The hydrolysis experiments were carried out at a substrate consistency of 10 gL-1 in 50 mM sodium acetate, pH 5, in a volume of 100 mL, and incubated in shake flasks with shaking (200 rpm) at different temperatures from 55 °C to 70 °C. Duplicate shake flasks were sampled (5 mL sample) at 2 h, 4 h, 6 h, 24 h, 48 h and 72 h from the start of the hydrolysis. Possible evaporation was checked by weighing and corrected when necessary by adding a correspond­ing amount of water. The release of hydrolysis products was followed during the hydrolysis.

The worldwide annual ethanol production via microbial fermentation amounted to ca. 40 Mt in 2005 (according to the Renewable Fuel Associa­tion; www. ethanolrfa. org) and is rapidly growing. Although bacteria such as Zymomonas mobilis and engineered Escherichia coli strains are capable of homoethanolic fermentation of sugars [17], the yeast Saccharomyces cere­visiae remains the organism of choice for large-scale industrial production of ethanol. Factors contributing to the popularity of S. cerevisiae as an industrial ethanol producer include its high ethanol tolerance, its ability to grow under strictly anaerobic conditions and — an important characteristic distinguishing it from prokaryotic organisms — its insensitivity to bacteriophage contamina­tions. Moreover, S. cerevisiae grows well at low pH, reducing problems with contamination of industrial processes with, for example, lactic acid bacteria.

Global concern about carbon dioxide emissions and climate change, deple­tion of oil reserves and geopolitical issues all contribute to a drive to increase the production of ethanol as a renewable transport fuel (see the contribution of Otero et al. in this volume). Presently, ethanol is exclusively produced from the starch or the sucrose fraction of a small number of (edible) agricultural crops such as corn, sugar cane, sugar beet and grain. To expand the feed­stock range for large-scale ethanol production and to improve productivity, it is of vital importance to enable efficient ethanol production from agri­cultural residues and other low-value sources of carbohydrates. Feedstocks such as corn stover, bagasse, wheat straw, non-recyclable paper or dedicated crops such as switchgrass represent an enormous potential in terms of avail­able carbohydrates. However, instead of starch and sucrose, the carbohydrates in these feedstocks consist of a complex matrix of cellulose, hemicellulose, pectin and lignin [69].

The use of lignocellulosic raw materials for ethanol production poses a number of major challenges compared to the use of conventional starch — or sucrose-based feedstocks:

(i) Release of monomeric sugars from lignocellulosic biomass requires a mix of physicochemical (extreme pH, high temperature, high pressure) and enzymic polysaccharide (hydrolases) treatments [19,37].

(ii) The resulting lignocellulose hydrolysates contain a wide variety of com­pounds that may inhibit the fermentation process. These compounds are either formed during the pretreatment process (e. g. furfural and hydroxymethylfurfural) or are biomass constituents that are released during hydrolysis (e. g. acetate, formate) [31,37,49,54].

(iii) Whereas starch — and sucrose-based feedstocks yield hexoses upon hydro­lysis, lignocellulosic biomass, and in particular its hemicellulose frac­tion, also contains large amounts of the pentose sugars D-xylose and L-arabinose. D-Xylose, generally the most abundant pentose, comprises up to 25% of the total sugar content in some hydrolysates [24,46,69].

Whereas S. cerevisiae spp. can rapidly ferment hexose sugars such as glucose, fructose, mannose and galactose, they cannot grow on nor ferment D-xylose or L-arabinose [7,69]. Given the importance of xylose fermentation for the efficient production of ethanol from lignocellulosic biomass [24,46,69], it is not surprising that introduction and optimisation of heterologous path­ways for xylose fermentation into S. cerevisiae has long been a hot topic in metabolic engineering of yeast.

Interestingly, it has long been known that S. cerevisiae is able to slowly metabolise the pentose sugar D-xylulose [30,71]. This keto-isomer of xylose is phosphorylated to D-xylulose-5-phosphate by xylulokinase (XKS1, [57]) and subsequently metabolised via the non-oxidative part of the pentose phos­phate pathway and glycolysis. It is therefore logical that strategies for convert­ing D-xylose into D-xylulose are an exhaustively studied topic in the quest for alcoholic fermentation of D-xylose by S. cerevisiae. These strategies will be briefly discussed in Sects. 1.2-1.4.

1.2

4.1

Cost Effective Growth Media

In order for ethanol production to be commercially feasible, the growth media cost should be kept at a minimum. In addition to engineering strains to require less nutritional supplementation, the design of simpler, and therefore cheaper, growth media is important for the expansion ofbioethanol production.

AM1 [76] and NBS mineral salts media [77] are two simple mineral salts media developed in our laboratory. Both have been shown to support high levels of cell growth and ethanol production. AM1 is a derivative of NBS, with a 65% reduction in salts. With low total alkali (4.5 mM) and total salts (4.2 gL-1), AM1 was able to support production of ethanol from xylose and lactate from glucose with average productivities of 18 -19 mmol L-1 h-1.

OUM1 medium contains corn steep liquor, mineral salts, and urea as sole nitrogen source; K. oxytoca BW21 produced over 40 gL-1 ethanol (0.47 g ethanol per gram glucose) in this medium within 48 h [57]. The use of urea as sole nitrogen source has the benefit of cost reduction while also reducing media acidification [78].

On-site preparation of crude yeast autolysate from spent yeast offers poten­tial synergy between grain-based and lignocellulosic processes. Preparation of this autolysate, optimization of the resulting media, and ethanol production by KO11 were demonstrated by [31], with ethanol yields comparable to LB.

K. oxytoca is able to utilize urea as sole nitrogen source, where urea has roughly half the cost of ammonium on an equivalent nitrogen basis. Addition­ally, because urea metabolism does not contribute to media acidification [79], the use of urea reduces the cost of pH control. With the goal of reducing the nutrient cost of K. oxytoca-based ethanol production, optimized urea medium (OUM1) was developed. In addition to containing urea as the sole nitrogen source, OUM1 contains corn steep liquor, mineral salts, and glucose [57].

4.2

The effluent from bioethanol production still contains a large amount of or­ganics that are not composed of carbohydrates. Anaerobic digestion (AD) has been used for a long time to treat organic waste streams with a high concen­tration of organic matter. The benefits of anaerobic treatment are stabilization of the waste stream, the high reduction of organic matter, and the production of methane, which can be used as energy source [25]. This gives an overall positive energy balance of the waste treatment process compared to aerobic waste treatment. The income from the methane produced after bioethanol production constitutes a value corresponding to a lowering of the ethanol production price by 34%.

The effluent from the fermentation step of bioethanol production con­tains low-molecular weight lignin degradation products primarily generated during the physical-chemical pretreatment. These aromatic compounds are generally difficult to degrade under anaerobic conditions. Furthermore, a re­peated reuse of the process water has the potential to cause a build up of these fermentation inhibitors. It is therefore important to achieve an anaero­bic purification technology that is able to remove these compounds from the process water. Experiments in our laboratory have shown that all problematic organic components can be removed in the anaerobic step. The low hydraulic retention times and the removal of organics are of great importance, looking at the overall process feasibility [26].

The Maxifuel concept has been implemented at pilot scale at the Techni­cal University of Denmark, DTU (Fig. 5) and the concept is planned to go into demonstration phase in 2008.

The plant is dimensioned to convert 150 kg dry biomass/day, and con­sists of 17 tanks (fermentation, reactors, and holding tanks). The ethanol fermentation takes place in two 2700 L fermenters. The plant includes all pro­cesses from straw to ethanol, and was brought into operation in the autumn of 2006.

4.6

The first xylose-utilizing strains of S. cerevisiae were generated by expressing the Pichia stipitis genes XYL1 [23] and XYL2 [24], encoding XR and XDH, re­spectively [46-48]. P stipitis was chosen as the source of the heterologously expressed enzymes because it produces ethanol from xylose with theoret­ical yield, albeit only under well-controlled oxygen limitation [47,49,50], while most other naturally xylose-fermenting yeasts produce considerable amounts of the by-product xylitol [50]. Xylitol formation is a consequence of the inability of the cell to oxidize reduced cofactors in the absence of oxygen [32]. Contrary to XRs from most xylose-utilizing yeasts, XRs from P stipitis, Pachysolen tannophilus, and Candida shehatae can use not only NADPH but also NADH as a cofactor [21], which permits recirculation of the cofactors between the first two steps of the xylose pathway (Fig. 1).

Nevertheless, the first S. cerevisiae strains expressing the P stipitis XR and XDH produced xylitol, and the ethanol yield from xylose was low [47,48]. This was ascribed to the preference for NADPH over NADH of the XR [23]. Much research has been devoted to developing metabolic engineering strate­gies to improve xylose fermentation by XR — and XDH-carrying strains, often guided by the early suggestions to express either a strictly NADH-specific XR activity [32] or to express a transhydrogenase activity [21]. Both approaches are further discussed in the following sections together with other metabolic engineering strategies. Kinetic modeling estimated that the conversion of xylose to xylulose required a ratio of 1:10 of the initial XR and XDH activ­ities [51], which has been experimentally supported by several independent investigations [51-54]. The higher level of XDH is necessary to “pull” the xy­lose toward central metabolism [55], especially since the equilibrium of the XDH reaction favors xylitol formation [56]. In addition, it has more recently been found that efficient xylose metabolism requires high activity of both XR and XDH [54,57].

3

Arabinose

3.1

Wheat straw is an abundant lignocellulosic crop residue with potential as a feedstock for ethanol production, especially in Canada and Europe. Wheat straw hydrolysate was therefore selected as one of the fermentation feed­stocks for evaluating the fermentation characteristics of S. cerevisiae RWB 218 under industrially relevant conditions (W. de Laat, unpublished data). Wheat straw was pretreated using steam explosion (Sunopta, Canada). The pulp thus obtained was then hydrolysed enzymically at pH 5.0 with cellulases and hemicellulases, yielding a hydrolysate that contained 50 g L-1 glucose, 20 g L-1 D-xylose, 6 gL-1 arabinose and 6 gL-1 of disaccharides (cellobiose, melibiose, maltose and sucrose, indicated as DP-2 in Table 2). The hydrolysate, which

also contained 3 gL-1 acetic acid and 0.3 gL-1 of lactic acid, was supple­mented with 0.4 gL-1 of (NH4)2PO4 as a combined source of nitrogen and phosphate. Fermentations were run at 32 °C, with an initial pH of 4.8.

When batch cultures on the wheat straw hydrolysates were inoculated with 1.5gL-1 of S. cerevisiae RWB 218, most of the available sugars were con­verted within 55 h (Table 2). The yield of ethanol on the consumed sugars was very high and, towards the end of the fermentation, even approached the theoretical maximum yield of 0.51 gg-1. This very high apparent yield might partially be caused by the additional hydrolysis of some oligosaccharides or by the presence of other sugars that were not identified in the analyses. Xylitol formation was not observed.

Even when a much lower initial biomass concentration of 0.1 gL-1 was used, S. cerevisiae RWB 218 reached the same degree of conversion in 80 h. Addition of vitamins, trace elements and/or the anaerobic growth factors Tween-80 and ergosterol [2,3] did not result in a faster fermentation. This demonstrates the modest nutritional requirements of S. cerevisiae during fer­mentation of hydrolysates of lignocellulosic materials, which often contain very low levels of nutrients required for microbial growth.

7.3

P. L. Rogers1 (И) • Y. J. Jeon1 • K. J. Lee2 • H. G. Lawford3

School of Biotechnology and Biomolecular Sciences, UNSW, 2052 Sydney, Australia p. rogers@unsw. edu. au

2School of Biological Sciences, Seoul National University, 151-742 Seoul, Korea 3Department of Biochemistry, University of Toronto, Toronto Ont., M5S 1A8, Canada

1 Introduction……………………………………………………………………………………………… 264

2 Development of Recombinant Strains of Z. Mobilis…………………………………………. 265

2.1 Increased Substrate Range Through Expression

of a Single Heterologous Gene……………………………………………………………….. 265

2.2 Strain Construction for Utilization of C5 Sugars………………………………………….. 266

2.3 NMR Analysis of Metabolic Characteristics of Recombinant Strains…. 269

2.4 Kinetic Characteristics of Recombinant Strains…………………………………………….. 269

2.5 Kinetic Model Development……………………………………………………………………….. 273

2.6 Effect of Inhibitors in Lignocellulosic Hydrolysates………………………………………. 274

2.7 Application to Industrial Raw Materials……………………………………………………… 275

3 Genome Sequence of Z. Mobilis…………………………………………………………………….. 278

4 Applications for Higher Value Products……………………………………………………… 278

4.1 Metabolites and Related Products………………………………………………………………. 278

4.2 Metabolic Engineering for Organic Acids and TCA Cycle Intermediates. . 279

4.3 Enzyme Based Biotransformations……………………………………………………………… 281

4.3.1 Sorbitol/Gluconate Production…………………………………………………………………… 281

4.3.2 Pharmaceutical Intermediates and Fine Chemicals………………………………………. 282

5 Discussion and Conclusions……………………………………………………………………….. 283

References…………………………………………………………………………………………………….. 286

Abstract High oil prices, increasing focus on renewable carbohydrate-based feedstocks for fuels and chemicals, and the recent publication of its genome sequence, have provided continuing stimulus for studies on Zymomonas mobilis. However, despite its apparent advantages of higher yields and faster specific rates when compared to yeasts, no com­mercial scale fermentations currently exist which use Z. mobilis for the manufacture of fuel ethanol. This may change with the recent announcement of a Dupont/Broin part­nership to develop a process for conversion of lignocellulosic residues, such as corn stover, to fuel ethanol using recombinant strains of Z. mobilis. The research leading to the construction of these strains, and their fermentation characteristics, are described in the present review. The review also addresses opportunities offered by Z. mobilis for higher value products through its metabolic engineering and use of specific high activity enzymes.

Keywords Ethanol production • Glycose/Xylose fermentations • Higher value products • Lignocellulosics • Metabolic engineering • Zymomonas mobilis

1

Introduction

Zymomonas mobilis has attracted considerable interest over the past decades as a result of its unique metabolism and ability to rapidly and efficiently pro­duce ethanol from simple sugars. An early paper by Millis [1] characterized the role which Zymomonas sp. play in causing cider sickness and a compre­hensive review by Swings and DeLey [2] provided much of the background for the subsequent stimulus in research activity in the early 1980s which fol­lowed the first of the “oil price shocks”. Further reviews over the ensuing decades [3-9] included extensive data on genetic and kinetic characteriza­tion of strains of Zymomonas mobilis capable of growing on an increasingly wide range of sugars. In a fine example of metabolic (pathway) engineering, recombinant strains of Z. mobilis were reported in 1995/6 from the National Renewable Energy Laboratory (NREL) Golden, CO, USA, that were capable of the efficient conversion to ethanol of the C5 sugars, xylose and arabinose present in lignocellulosic hydrolysates [10,11]. Most recently, the reporting of the complete genome sequence of Z. mobilis ZM4 (ATCC 31821) [12] has opened up further potential for strain enhancement and for its use for higher value products.

Table 1 provides an outline of the key research milestones which have oc­curred for Z. mobilis over the past three decades with the present review focusing particularly on those developments which have been reported over the past 5-10 years.

2

The oldest example of widespread biofuel development is found in Brazil, which produces bioethanol from sugar- or starch-based material in the form of sugarcane and sugarcane residues. Because of Brazil’s optimal climate, two seasons of sugarcane growth can be achieved, adding greatly to the poten­tial production of both sugar and bioethanol products. In response to the first oil crisis of the 1970s, Brazil invested heavily in fuel alcohol primarily as a means of increasing fuel security and saving foreign currency on petroleum purchases. The original policy choice was to create direct funding sources to create biofuel capacity. In 1975, a diversification program for the sugar in­dustry called Proalcool was created with large public and private investments supported by the World Bank, allowing expansion of the sugarcane plantation area and construction of alcohol distilleries, either autonomous or attached to existing sugar plants [19].

The second group of policies introduced in Brazil provided a subsidy for bioethanol use. Two related financing schemes were organized to guarantee fuel sale price; the FUPA program guaranteed US $ 0.12 L-1 for E22 (a blend of 22% ethanol in gasoline), while the FUP program provided US$ 0.15 L-1 for E100 (or pure, anhydrous ethanol) fuel. By 1996/97, the total subsidy de­livered via these programs reached about US $ 2 billion year-1 [19].

The presence of a renewable fuel standard and of strong subsidies to E100 production, combined with the second oil shock of the early 1980s, resulted in the successful adaptation of engines to E100 fuel use. By 1984, E100 vehicles accounted for 94.4% of domestic automobile manufacturers’ production, and in 1988 participation in the E100 program reached 63% of total vehicle use in the country [20]. The upward trend ended, however, when high global sugar prices led to a crash in availability of fuel alcohol, resulting in a consumer shift away from E100 vehicles.

From 1989 to 1996, the sugar export market was very strong, and thus the cost of sugar to the bioethanol industry soared and fuel bioethanol short­ages resulted. In response, the Brazilian government made a failed attempt to restrict sugar exports, and then announced that the fuel market would be deregulated as of 1997. While deregulation began with E100 fuels, subsi­dies for blended fuels remained in place for an additional period, which had the effect of increasing overall alcohol production at the time. When price controls on E22 were removed in 1999, however, the prices for bioethanol collapsed [19].

Faced with an excess of bioethanol and collapsed prices at home, major producer groups joined together to form Brasil Alcool SA in March 1999, and made the decision to export excess bioethanol at any price. Later that year, a mechanism to create a monopoly on fuel bioethanol named Bolsa Brasileira de Alcool Ltda was created by the founders of Brasil Alcool. This monopoly drove a dramatic increase in bioethanol export prices for a period after its inception, with prices doubling within a year [20]. Since 1999, the total production of bioethanol in Brazil has risen; this trend has been driven by the expansion of export markets for bioethanol, rising world prices for oil, and an increase in domestic oil supply. The Brazilian industry today follows a simple biorefinery model, where the production of a combination of prod­ucts, including refined sugar, bioethanol, and energy from the combustion of sugarcane residues (bagasse) improves both economic and environmen­tal performance. Brazil controls more than 75% of the world’s export market, with primary exports going to the USA, Europe, Korea, and Japan; Brazil’s es­timated total exports will be approximately 3.1 billion L in 2006 [12]. Many countries that lack significant biomass resources, such as Japan, have made Brazilian bioethanol a part of their renewable fuel strategies.

Brazil’s domestic market still utilizes the single largest portion of fuel bioethanol capacity in the country. The presence of a Renewable Fuel Stan­dard means that all Brazilian gasoline has a legal alcohol content requirement that has ranged between 20% and 25% (currently 23%, as of 20 November 2006) [21]. Most vehicles are being run on E20 or E22, but sales of flex-fuel vehicles capable of operating on E85 blends are strong. Brazil has developed a unique distribution infrastructure for this fuel, with a network of more that 25 000 gas stations with E20 pumps.

Today, Brazil remains a dominant bioethanol producer and the single larg­est exporter of this fuel, with shipments expected to hit a record 3 billion L in the 2006-07 harvest. Rising demand for bioethanol — in part caused by poli­cies in other countries — has created an impetus for new product capacity. Recently, it was reported that UNICA plans to open 77 new bioethanol plants by 2013, adding to the existing 248 plants. When complete, this will raise the country’s production capacity to about 35.7 billion L [21].

2.2

The P. stipitis XR, which converts xylose to xylitol, prefers the cofactor NADPH over NADH by a factor of approximately 100 [23]. In yeast, NADPH is primarily formed in the oxidative PPP converting glucose-6-phosphate to ribulose-5-phosphate. Therefore, genes coding for enzymes in the oxidative PPP were deleted in order to decrease NADPH concentration in the cell and

thus force XR to use NADH instead of NADPH, which was demonstrated by the deletion of ZWF1, coding for glucose-6-phosphate dehydrogenase (G6PDH) [114] (strain TMB3255, Table 2), [115] (strain H2723, Table 1). In­creased ethanol formation at the expense of not only xylitol formation but also the xylose consumption rate was observed [114] (strains TMB3001 and TMB3255, Tables 2 and 3). In a follow-up study, the G6PDH activity was in­stead fine-tuned, which enabled the design of strains with increased ethanol yield and reasonable xylose consumption rate [116] (strains TMB3256 and TMB3037, Table 2, Fig. 4). However, in an industrial context, it is worth notic­ing that the ZWF1 deletion increases the sensitivity toward lignocellulose hydrolysates, possibly due to the limited intracellular NADPH concentration, which is important for inhibitor tolerance [116,117].

4.5.2

Historically, Saccharomyces has served as the main biocatalyst for commer­cial ethanol production. Considering that Saccharomyces and Z. mobilis are naturally ethanologenic, these organisms are obvious candidates for ethanol production. However, both organisms lack the native ability to utilize pentose sugars, the major component of the hemicellulose fraction of biomass [9,10]. Though E. coli lacks the native ability to produce ethanol as the major fer­mentation product, it utilizes both hexose and pentose sugars [11] and the uronic acid constituents of pectin [12]. The breadth of carbohydrates metab­olized, extensive background of knowledge, and ease of genetic manipulation made E. coli an obvious choice for metabolic engineering of a microbial bio­catalyst for production of ethanol from lignocellulose.

2.1