Other major biofuel producers include China, which has grown its bioethanol production sector rapidly since 2000 to become the third-largest single bioethanol producer after the USA. Total capacity from four plants in 2005 was about 1.3 billion L, but continued high prices for international oil has led the National Development and Reform Commission to announce that biofuel production will increase dramatically, providing China with the ability to re­place about 2 million t of crude oil by 2010, and 10 million t by 2020 [63]. The Commission also announced that China would begin shifting to non-grain feedstocks, including sweet sorghum, for bioethanol production [63]. Jilin Fuel Alcohol remains the world’s largest corn-based bioethanol plant with a current capacity in excess of 350 million Lyear-1 [64]. The biofuel industry in China has been subsidized, mostly in terms of funds to construct biofuel plants. Some Chinese provinces have announced biofuel mandates, although the national government has not yet made any decision about legislating bio­fuel use [63].

A country poised to be a major biofuel producer is Canada, which cur­rently produces about 250 million L annually [15]. Much of the funding being made available to fund research and development in biofuels in Canada has depended upon the federal government’s environment strategy. This strat­egy has evolved significantly with the ascension of a Conservative minority federal government in 2005, who made a campaign promise to introduce a 5% biofuels mandate. An agreement with provincial governments on the 5% mandate was reached in May, 2006, which will see this mandate take full effect by 2010 [65]. Recently, the federal government announced the proposed Clean Air Act, which was tabled on 19 October 2006 [66]. Unfortunately, the pro­posed Act does nothing to codify the government’s biofuels target, and does not provide concrete policy incentives for additional biofuel use. To help spur some biofuel development, Agriculture and Agri-Food Canada is providing CAD $ 10 million (approximately US $ 8.7 million) in the fiscal year 2006/2007 through the Biofuels Opportunities for Producers Initiative (BOPI). The ob­jective of the Initiative is to help agricultural producers develop business plans for new biofuels projects [67].

Previous governments have provided more substantial support to biofu­els, including a cumulative investment of CAD $ 2.7 billion (US $ 2.34 billion) into the implementation of the former Climate Change Plan for Canada [68], which included incentives for the development and use of environmentally — friendly technologies including bioethanol. The federal Canadian govern­ment provided direct funding for the industry through the Ethanol Expansion Program, which in 2004 and 2005 provided a total of CAD $ 118 million (US$ 102 million) in direct funding for 11 projects, six of which are cur­rently in active development [69]. The federal government provides an excise tax exemption for biofuels, as do the provinces of Manitoba, On­tario, and Alberta [70]. Most recently, the Alberta government has an­nounced a commitment of CAD $ 239 million (US $ 207 million) to expand the province’s bioenergy sector by encouraging products including biofuel development [71]. Other nations with biofuel-friendly policies include Aus­tralia, where a bioethanol production subsidy is in place that replaces excise tax exemptions at a rate of approximately US$ 0.21 L-1 produced. Capital subsidies have been provided for two bioethanol production plants [64]. In Thailand, excise taxes are waived for bioethanol. In Latin America, produc­tion schemes in Peru and Columbia have been linked to urban renewable fuel standards in Columbia [64]. In a move designed to utilize surplus pro­duction, the sugar industry in India has successfully lobbied the government for state-level E5 fuel mandates, which were passed in September 2002 and which apply to nine states and four territories. In order to support these man­dates, an excise tax exemption was granted and bioethanol prices have been fixed by a Tariff Commission [72]. Production from other nations will become more important as capacity comes on-line and the international market for bioethanol continues to develop.

3

Birgitte K. Ahring (И) • Peter Westermann

Bioscience and Technology, BioCentrum-DTU, Technical University of Denmark,

Building 227, 2800 Lyngby, Denmark

bka@biocentrum. dtu. dk

1 Introduction……………………………………………………………………………………………… 290

2 Hydrogen Production………………………………………………………………………………… 291

3 Methane Production…………………………………………………………………………………. 291

4 Production of Biofuels Using the Maxifuel Concept……………………………………… 292

4.1 Pretreatment…………………………………………………………………………………………….. 295

4.2 Hydrolysis………………………………………………………………………………………………… 296

4.3 Separation………………………………………………………………………………………………… 296

4.4 Fermentation…………………………………………………………………………………………….. 296

4.5 Waste Water Treatment…………………………………………………………………………….. 297

4.6 Bio/Catalytic Refineries……………………………………………………………………………… 299

4.7 Integrating Conventional and Bio/Catalytic Refineries………………………………….. 299

5 Conclusion……………………………………………………………………………………………….. 301

References……………………………………………………………………………………………………. 301

Abstract Large scale transformation of biomass to more versatile energy carriers has most commonly been focused on one product such as ethanol or methane. Due to the nature of the biomass and thermodynamic and biological constraints, this approach is not optimal if the energy content of the biomass is supposed to be exploited maximally. In natural ecosystems, biomass is degraded to numerous intermediary compounds, and we suggest that this principle is utilized in biorefinery concepts, which could provide dif­ferent fuels with different end use possibilities. In this chapter we describe one of the first pilot-scale biorefineries for multiple fuel production and also discuss perspectives for further enhancement of biofuel yields from biomass. The major fuels produced in this refinery are ethanol, hydrogen, and methane.

We also discuss the applicability of our biorefinery concept as a bolt-on plant on conventional corn — or grain-based bioethanol plants, and suggest that petroleum-base re­fineries and biorefineries appropriately can be coupled during the transition period from a fossil fuel to a renewable fuel economy.

Keywords Biorefinery • Fuel cells • Hydrogen • Methane • Reforming

1

Introduction

Traditionally, the development of biological processes to transform biomass to more versatile energy carriers has focused on the production of one en­ergy carrier, either hydrogen, methane, or ethanol. Among these products, only methane is released from the conversion of organic matter in nature; both hydrogen and ethanol are intermediates during anaerobic degradation and are further metabolized to methane in nature [1]. The production of these two energy carriers, therefore, demands a physical separation of indi­vidual processes in the anaerobic degradation chain, or the use of defined microbial cultures under controlled conditions. This can be carried out in a biorefinery, which is a facility that integrates biomass conversion processes and equipment to produce fuels, power, and chemicals from biomass [2,3]. The biorefinery concept is analogous to today’s petroleum refineries, which produce multiple fuels and products from petroleum.

Instead of concentrating on the biological production of only one en­ergy carrier, the simultaneous production of hydrogen, methane, and ethanol leaves the possibility to optimize the exploitation of the specific energy car­riers to suit specific needs, corresponding to the current use of specific fossil fuels for specific purposes. Hydrogen can for instance be used in fuel cells for urban transportation. Ethanol can be used in fuel cells in rural areas, and me­thane can be used in fuel cells for local electricity and heat production in fuel cells or micro-turbines [4]. Although the fuel cell technology was developed initially for molecular hydrogen, this technology is in rapid progression, and fuel cell systems dealing with more complex compounds such as ethanol are currently being developed [5,6].

Despite the obvious advantages of combining the production of different energy carriers, only a few concepts have been published. Common to the known concepts is a much better exploitation of the biomass by suiting spe­cific microbiological processes to the conversion of different fractions of the substrates to different fuels. The different processes are thereby exploited in an additive sequential fermentation, transforming most of the energy avail­able in the substrate to usable energy carriers. Furthermore, biorefineries might be considered as more environmentally friendly processes since pro­cess water and nutrients from the different processes can be recirculated, and waste production can be kept minimal [4].

By producing multiple products, a biorefinery can also take advantage of the differences in biomass components and intermediates and maximize the value derived from the biomass feedstock. A biorefinery might, for ex­ample, produce one or several low-volume, but high-value, chemical products and a low-value, but high-volume liquid transportation fuel, while generat­ing electricity and process heat for its own use and perhaps enough for sale of electricity. The high-value products enhance profitability, the high-volume fuel helps meet national energy needs, and the power production reduces costs and avoids greenhouse-gas emissions.

2

Petrochemicals and petroleum-based products such as plastics are widely integrated into our lifestyles and make a major, irreplaceable contribution to virtually all product areas. Increasing petroleum costs have provided an opportunity for a number of renewable bio-based chemicals or plastics, in addition to the bio-based fuels, to become economically competitive. How­ever, full commercialization of renewable commodity chemicals to replace the currently exploited petrochemicals is critically tied to production cost. Therefore, development of low cost fermentation routes, increased microbial biocatalyst efficiency and productivity and increased final fermentation titer are desired.

5.1

Flowsheeting programs, e. g. Aspen Plus, HYSYS and ChemCad, may be used to perform rigorous material and energy balance calculations, with the use of detailed equipment models, to determine the flow rates, composition and energy flow for all streams in the process. Because of their flexibility, the programs have many advantages when comparing different process configu­rations or scenarios in terms of overall efficiency, minimum energy demand or lower production cost. Also, they serve as a powerful tool when performing sensitivity analyses, due to the ease of changing a certain parameter. All flow­sheeting programs are based on a modular approach where each module is a mathematical model of a unit operation. The fundamental equations needed to accurately describe standard process equipment, such as columns (distil­lation, absorption, etc.), heat exchangers, pumps, reactors and splitters, are normally available as part of the program. The actual simulation is performed by arranging different unit operation modules into a complete flowsheet that represents the process to be simulated.

Construction of a process model in a flowsheeting program can be sum­marized in the following three steps.

• Flowsheet definition: The flowsheet defines the process configuration. It shows all streams entering the system as well as all unit operations and their interconnecting streams. The flowsheet also indicates all product streams that will be determined by the simulation program.

• Chemical components: The user must specify all the chemical compo­nents to be used in the system. All necessary physical and thermodynamic properties must be defined for each component. Normally, a database con­taining these properties for a large number of chemical compounds is included in the flowsheeting software. In general, the size of this database, which varies greatly between different simulators, determines the cost of the flowsheeting program. If data for some compounds are missing the user has to define them.

• Operating conditions: For every unit operation the user has to specify the operating conditions, such as temperature, pressure, heat duties, etc. In addition, all input streams have to be completely defined. Enough infor­mation has to be provided to result in a single steady-state solution based on material and energy balances coupled with phase equilibrium equa­tions.

2.1

Detailed kinetic studies have been reported in the literature for several re­combinant strains of Z. mobilis from NREL capable of utilizing both glucose and xylose. The initial evaluation by Zhang et al. [10] involved the batch culture growth of the strain CP4 (pZB5) on medium containing 25 g L-1 glu­cose and 25 gL-1 xylose. Batch and continuous culture studies on strain 39676 (pZB4L) were reported subsequently by Lawford et al. [31,33,34]. This strain was derived from the host ATCC 39676 transformed with a plasmid derived from pZB4. Final product values for 40 gL-1 glucose/40 gL-1 xylose medium included 4.04 g L-1 xylitol as well as 36.6 g L-1 ethanol [49] although it should be noted that xylitol levels with this particular recombinant strain were unusually high. Further studies reported by Lawford and Rousseau [35] focused on kinetic and energetic evaluations of strain CP4 (pZB5) in batch and fed-batch fermentations. Kinetic characterization of the chromosoma­lly integrated xylose/arabinose strain AX101 (derived from ATCC 39676) was also reported [37,38].

To determine which of the strains was likely to be most suitable for larger scale ethanol production, a comparative evaluation in batch and continuous

culture of strains CP4(pZB5) and ZM4(pZB5) was carried out by Joachimsthal et al. [28]. From the results it was found that ZM4(pZB5) was capable of con­verting a mixture of 65 g L-1 glucose and 65 g L-1 xylose to more than 60 g L-1 ethanol in 48 h in batch culture with an ethanol yield of 0.46 gg-1, with this latter strain demonstrating superior specific sugar uptake and ethanol pro­duction rates. The results for ZM4(pZB5) are shown in Fig. 3 together with the values of comparative kinetic parameters in Table 2. Higher sugar con­centrations (75 gL-1 each sugar) resulted in incomplete xylose utilization (80 h) presumably due to increasing ethanol inhibition of xylose assimila — tion/metabolism at ethanol concentrations of 65-70 gL-1.

The results for continuous culture with ZM4 (pZB5) and medium contain­ing 40gL-1 glucose and 40 gL-1 xylose are shown in Fig. 4 [28]. While the concentration of glucose was close to zero at dilution rates up to D = 0.15 h-1, increasing residual xylose at dilution rates higher than 0.08 h-1 indicated that the maximum volumetric rate of xylose uptake for the culture had been exceeded. The maintenance energy coefficient (m) under these conditions was estimated by extrapolation as 1.6±0.2gg-1 h-1 (within 95% confidence limits) based on linear regression analysis of the data from Fig. 4a for the maximum specific sugar uptake rate (glucose and xylose) vs. dilution rate (D) (Fig. 4b). A “true biomass yield” of 0.044 gg-1 was determined from the inverse of the gradient of this linear plot. For similar experimental con­ditions, closely related values were observed by Lawford and Rousseau for strain CP4 (pZB5) [34]. However, Lawford and Rousseau noted, when ob-

Mm: maximum specific growth rate (h-1)

(qs)m: maximum specific sugar uptake rate (gg-1 h-1)

(qp)m: maximum specific ethanol production rate (gg-1 h-1) (Yx/s): overall cell yield (based on total sugar utilized) (gg-1) (Yp/s): overall ethanol yield (based on total sugar utilized) (gg-1)

served over the lower dilution rate range of D = 0.04-0.08 h-1, that both strain CP4 (pZB5) and a biomass hydrolysate adapted variant of 39676(pZB4L) exhibited values of m and “true biomass yield” that were significantly lower [35].

Results with a potentially high productivity cell recycle system using a membrane bioreactor are shown in Fig. 5 [29]. From Fig. 5(a), at sugar con­centrations of 50 gL-1 glucose and 50 gL-1 xylose and D = 0.1 h-1, an ethanol productivity of 5 g L-1 h-1 was achieved with an ethanol yield based on total sugars utilized (Yp/s) = 0.50 gg-1. No decline in specific ethanol productivity was evident up to 70 h, however as shown in Fig. 5(b), a decrease in total vi­able cells was observed after an initial steady state (40-50 h). This indicates that for effective longer term operation, high cell concentrations should be

— 2.5

— 2 ^ Li 3

CO со ш

Є о

-1 s

— 0.5

0. 02 0.04 0.06 0.08 0.1

Dilution rate (h’1)

Fig.4 (a) Kinetics of ethanol production by Z. mobilis ZM4 (pZB5) in continuous culture on medium containing 40 gL-1 glucose and 40 gL-1 xylose (T = 30 °C, pH = 5.0). Sym­bols: biomass •; glucose ♦; xylose □; ethanol ▲ (b) Effect of dilution rate on specific rates of total sugar uptake (qs) and ethanol production (qp). Estimation of maintenance energy (m) value at D = 0 by extrapolation. Symbols: qs °; qp A achieved by less stressful methods than membrane-based cell recycling (e. g., by use of flocculent cells and cell settling).

2.5

As seen in the review of major biofuel producers, a common policy instru­ment used to support the industry is direct government program funding, in the form of contracts, loans, grants, or fiscal guarantees. It is difficult to evaluate the effectiveness of direct funding by comparing different countries, where synergistic policies (such as renewable fuel mandates, excise tax ex­emptions, etc.) or simply more favorable market conditions may play a role in determining capacity. However, within a single country it may be easier to see the impact of direct funding on the establishment of biofuel cap­acity. The bioethanol industry in the USA has been chosen for an analysis of the effectiveness of direct funding towards establishing biofuel produc­tion capacity. For the purpose of this study, direct funds are considered to be funds earmarked for all aspects of research, development and demon­stration, including all biofuel production as well as biomass production for general energy purposes. When different funding sources were considered, the only real criteria applied to warrant their inclusion in this study were (1) that the funds be applicable to research, development, and demonstra­tion (RD&D) projects for bioethanol, including construction or modification of production facilities, and (2) that bioethanol is accounted as an eligible product. Funding sources that recognized bioethanol as a co-product of mate­rial or bioenergy generation were also included. Estimates of the cumulative, total funding available to support the bioethanol industry are shown in Fig. 2. Canada is included in this graphic for comparison’s sake.

In Fig. 2, direct funds available in each state are indicated by the shading on the map, from blue (base levels of cumulative funding provided by the federal government as of 2005) to light or dark red (additional state fund­ing, depending upon the cumulative amount of funds available as of 2005). Existing bioethanol production capacity for 2005 is indicated by the yel­low circles, logarithmically sized according to the scale indicated. Additional bioethanol production capacity expected to be online as of 2007 is indicated by the dark orange circles, again plotted logarithmically. The graph indicates that bioethanol production is likely to be found where funding is available for infrastructure development, biomass procurement, and plant operation. Each of the major bioethanol-producing states has followed a different ap­proach in creating these incentives. Each approach represents a successful strategy for attracting the industry and expanding bioethanol production capacity.

In Illinois, the primary incentive offered to bioethanol producers is the Illinois Renewable Fuels Development Program, which offers up to US $ 5.5 million per facility in grants for the construction or retrofitting of renewable fuels plants, provided that they are a minimum of 114 million L in capacity and that the total grant award does not exceed 10% of total construc­tion costs, or US $ 0.026 L-1 of additional biofuels capacity created [73]. Both bioethanol and biodiesel production facilities are currently the primary re­cipient of these funds. In addition, the Renewable Energy Resources Program offers funding at various levels to promote the development and adoption of renewable energy within the state. With two new plants under construction in 2006, the total funding available to the bioethanol industry is estimated at approximately US$ 30.15 million [23]. Currently, Illinois has five operat­ing facilities with a capacity of 5.1 billion Lyear-1, while two new facilities are under construction [18].

In Iowa, a number of innovative programs are in place. The Iowa Re­newable Fuel Fund’s Financial Assistance Program offers a combination of forgivable and traditional low-interest loans for projects involving biomass and alternative fuel technologies, while the Alternative Fuel Loan Program of­fers zero-percent interest loans for up to half the cost of biomass or alternative fuels related fuel production projects, up to a maximum of US $ 250 000 per facility [74]. Approximately 20% of the money awarded under this program is in the form of forgivable loans, while the remaining 80% are low-interest loans. A number of other incentives, including the Ethanol Infrastructure Cost-Share Program, provide incentives for installation or conversion of E85 refueling stations [75].

In Minnesota, the chief incentive is the Ethanol Production Incentive. Originally, this incentive provided direct payments to producers at a rate of approximately US $ 0.052 US L-1 bioethanol, although the passage of bill SF 905 (2003) has reduced this amount to US $ 0.034 L-1 from 2004-2007. In 2007, the original incentive will be restored and producers may be reim­bursed for lost incentive if funds are available. The total fund available is US $ 37 million, although there is a cap of US $ 3 million per producer, which essentially means that producers of more than 15 million L year-1 are ineli­gible for extra incentive [76]. Perhaps due to this restriction in funding, the program has resulted in the establishment of 15 individual facilities by 2006 with a total production capacity of 1.9 billion Lyear-1 [18]. The Ethanol Pro­duction Incentive expires June 30, 2010 [77]. Ethanol infrastructure grants are also available to help upgrade service stations for dispensation of E85 fuels [23]. Minnesota has also enacted legislation for a bioethanol blend man­date, currently enforcing a 10% bioethanol blend for consumers (to increase to 20% bioethanol in 2013) [77].

In South Dakota, the Ethanol Production Incentive is designed as a dir­ect payment of US $ 0.052 L-1, with a maximum of US $ 1 million annually or US $ 10 million in total to any single facility. Unlike the incentives described for Minnesota, Illinois, or Nebraska, this particular program is targeted spe­cifically at bioethanol from cereal grains and expires this year [22]. While this level of support is lower than in many other states, South Dakota also has an excise tax exemption on bioethanol which provides additional financial in­centive for production. Currently, South Dakota has 11 operating facilities, with four additional plants under construction and a total production cap — acityof 2.2 billion L year-1 [18].

In Nebraska, the main program is the Ethanol Production Incentive, which offers a tax credit of US $ 0.048 L-1 bioethanol for up to 60 million L of annual production per facility, or 473 million L in total production over the course of a 96-month consecutive period [78]. This credit, which will expire in 2012, is limited to a total of US $ 22.5 million. As a tax credit, these funds can be con­sidered to be defrayed costs in direct support of the industry [23]. Nebraska currently has a production capacity of 1.8 billion L annually in ten facilities, with three new installations currently under construction [18].

As these examples demonstrate, a range of policy tools have been deployed in areas with significant bioethanol production capacity. The tools of pro­duction incentives, tax exemptions, direct loans, and cost-share schemes are shown to be effective in attracting capacity to individual jurisdictions, and the tools are shown to be flexible in achieving different results. The Min­nesota example, in particular, shows the potential impacts of small changes to policy. By limiting the capacity to which the incentive applied, the state gov­ernment was able to spur the creation of many individual facilities, which will in turn have a direct impact on jobs and the local economy. It is important to remember, however, that each of these strategies build upon the US fed­eral government’s strong commitment to research and development. Without that commitment, the rapidly improving technology that makes these facili­ties possible would not exist. However, it is interesting to note the differences that small amounts of local funding might have on productivity.

In Fig. 3, the relation between state funding for biofuels is compared to ac­tual bioethanol production capacity, using the funding data and bioethanol production capacities for 2003 and 2005. The two years of data are dif­ferentiated by the shaded and white circles. In 2003, a strong correlation was found between state-level funding and bioethanol production capacity (r2 = 0.85). This indicates that direct funding likely played a role in attract­ing new bioethanol capacity, and thus it could be concluded that this is an effective policy tool. By 2005, the changes in production capacity and dir­ect funding levels in many states has reduced this correlation significantly (r2 = 0.64). It may be postulated that a shift is taking place, in which the amount of funding available to capital projects has become less import­ant in relation to some other factor, such as feedstock availability or mar-

ket influences. Indeed, follow-up analyses using corn production data [79, 80] indicate that in the same period, the relation between bioethanol pro­duction capacity and corn harvest figures on a state level show the op­posite trend. In 2003, the correlation between the two was fairly weak (r2 = 0.58), while in 2005, this correlation had grown stronger (r2 = 0.83). In 2003, availability of corn seemed to be less important than direct fund­ing for bioethanol facilities. It may be postulated that the rapid growth in bioethanol capacity seen to 2005, coupled with strong prices for bioethanol, has made feedstock availability more important than funding for construc­tion purposes.

4

Biological pretreatment can be performed by applying lignin-degrading mi­croorganisms, such as white — and soft-rot fungi, to the lignocellulose mate­rials [44,47]. The method is considered to be environmentally friendly and energy saving as it is performed at low temperature and needs no use of chemicals. However, the rate of biological pretreatment processes is far too low for industrial use, and some material is lost as these microorganisms to some extent also consume hemicellulose and cellulose, or lignin [42]. Never­theless, the method could be used as a first step followed by some of the other types of pretreatment methods.

4

The total production cost for cellulosic ethanol must still be substantially re­duced to enable large scale commercialization, and at least a portion of this reduction must come from enzyme cost. Realistically, enzyme cost targets in the range of $0.30/gallon at the commercial scale should be achievable in the near future by avoidance of transportation and formulation costs. In such a scenario, on-site or near-site enzyme production is essential, where enzymes are produced using reduced-cost feedstocks, transported short dis­tances, and not stored for extended periods of time. The least expensive alternative in this situation involves the direct use of whole fermentation broth (including cell mass) to circumvent expensive cell removal and en­zyme formulation steps. To investigate this possibility, we compared the use of whole fermentation broth and cell-free broth as catalysts for PCS hydro­lysis in microtiter-scale reactions at 50 °C, pH 5.0, for up to 120 h. The results of this study strongly suggest that both preparations, dosed at equal volumes, give comparable yields of reducing sugars from PCS, suggesting that costly cell removal may not be required.

6

Conclusions

The development of cost-effective enzymes for the widespread utilization of lignocellulosic biomass will require continued research and development to be successfully deployed. Although great progress has been made in identi­fying new enzyme mixes with improved catalytic efficiency, improvements in enzyme yield, and improved enzyme production economics, much work remains. There are thousands of organisms involved in the natural decom­position of plant material in our biosphere, and only a fraction of those have been isolated or investigated. Since these organisms work collectively to de­grade biomass, better enzymes, with greater synergies, will be uncovered with additional work. Future efforts will also likely require the use of directed evolution techniques to collectively optimize enzymes to perform under con­ditions more compatible with the fermentation organisms used to produce ethanol and other products. In the short term, there are also great strides to be made in the area of process integration. Here, closely coupling the steps of pretreatment, hydrolysis, and fermentation has the potential to significantly increase overall process efficiency and reduce cost.

Acknowledgements Our thanks to the members of the National Renewable Energy Lab­oratory who kindly supplied acid pretreated corn stover, numerous methods of analysis, and many helpful discussions over the course of our collaboration and to the US Depart­ment of Energy for funding much of the work described.

Metabolic engineering strategies for pentose fermentation are developed to fi­nally generate strains that ferment pentose sugars to ethanol under industrial conditions, which may include suboptimal pH and an array of compounds which inhibit cellular metabolism. Industrial strains of S. cerevisiae, including baker’s yeast, generally out-compete most other microorganisms with regard to the properties required in industrial ethanol production [9,10,133-135], including ethanol productivity, ethanol tolerance, lignocellulose hydrolysate tolerance, and tolerance to low pH [136].

5.1

To eliminate the dependence of KO11 and LY01 on costly nutritional supple­mentation, a new ethanologenic E. coli strain was constructed. The starting strain SZ110, a derivative of KO11 modified for production of lactic acid in mineral salts medium (see Sect. 5.1), was re-engineered for ethanol produc­tion.

2.2.1

Conversion of SZ110 to LY168

Strain SZ110, a derivative of KO11, was engineered and metabolically evolved to produce lactic acid, as described in detail below [39]. Evolved derivatives of SZ110 produced D-lactate at 92% yield from 100 gL-1 glucose in inexpen­sive mineral salts media. Since this cheap and efficient utilization of large amounts of sugar is the desired biocatalyst behavior, strain SZ110 was chosen as the starting point for re-engineering of ethanologenic E. coli(Yomano et al., submitted). Conversion of this strain from lactic acid production to ethanol production involved several steps, beginning with deletion of the lactic acid production gene IdhA. The Z. mobilis PET operon, inserted at the pfl locus in KO11, was removed during engineering of SZ110 for lactic acid production by deletion of the entire focA-pflB region [39]. Since elimination of ackA and adhE prevents undesirable carbon loss, deletion of pflB is unnecessary and possibly limits acetyl-CoA levels. Therefore, the native pfl gene was restored in the re-engineered ethanologenic E. coli. To select for optimal integration of the Z. mobilis homoethanol pathway, a promoterless operon containing pdc, adhA, and adhB was randomly inserted by transposon.

Specific growth requirements of both the donor and recipient strains en­abled direct functional selection in minimal medium without antibiotics. Candidate ethanologenic strains were enriched by serial transfers in mineral salts medium. One clone was selected and designated LY160. Further evolu­tion of strain LY160 by serially subculturing into fresh mineral salts medium every 24 h for 32 days led to strain LY160im, an intermediate strain with con­tinued improvement in performance. It was determined that the Z. mobilis ethanol pathway in LY160im was integrated within rrlE, a 23S ribosomal RNA subunit, concurrent with the direction of transcription. The complex regu­lation of ribosomal RNA transcription is reviewed in [40,41]; the presence of two promoters results in high expression at high growth rates and basal expression at low growth rates and during stationary phase, making rrlE an excellent site for PET integration. The Pseudomonas putida short chain es­terase estZ gene was also integrated into the microbial biocatalyst to lower ethyl acetate levels in the broth. The final strain was designated LY168.

2.2.2