consist of two stripper columns (25 trays each) and a rectification column (35 trays) heat integrated by operating at different pressures. The inlet feed temperature was increased from 80 °C to the boiling temperature before entering each stripper column feed concentration, as shown in Fig. 6. The distillation step normally consists of a stripper column, in which the ethanol is separated from all solid and non-volatile compounds, and a rectification column, in which the ethanol is concentrated close to the azeotropic point. The implementation of heat inte­gration, for instance by using the overhead vapor from the stripper as the heat
source in the reboiler of the rectification column, significantly reduces the en­ergy demand. Nevertheless, it is of great importance to obtain a high ethanol concentration in the distillation feed. In a starch-based process the ethanol concentration in the stream entering the distillation step is normally above 8% (w/w). In a lignocellulose-based process, however, the aim has been to reach at least 4-5% (w/w) ethanol. In addition, a high ethanol concentration results in a high concentration of non-volatile compounds, which also leads to a decrease in energy demand in the evaporation step.

Recirculation of process streams is one way of reducing the overall energy demand, which results in a decrease in overall production cost, as shown by Wingren et al. [38]. Recirculation of part of the stream after distillation back to the fermentation step would result in an increased concentration of non­volatiles and thus a reduction in the energy demand in the evaporation step. Recirculation of part of the stream before distillation would also result in an increase in the ethanol concentration and thus a reduction in the energy de­mand in both the distillation and evaporation steps. This is true for both the SSF and SHF configurations. However, in the same study it was shown that it is even more beneficial to increase the substrate concentration in the SSF step. This would affect not only the costs related to distillation and evaporation, but also the cost of SSF. On the basis of this fact, one of the main objectives of several experimental studies performed during recent years has been to increase the substrate concentration in SSF [40-43]. This results in reduced water consumption, which greatly reduces the energy demand for distillation and evaporation, provided that the ethanol yield is maintained at a high level. In Fig. 7, the process heat duty (in MJ L-1) and the overall production cost

(in US$ L-1) are presented as functions of the WIS concentration in SSF. The ethanol yield and the amount of yeast (NB: not the yeast concentration), were the same as in the 10-% WIS case when varying the WIS concentration. The reduction in production cost is due to an increase in co-product credit and a reduction in the fixed capital cost.

Process simulations clearly demonstrate the potential reductions in pro­duction cost and energy demand that can be obtained by running SSF at higher substrate concentrations. However, given the large number of com­pounds involved, and due to the fact that they may act synergistically, it is impossible to predict the impact of increased concentrations on the perform­ance of the yeast and enzymes using process models. Effects on parameters such as productivity (yield, residence time), yeast and enzyme dosages have to be determined experimentally, preferably on pilot scale.

Savings in energy demand can also be accomplished by changes in the process design. Evaporation is the traditional, but energy-demanding, way to concentrate the water-soluble, non-volatile components in the stillage stream. To reduce the energy requirements for evaporation, multiple evaporation ef­fects are used. This has a significant effect on the overall process heat duty, as shown in Fig. 8. (In the simulation results presented in Figs. 6 and 7, evapo­ration was carried out with five effects.) The energy savings have, of course, to be weighed against the increase in capital cost. Also shown in Fig. 8 is a case where the use of mechanical vapor recompression (MVR) has been implemented in the evaporation unit. In a traditional multiple-effect evap­orator system, a large proportion of the energy supplied ends up as latent heat in the vapor phase leaving the last effect in the evaporator. This vapor is

§

normally condensed using cooling water. Another option is to compress the vapor, thereby raising the temperature to a level at which the latent heat can be utilized. The vapor can then be used as a heating medium to replace most of the primary steam. When compression is carried out by aid of a mechanical compressor the process is referred to as MVR. An electrical motor or a steam turbine provides power to the compressor. The overall process heat duty was reduced from 15.1 (base case configuration) to 10.3 MJ L-1 when MVR was applied to the evaporation step (Fig. 8), while the overall electric power re­quirement was estimated to increase from 2.2 (base case configuration) to

2.8 MJ L-1 (data not shown).

It has also been proposed that the entire evaporation step be replaced by an anaerobic digestion step, in which most of the organic material (unfermented sugars, acids, yeast, etc) is converted to biogas mainly consisting of methane and carbon dioxide. This was estimated to reduce the production cost by about 7%. The performance of such a system is dependent on a number of parameters such as the composition of the feed, residence time, temperature, etc. A crucial question is also how to handle the sludge from the anaerobic digestion. Further investigation is required since very limited data regarding the performance of this kind of system have been published.

3.2

Several studies on ethanol production by wild-type strains of Z. mobilis on industrial starch-based raw materials have been reported. Bringer et al. [69] investigated an industrial-scale process and Poosaran et al. [70] evaluated a cassava-derived starch hydrolysate. In the latter case in a batch culture at controlled T = 30 °C and pH = 5.0, fermentation using Z. mobilis ZM4 gave an ethanol yield of 95% theoretical, a productivity of 6 gL-1 h-1 and a final ethanol concentration of 114 gL-1. Under the same conditions, a strain of Sac — charomyces uvarum gave an ethanol yield of 90% theoretical, a productivity of 4gL-1 h-1 and a final ethanol concentration of 106gL-1 for a cassava starch suspension (23% glucose equivalent). A comparative batch and continuous culture study with starch hydrolysate using yeast and Z. mobilis 29191 has also been reported by Beavan et al. [71].

Extensive studies with various strains of Z. mobilis have been reported by using sugar cane syrup and molasses [72-76] and for sugar beet mo­lasses [77,78] with evidence of yield reductions on sucrose based media due to production of the fructose polymer levan as by-product [3,6] and rate reductions due to high salt concentrations in the molasses. Improved produc­tivities were reported following membrane desalting of high salt-containing sugar cane molasses [72].

Most recently, Davis et al. [79] studied the fermentation of a hydrolyzed waste starch stream from flour wet milling using both Z. mobilis ZM4 and an industrial ethanol-producing strain of S. cerevisiae. With glucose concen­trations in the range 80-110gL-1, Z. mobilis ZM4 demonstrated superior fermentation characteristics. In a repeated batch process (five cycles), rapid concentration of the cells and increased productivities were achieved by cell settling between batches using the flocculent strain Z. mobilis ZM401 (ATCC 31822) as characterized by Skotnicki et al. [80]—see Fig. 6.

Similar flocculent mutants of wild-type Z. mobilis strains CP4 and ATCC 29191 have been isolated by Lawford et al. [16] and Fein et al. [81] using a spe­cially designed chemostat. These strains were deposited with the ATCC as strains 35 000 and 35 001, respectively. The use of such flocculent cultures was demonstrated to increase volumetric productivity by as much as ten-fold [82]

and may have considerable potential in future large-scale processes for more stable fermentations.

Recombinant strains of Z. mobilis developed for xylose utilization have been evaluated on various agricultural residues including oat hull hydrolysate produced by the Iogen process [40]. Oat hull hydrolysate contains glucose, xylose and arabinose in a mass ratio of 8 : 3 : 0.5. Synthetic hydrolysate (6% w/v glucose; 3% w/v xylose; 0.75% w/v acetic acid) at initial pH 5.75 was mixed with either 2 ml L-1 corn steep liquor (CSL) or 1.2 g L-1 di-ammonium phosphate as N source and used for evaluation of ethanol production. From the results it was concluded that the highest productivity was achieved with Z. mobilis ZM4 (pZB5). In this and other studies, CSL was also found to be an effective nutrient source to replace yeast extract in the fermentation media for Z. mobilis [83-85].

Further studies were reported by Mohagheghi et al. [41] with an integrant strain (designated Z. mobilis Fig. 8b) derived from ZM4 (pZB5) using over­limed corn stover hydrolysate. The hydrolysate contained 16 gL-1 glucose, 69gL-1 xylose and 11 gL-1 acetic acid at pH = 5.0. This medium was sup­plemented with 100 gL-1 glucose and diluted to various concentrations prior to fermentation. The authors found that up to 50 g L-1 ethanol was produced by the integrant strain with diluted 80% corn stover hydrolysate. Yields of 83-87% theoretical (based on sugars utilized) were reported.

One of the potential issues for large-scale Z. mobilis fermentations is whether or not contamination control is needed particularly in the presence of ethanol-tolerant strains of Lactobacilli. Such contamination constitutes a problem in many yeast-based processes and can reduce yields by an es­timated 2-5%. However, its impact is reduced as pH decreases to 3.0-3.5 towards the end of batch fermentation (in the absence of pH control). “Acid washing” of the residual yeast at this pH or lower is often used to minimize contamination in yeast subsequently used in a repeated batch process. Z. mo­bilis is more sensitive to low pH than S. cerevisiae and contamination was identified as a problem by Bringer et al. [69] in their study on an industrial — scale process for conversion of starch to ethanol using Z. mobilis although Lawford and Rousseau [63] demonstrated that lactic acid in such circum­stances is not likely to be inhibitory to Z. mobilis. Interestingly, although rarely observed in Z. mobilis fermentations due to the usual high metabolic flux rates in the ED pathway, conditions have been reported which can pro­mote lactic acid synthesis in Z. mobilis [37,85].

The issue of contamination control was addressed directly by Grote et al. [86] in which a continuous culture of Z. mobilis ZM4 was directly contaminated with a 10% (v/v) inoculum of Lactobacillus sp. isolated as an ethanol-tolerant contaminant from an industrial plant (Grain Processing Corporation, Muscatine, Iowa). It was found at D = 0.1 h-1 under condi­tions of glucose limitation, pH control at 5.0 and ethanol concentrations of 60-65 gL-1, that the addition of the contaminant caused only a temporary disturbance in the process. Steady state conditions with no evidence of sus­tained contamination were regained within five to six generations. These results suggest that contamination is not likely to be a significant problem once an active culture of Z. mobilis is established providing that the pH is maintained above 3.5-4.0. A similar conclusion was reached in a recent study [87] using an acid-tolerant strain of Z. mobilis under non-sterilized feed and operating conditions.

3

Recombinant expression of the Z. mobilis homoethanol pathway has been the cornerstone of E. coli ethanologenesis. However, recent progress has en­abled ethanol production by a mutant E. coli strain lacking foreign genes [46]. Due to the inability to regenerate NAD+ and maintain redox balance, wild — type E. coli is unable to grow anaerobically in the absence of both IdhA and pflB [47]. Chemical mutagenesis was used to isolate AldhA/ApflB deriva­tives capable of anaerobic growth. The resulting strain SE2378 fermented glucose and xylose to ethanol with 82% yield. Further analysis of SE2378 revealed an essential mutation within the pyruvate dehydrogenase (PDH) operon. In native strains, pyruvate formate-lyase is primarily responsible for production of acetyl-CoA during anaerobic growth; PDH is reportedly inactive [48] or weakly active [49] under these conditions. The essential mu­tation in the pdh operon restored function during anaerobic growth and produced an additional NADH for each pyruvate. This additional NADH al­lowed the balanced production of 2 moles of ethanol per mole of glucose by a novel pathway not previously known in nature. The anaerobic spe­cific growth rate of SE2378 was reduced approximately 50% relative to the parental strain in rich media and no growth was observed in glucose mini­mal media without acetate, glutamate, or corn steep liquor supplementation. Despite growth challenges, the maximum specific productivity of SE2378, 2.24 g ethanol h-1 gcells-1, is comparable to KO11 and ethanol was pro­duced from 50 g L-1 glucose and xylose at greater than 80% of the theoretical yield.

2.5

Ethanol production from lignocellulosic biomass has to include a pretreat­ment more intensive than those used in processing sugar and starch-rich biomass in order to release the sugar compounds contained in the biomass. Agricultural residues like wheat straw or other types of biomass derived from plant material contain lignin, which is constructed to resist microbial attack and to add strength to the plant. Pretreatments are used to open the biomass by degrading the lignocellulosic structure and by partially hydrolysing the substrate. Current pretreatment methods, however, contribute to 30-40% of the total costs of bioethanol production from lignocellulosic biomass. The National Renewable Energy Laboratories (NREL) estimates that in an Nth generation plant (mature technology), feedstock handling and pretreat­ment would account for approximately 20% of the total ethanol production costs [15].

Several pretreatment methods have been developed [16] (see also Zac — chi, this volume). However, in all methods the biomass concentrations need to be higher than 20% dry weight to ensure a suitable ethanol concen­tration for the subsequent distillation process. A new patented pretreat­ment process, Wet-ox explosion (WE), has been developed in our labora­tory combining steam-explosion and wet oxidation using small amounts of oxygen [17]. The optimal combination of process parameters such as tem­perature (170-200 °C), pressure (12-30 bar), amount of oxygen addition, and residence time (2-15 min) has been tested. Depending on the biomass mate­rial used, the method will yield variable sugar yields but overall the results show that the method will be efficient and cost-effective for opening of most major biomass materials such as straw, corn stover, bagasse, and woody materials. Table 1 shows the apparent advantages and disadvantages of this pretreatment method.

Table 1 Advantages and disadvantages of Wet-Ox-Explosion

Disadvantages

Fast and efficient Requires water supply

No emission products Advanced technology

Low heat consumption No standard equipment

No detoxification Only tested on pilot scale

Easily convertible substrates No waste products

4.2

Succinate, a natural E. coli fermentation product, can serve as substrate for the production of many compounds currently derived from petroleum [112]. Although there have been numerous reports of succinate production by E. coli and other biocatalysts (for example [147,148]), these processes often involve undesirable nutritional supplementation, multiple steps and low product titers.

Due to our success, described above, in using a combination of directed engineering and metabolic evolution to design ethanol and lactic acid mi­crobial biocatalysts, we have used a similar approach to develop a succinate — producing microbial biocatalyst that attains high product titers in simple mineral salts media [149]. Directed engineering consisted of elimination of the lactate, acetate, and ethanol-forming pathways (IdhA, ackA, adhE), leaving succinate production as the primary route of NADH oxidation. The poor growth and fermentation of the resulting strain in mineral salts media were improved by metabolic evolution. Further directed engineering (focA, pflB, mgsA) reduced co-product formation. The resulting microbial biocat­alysts, KJ060 and KJ073 (poxB), produced nearly 700 mM succinate from glucose with a molar yield of 1.2-1.6; the maximum theoretical molar yield is 1.71 (Jantama et al., unpublished results). KJ060 and KJ073 produced 250 and 183 mM acetate, 39 and 118 mM malate, 0 and 5 mM pyruvate, and 2 and 0 mM lactate as co-products.

5.5