Enzymatic hydrolysis can be facilitated by chipping, milling and grinding the biomass into a fine powder to increase the surface area of the cellulose. In most cases the power consumption is forbiddingly high to reach high digestibility in the enzymatic hydrolysis. It can be even higher than the theor­etical energy content that is available in the biomass [21]. However, physical treatment in an extruder combined with heating and addition of chemicals could be an interesting option [22]. Another method that has been suggested is irradiation of cellulose by gamma rays, which cleaves the ^-1,4-glycosidic bonds, thus giving a larger surface area and a lower crystallinity [23]. This method is, however, far too expensive to be used in a full-scale process. It is also doubtful that it can be used in combination with technologies supposed to be environmentally friendly.

3.2

In nature, microbes can efficiently degrade biomass by secreting an array of synergistic enzymes, including cellulases, often from numerous microbes in­termingled in their growth. In an effort to identify new proteins that could work synergistically with those secreted by T. reesei, we conducted mixing ex­periments by supplementing Celluclast 1.5 L with broth from a wide array of cellulolytic fungi grown under cellulase-inducing conditions. By comparing the saccharification of acid pretreated corn stover using equal protein load­ings of either Celluclast alone or mixtures of Celluclast with these broths, fungi secreting components that could work synergistically with the T. ree­sei cellulases could be detected. In Fig. 7, an example of such an experiment shows that a mixture of T. reesei broth and Thielavia terrestris broth has the same level of hydrolyzing activity as twice as much T. reesei or T. terrestris broth alone. These results suggested that some activity present in the T. ter — restris broth was working in synergy with the cellulases present in T. reesei broth to more efficiently degrade the cellulose in the corn stover.

In order to identify the protein or proteins responsible for the observed synergism with the T. reesei cellulases, the T. terrestris broth was fraction­ated and individual fractions were assayed for synergism similarly. Fractions displaying synergism were separated on one — and two-dimensional polyacry­lamide gels, individual proteins were isolated by removal from the gels and subjected to sequencing by tandem mass spectrometry. Several independent chromatographic fractions contained proteins with homology to glycosyl hydrolase family 61A, a protein previously identified in a number of cellu­lolytic fungi. When purified to homogeneity, a number of these proteins were demonstrated to significantly enhance the activity of the T. reesei cellulases in synergism assays. Inclusion of these proteins at less than 5% of the total enzyme dose in some cases could reduce the required cellulase loading by as much as twofold. These results suggested that the GH61 family proteins were the major components responsible for the enhancement of Celluclast 1.5 L activity by crude T. terrestris fermentation broth.

We also tested the cellulase-enhancing effect of GH61 proteins on a var­iety of other lignocellulosic substrates from a variety of pretreatments when

combined with T. reesei cellulases. Those GH61 proteins capable of enhanc­ing hydrolysis of acid pretreated corn stover also enhanced hydrolysis of other substrates, although they differed in their effectiveness by varying amounts. None of the GH61 proteins were able to enhance the hydrolysis of pure cel­lulose in the form of filter paper. This lack of enhancement was also shown with other pure cellulose substrates such as Avicel, phosphoric-acid swollen cellulose, and carboxymethyl cellulose.

The GH61 proteins by themselves showed no significant detectable hy­drolytic activity on PCS or any other lignocellulosic substrate tested, indi­cating that the enhancement was not likely to be the result of any intrinsic endo — or exoglucanase activity of the GH61 proteins. The hydrolytic activity of several GH61 proteins was tested on a variety of model cellulose and hemi — cellulose substrates, but little or no activity was found. These results suggest that the enhancement of cellulolytic activity by GH61 is limited to substrates containing other cell wall-derived material such as lignin or hemicellulose, although there is no clear correlation between the proportions of these ma­terials and the degree of enhancement observed. These findings could be of significant interest for not only the elucidation of the physiological functions of the GH61 protein family, but also the development of a viable enzymatic system to convert biomass to simple platform sugars.

Several of the GH61 genes were transformed into T. reesei, resulting in transformants expressing GH61 at various levels, depending on the number of inserts and site of integration. Fermentation broths produced by these trans­formants were assayed for PCS hydrolysis at various protein loadings to assess their improvement in specific performance relative to control strains not ex­pressing non-native GH61 proteins. The results confirmed that certain GH61 proteins expressed at relatively low levels are capable of significantly enhanc­ing the hydrolysis of cellulose in PCS. For example, expression of T. terrestris GH61B in T. reesei allows for a reduction in protein loading of 1.4-fold to reach 90% conversion of cellulose to glucose in 120 h. The protein loading reduction made possible by GH61 addition becomes more pronounced at longer incubation times and higher levels of hydrolysis, and higher solids loadings.

4.2.3

By selectively choosing pretreatment conditions it should be possible to cre­ate substrates with characteristics (hemicellulose and lignin content, particle size, available surface area etc.) that greatly enhance subsequent enzyme — mediated hydrolysis. For example we can adjust the time, temperature and SO2 concentration of steam pretreatment with consequential effects on over­all product recovery, ease of hydrolysis, etc. In addition to adjusting pretreat­ment parameters, it has been shown that the inherent physical properties of the biomass can be used to anticipate the performance of the pretreated sub­strate in subsequent hydrolysis experiments [45-47]. The particle size, purity, moisture content, and the internal variations in the biomass feedstock, such as the presence of compression or tension wood can all have significant ef­fects on the efficiency of pretreatment. As mentioned earlier, a reduction in the substrate particle size prior to pretreatment is a common practice used in some pretreatment processes [22]. However, size reduction through milling or grinding requires a substantial input of energy, and adds significantly to the total cost of the pretreatment [47]. Therefore, in the case of woody biomass, it is desirable to utilize a biomass particle size that can be produced economically at a large scale with existing equipment, such as the wood chips used in the pulp and paper industry [48].

For the operation of a large-scale bioconversion process a suitable wood chip size should be selected based on a compromise between the energy/cost of producing the chips and the subsequent effectiveness of the pretreatment and product recovery. Some of our previous work has shown that chip size and moisture content have a profound effect on the ease of hydrolyzability of the resulting substrate [46]. For example, by increasing the chip size of Douglas-fir (Pseudotsuga menziesii (Mirb.)) from 0.42 mm2 to 5 cm2 prior to SO2-catalyzed steam pretreatment, greater recovery of the solids and a re­duction in the production of inhibitors could be observed. This could be attributed to a decrease in the “relative severity” of pretreatment undergone by the larger chip at equivalent pretreatment conditions. It was also shown that the lignin present in the larger chips (5 x 5 cm) experienced less con­densation and was therefore more amenable to subsequent alkaline peroxide delignification. This material from the larger chips consequently exhibited a 10-15% increase in hydrolysis yield over that obtained with the mate­rial originating from the smaller chips. Similarly, by increasing the moisture content of the chips from 12 to 30% an improved recovery of glucose and hemicellulose-derived sugars could be achieved. The increased recovery of sugars by raising the moisture content of the chips could be explained by a similar mechanism as it was observed with the increase in chip size. This was due to the additional moisture adsorbing the heat applied during steam pretreatment, resulting in a decrease in the severity of the treatment under­gone by the chips.

In addition to the inherent variations in the properties of incoming biomass, pretreatment schemes must also deal with the fluctuating “purity” of the lignocellulosic substrate, as woody biomass can be expected to contain “con­taminants” such as bark, needles, leaves, branches, etc, that differ significantly in their chemical composition from “white wood” [49]. It is likely that future wood-based bioconversion facilities would involve large amounts of biomass being sent to a chipping unit without careful control of debarking or branch removal. Similarly, it is unlikely that higher-value wood chips will be used extensively in commercial bioconversion facilities due to competition from traditional pulp and paper mills. Therefore a bioethanol facility’s feedstocks are likely to be either coppiced whole plants such as willow or, in the short term, residues from saw and pulp mills such as sawdust, shavings and hogfuel that contain significant amounts of bark, ash and lipophilic extractives. Tree thinnings such as branches have been pretreated using dilute acid during inves­tigations assessing their viability as a biomass feedstock for bioconversion [50], where a two-stage pretreatment was required to hydrolyze 50% of the cellulose while the remaining cellulose was readily hydrolyzed by cellulases.

In similar work, utilizing feedstocks with high bark content, the liquid stream from the steam pretreatment (SP) of a Douglas-fir chip furnish con­taining 30% bark (SP-DFB) was shown to contain lower amounts of total sugars, furfural and 5-hydroxymethylfurfural compared to the liquid stream (prehydrolyzate) isolated from the pure whitewood (SP-DF) [51]. Although the concentration of lipophilic extractives increased in the bark-laden water — soluble stream, subsequent fermentation by Saccharomyces cerevisiae re­sulted in a complete utilization of the hexose sugars within 48 h with compa­rable ethanol yields regardless of whether bark was present or not. Although these results looked encouraging, with regard to the “robustness” of the over­all SP process, the solid fraction of the pretreated Douglas-fir containing bark showed significant differences when compared to the whitewood. The addition of 30% bark to a Douglas-fir chip furnish prior to SP resulted in a significant increase in the lignin detected in the water-insoluble fraction. The SP-DFB solids fraction also contained 56% lignin compared to only 44% lignin in the case of the pure whitewood (SP-DF) sample. Consequently, the lignin content decreased to only 18% for the SP-DFB fraction as compared to 9% in the case of SP-DF upon subsequent alkaline peroxide delignifica — tion [52]. Although the alkaline peroxide delignified SP-DFB had a higher lignin and phenolic extractive content, it resulted in a similar hydrolysis yield to the SP-DF fraction, most likely due to the removal of surface lignin dur­ing the alkaline peroxide delignification stage [53]. Although hydrolysis was not performed in the absence of the delignification step, it can be expected that, due to its higher lignin content, the SP-DFB fraction would be more re­sistant to hydrolysis than was the SP-DF substrate. It is apparent that, during SP of lignocellulosic feedstocks such as Douglas-fir, the inherent properties of the wood have a significant effect on the downstream partitioning of the cellulose, hemicellulose and lignin components.

3

Biological conversion of biomass to hydrogen either proceeds through photo­fermentation or dark fermentation. In dark fermentation the yield is only 10-20% of the potential hydrogen amount that theoretically can be de­rived from organic matter ([7] and Westermann P, J0rgensen B, Lange L, Ahring BK, Christensen CH (2007) Int J Hydrogen Energy (accepted for pub­lication)). Typical hydrogen yields are from 0.52 mol H2/mol hexose, when molasses was the substrate in a batch culture of Enterobacter aerogenes [8], to 2.3 mol when glucose was the substrate in continuous culture of Clostrid­ium butyricum [9]. Besides the low hydrogen yield, a major problem of fermentative hydrogen production is hydrogen-consuming microorganisms such as methanogens and acetogenic bacteria. In these processes, hydrogen is inevitably converted into methane or acetate, respectively, unless the re­sponsible microorganisms are excluded by sterilization of the biomass before fermentation and inoculation with specific hydrogen-producing microbes, or the process is carried out under conditions adverse to the hydrogen utilizers. A combination of biohydrogen production with fuel cell technology is, how­ever, rather straightforward since the fuel cell technology is available [10]. An upgrading of produced gases might be necessary before they are introduced into the fuel cells [11].

As a stand-alone process, fermentative hydrogen production from biomass is currently not feasible due to the low yield attained.

3

The use of polylactic acid (PLA) as a biodegradable carbohydrate-based plas­tic is rapidly expanding in many areas such as food packaging, drug deliv­ery, textiles, medical implants, and cosmetics [111-114]. Both the physical properties and the rate of biodegradation can be controlled by adjusting the ratio of the blended enantiomers, D(-)-lactate and L(+)-lactate [115]. For decades, lactic acid bacteria have been used to produce optically pure d(-)- and L(+)-lactate. However, high costs due to the need for complex media and the inability to ferment a broad range of sugars have con­strained the use of PLA to the manufacture of medical grade sutures and implants. Alternative biocatalysts from a variety of organisms are currently being investigated for efficient and inexpensive production of optically pure isomers [116-121].

Biocatalysts derived from E. coli K-12 had been previously engineered to produce D(-)-lactate but these were not able to metabolize 10% glucose or sucrose to completion in rich or minimal media [122-124]. The success and robustness of E. coli W derived-ethanologenic KO11 prompted the redirection of metabolism in this organism from ethanol to D(-)-lactate production [39]. Elimination of adhE, ackA, and the Z. mobilis homoethanol pathway from KO11 yielded strain SZ110 [39]. SZ110 was subjected to metabolic evolution in LB 100 g L-1 glucose [39], mineral salts medium with 100 g L-1 sucrose, and mineral salts medium with 100 gL-1 glucose, along with genetic manipula­tions to reduce co-product formation and remove foreign genes, to ultimately generate D(-)-lactate-producer SZ194 [125]. Replacement of the native SZ194

IdhA gene with the Pediococcus acidilactici IdhL gene and further metabolic evolution in mineral salts medium with glucose resulted in L(+)-lactate pro­ducer strain TG103 [121]. Both SZ194 and TG103 produced 1.2 M lactate from 12% glucose in mineral salts medium supplemented with 1 mM betaine. How­ever, lactate optical purity decreased from 99.5% to 95% in the presence of betaine [42,121]. This chiral impurity was associated with high glycolytic flux rates; spillover of carbon to lactic acid through the methylglyoxal pathway was the source of the contamination [126-128]. Elimination of the first com­mitted enzyme of the methylglyoxal pathway (mgsA) restored the product optimal purity to close to 100% [121]. The resulting E. coli strains, TG114 and TG108, consistently produced high titers of greater than 99.9% chirally pure d(-)- and L(+)-lactate, respectively, from 12% glucose at greater than 95% of the theoretical yield [121]. The lactate titer, yield, and optical purity attained by TG114 and TG108 are the highest compared to other lactate-producing organisms and were achieved in simpler fermentation medium and condi­tion, making these microbial biocatalysts some of the most efficient lactate producers.

5.2

Process simulations cannot replace experiments, but constitute a useful tool in the planning and evaluation of experiments. Furthermore, they highlight factors that are sometimes neglected in experimental studies, for example, the amounts of chemicals needed in the process (catalyst in pretreatment, acid/base for pH adjustment, nutrients and not least enzymes and yeast), which constitute a significant contribution to the production cost. The over­all demand of steam, process water and cooling water are other important factors.

Optimization of ethanol production from lignocellulosic feedstock requires a model that includes all the major process steps, since changing the condi­tions in one process step is likely to affect other parts of the process. Although no full-scale plant based on enzymatic hydrolysis has yet been built, most of the process steps (e. g. distillation, evaporation, drying and incineration) are considered to be technically mature, i. e. their operational performance is well known. Of course, the application of these unit operations in a lignocellulose — to-ethanol plant still requires to be verified on pilot scale before a full-scale plant can be constructed. However, the ethanol process includes other process steps, which are associated with greater uncertainties regarding design and performance on full scale. This is definitely true for the pretreatment step, ir­respective of the pretreatment method chosen, or how it is configured (Galbe, in this volume). It also applies to enzymatic hydrolysis or SSF at high solids concentrations, as well as solid-liquid separation of the stillage.

The modeling of a lignocellulose-to-ethanol process poses a number of unique challenges. In contrast to well-defined systems, such as pure ethanol — water systems, it involves not only vapor and liquid phases but also a solid phase, including atypical compounds like cellulose, lignin and yeast. There­fore, when simulating such a process it is necessary to use a flowsheeting program that is able to handle solid components. In most techno-economic evaluations of the lignocellulose-to-ethanol process that have been performed during the past 10 years, Aspen Plus from Aspen Technologies has been used [16—20]. In Aspen Plus, a separate solid stream is used that does not interact with the liquid phase and never ends up in the vapor phase. There is thus no need to estimate vapor phase data such as heat of vaporization or vapor pressure for components treated as solids (lignin, glucan, yeast, etc.).

3

On the basis of earlier kinetic modelling for the conversion of glucose to ethanol by wild-type Z. mobilis [59], a further model has been developed for the fermentation of glucose/xylose mixtures by ZM4 (pZB5) [60]. A two — substrate model was constructed based on Monod kinetics for substrate lim­itation, as well as functions for product (ethanol) inhibition and substrate inhibition at the higher glucose and xylose concentrations. The model simu­lation data for various glucose/xylose concentrations were compared with the experimental results using a Microsoft Excel-based program and statistical analysis for error minimization. Using this approach, it was established that the model (with relevant values of the constants) provided good agreement with the experimental batch culture data for 25/25, 50/50 and 65/65 g L-1 glu — cose/xylose media. It should be noted that the model did not include any repression of xylose uptake by glucose as experimentally both glucose and xylose are taken up simultaneously even at the high initial glucose concen­trations. However, this does not preclude the possibility that some glucose repression of xylose might be occurring. The results indicate that ethanol inhibition of xylose utilization is likely to be the more dominant factor in influencing its kinetics.

2.6

Another common policy instrument to promote biofuel use and consump­tion is exemption from excise taxes or mineral spirits taxes. Excise taxes are commonly used in the transportation sector and are designed to fill the gap between property and income taxes. These types of taxes can be imposed on the sale or use of certain articles, including fuels, and on certain transac­tions and occupations. In many cases, these taxes are not itemized in sales receipts and cannot be easily detected, and thus result in a hidden cost to the consumer [81,82]. As shown previously in Table 1, excise tax rates for the countries under consideration range considerably.

In North America, excise taxes have been used as a tool to support renew­able biofuels for some time. The federal governments of both Canada and the USA offer an exemption on bioethanol, which results in a slightly reduced tax rate for E10 blends. In addition, some state and provincial governments also offer exemptions. The largest North American exemption on excise taxes is currently offered in Manitoba, although that status is dependent upon the value of Canadian and American currency.

In Fig. 4, the excise tax exemptions are shown for the USA, and are re­lated to bioethanol production capacity. The federal and state exemptions are illustrated by the shading on the map, with blue indicating the base fed­eral exemption, and shades of red from light to dark indicating increasingly higher state-level exemptions. Bioethanol production capacity in 2005 is in­dicated by the size of the yellow circles, increasing on a logarithmic scale as shown in the legend. Expected additional bioethanol capacity for 2007 is shown by the dark orange circles.

In the USA, Idaho offers the largest combined exemption on E10 fuels at US $ 0.021 L-1, but has no active production of bioethanol. Of the largest bioethanol-producing states, South Dakota and Iowa are the only two produc­ing states that offer an additional exemption on state excise taxes. It may be inferred that excise tax exemptions provide a benefit for producers, but are

■ >$0 20 SO 16-SO 20 $0 14-SO 16

SO 14 (base US Federal exemption)

■ SO 073 (base Canadian Federal exemption)

Fig. 4 Geographic distribution of North American federal and state/provincial-level ex­cise tax exemptions (2005), existing bioethanol production capacity (2005), and projected bioethanol production capacity (2007) [15,17,21,22,69,81] not the deciding factor in determining where to install capacity for produc­tion.

Similarly, exemptions on excise taxes cannot be simply related to bio­ethanol production in Canada or Europe. In Canada, Manitoba offers com­bined exemptions that are higher than any offered in the USA. Combined federal and provincial excise tax exemptions on E10 reach as high as US$ 0.0256 L-1 in Manitoba, as compared to US$ 0.0181 L-1 in Ontario. At the current time, however, Ontario continues to lead Canada in the amount of bioethanol produced, while Manitoba currently lags behind jurisdictions such as Saskatchewan (which has individual incentives) and Quebec (where exemptions are limited to the federal level). In Europe, high excise taxes mean that exemptions for bioethanol (and other biofuels) are very significant and orders of magnitude larger than those found in North America. France offers the largest incentive in the form of tax exemptions, but has focused pro­duction of biofuels on ETBE, while Spain produces a significant amount of bioethanol under a significantly lower excise exemption regime, as indicated in Table 1 [35,43].

In Fig. 5, the level of excise tax exemptions are plotted against bioethanol production capacity and the correlation between the two is examined, using bioethanol production capacities for 2003 and 2005. The two years of data are differentiated by the shaded and white circles. In 2003, no correlation was found between state-level excise tax exemptions and bioethanol pro­duction capacity (r2 = 0.01). This may be evidence that the federal level exemption, which applies to all states, is a sufficient incentive for produc­ers, and that additional incentives are not required to spur development of bioethanol capacity. It could thus be concluded that this is a less effective policy tool for state-level planners. By 2005, the changes in production cap­acity has slightly changed this correlation, but not to any significant extent (r2 = 0.04). It may be postulated that excise tax exemptions have far less in­fluence over the development of bioethanol capacity than does the amount of funding available to capital projects, feedstock availability, or other market influences. Follow-up analyses compared excise tax exemptions to estimates of bioethanol consumption [83,84] and indicate that in the same period, no correlation (r2 < 0.01) could be found between the use of bioethanol in gaso­line blends and state-level excise tax exemptions. This indicates that excise tax exemptions do not serve as a particularly effective tool in enforcing the use of renewable fuels, and that there is no clear cause-and-effect relationship between the level of these exemptions and the establishment of the industry within individual jurisdictions. While excise tax exemptions are undoubtedly an important economic component of a bioethanol producer’s business plan,

they would seem to have less effectiveness as a policy tool to create biofuel capacity or increase its consumption.

5

In open anaerobic ecosystems where biomass is not sterilized, the degra­dation carried out by ubiquitous microorganisms normally follows a rather well-defined pathway as shown in Fig. 1. If no inorganic electron acceptors such as sulfate or nitrate are present, methane is the inevitable terminal biofuel product since all intermediates from the fermentative bacteria can be degraded to methane, carbon dioxide, and water. The natural end prod­ucts of the fermentative bacteria in such open systems are short-chained volatile fatty acids, hydrogen, and carbon dioxide. Alcohols are only formed in small amounts. Approximately 90% of the energy of the converted biomass is conserved in the end products, and only 10% is used by the fermentative bacteria [1]. In the terminal formation of methane from the fermentation

products, the biomass carbon is sequestered completely to the most oxi­dized (CO2) and the most reduced (CH4) states. Only 4% of the original biomass energy is utilized by the terminal link, leaving 86% of the ori­ginal energy content in the formed methane (Fig. 1), which constitutes the sound energetic rationale for the extensive exploitation of the biogas pro­cess. The obligate biology leading to methane formation has an intrinsic stability governed by thermodynamics, which ensures that methanogenesis proceeds within a wide spectrum of physical and chemical conditions. In most methanogenic fermentations the methane yield lies close to the theor­etical maximum of 3 mol of methane per mol of glucose, calculated from the Buswell equation [12,13]:

CaHbOc + (a — b/4 — c/2)H2O ^

(a/2 — b/8 + c/4)CO2 + (a/2 + b/8 — c/4)CH

4

During oxidative growth, roughly half of the sugar carbons can be diverted into cell mass and CO2 [129,130]. For this reason, bacterial production of commodity chemicals has traditionally focused on generating reduced end — products using anaerobic conditions, in order to minimize the loss of carbon as cell material or CO2. Current biological production of acetate involves complex growth conditions consisting of two separate organisms: an ini­tial fermentation of sugars to ethanol by Saccharomyces and subsequent oxidation to acetate by Acetobacter under aerobic conditions [131-133]. Strain TC36, an E. coli W3110 derivative, was engineered to merge aspects of both fermentative and oxidative metabolism for the production of acetate via a single microbial biocatalyst [77]. TC36 contains multiple chromosomal gene deletions to eliminate production of formate focA-pflB), succinate frdBC), lactate (ldhA), and ethanol (adhE), to disrupt the tricarboxylic acid cycle (sucA) and, most notably, to inactivate oxidative phosphorylation (atpFH) in order to direct the flow of carbons from sugar to acetate with minimal car­bon loss to other fermentation products, CO2, and cell mass. A maximum of 878 mM acetate was produced by TC36 in mineral salts medium. Though this is a lower titer than that achieved during ethanol oxidation by Aceto — bacter, TC36 has a twofold higher production rate, can metabolize a wide range of sugars, and requires a simple, single step process in mineral salts medium.

Pyruvate is used as a food additive, nutriceutical, weight control supple­ment, and starting material for the production of amino acids and acetalde­hyde [15,134]. Pyruvate can be produced by either chemical or biological processes. Chemical synthesis from tartrate entails the use of toxic solvents, requires a great deal of energy, and is very costly [135]. Biological produc­tion involves two auxotrophic microorganisms that require costly nutritional supplements and strict regulation of media composition [134,136], or an E. coli strain that produces pyruvate from glucose and acetate in complex medium [137]. More recently, a microbial biocatalyst has been developed for the efficient synthesis of pyruvate from sugar requiring only inexpen­sive mineral salts medium [138]. Strain TC36, an E. coli W3110 derivative described above, was used as a platform to generate the pyruvate pro­ducer TC44. This strain encompasses two additional chromosomal deletions, ackA and poxB, to allow pyruvate accumulation and eliminate acetate pro­duction. TC44 yields (0.75 g pyruvate per gram of glucose), titer (749 mM maximum) and production rate (1.2 g of pyruvate L-1 h-1) in mineral salts medium were comparable to or better than the previously described bio­catalysts, which required costly nutritional supplements and complex me­dia [138]. This strain improves the cost of pyruvate production by reduc­ing the costs of materials, process controls, product purification, and waste disposal.

5.3