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Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC 19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The "Iogen Process" for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biom-ass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory’s metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN’s feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN’s biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L·h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L·h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of D- and L-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of DL-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, D-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. D-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.

Sphaerotilus natans is a sheathed bacterium existing in the activated sludge of wastewater treatment plants. It is one of the filamentous bacteria causing the bulking and foaming difficulties of activated sludge. Isolating the strain and culturing it in an axenic environment could not only provide the metabolic knowledge of the strains that would be useful in the development of wastewater treatment methods, but also could enable us to gain an understanding of the mechanism by which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly[3-HB-co-3-HV]) is produced by this strain. This article reports the screening and isolation of the strain from the activated sludge using the Nile blue staining method together with Fourier transform infrared analysis. We investigated the ability of the selected strain to produce poly(3-HB-co-3-HV) copolymer using glucose and peptone, or by adding valeric acid or sodium propionate as precursor. Proper precursor feeding could dramatically enhance its 3HV content in the copolymer P(3HB-co-3HV). By controlling the different feeding times in fed-batch fermentation, different desired copolymers were obtained with 15, 40, and 70% 3HV mole fraction of the copolymer. Polymer properties were analyzed by gas chromatography, differential scanning calorimetry, thermo-gravimetry, and nuclear magnetic resonance analysis.

Plastic wastes are considered to be severe environmental contaminants causing waste disposal problems. Widespread use of biodegradable plastics is one of the solutions, but it is limited by high production cost. Biologic wastewater treatment generates large quantities of biomass as activated sludge. Only a few reports focus on the potential of utilizing resident Bacillus species from activated sludge in polyhydroxbutyrate (PHB) production as well as the production of PHB from food wastes. They have attractive properties such as short generation time, absence of endotoxins, and secretion of both amylases and proteinases that can well utilize food wastes for nutrients, which can further reduce the cost of production of polyhydroxyalkanoates (PHAs). Two PHA-producing strains, HF-1 and HF-2, were isolated from activated sludge. HF-1 outperformed HF-2 in terms of growth and PHB production in hydrolyzed soy and malt wastes. The isolated bacteria was characterized by DNA sequence alignment. Cell extracts of HF-1 were also compared to Bacillus megaterium cell extracts on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The biopolymers accumulated were analyzed by gas chromatography, nuclear magnetic resonance, and Fourier transform infrared methods.

In pH-controlled batch fermentations with pure sugar synthetic hardwood hemicellulose (1% [w/v] glucose and 4% xylose) and corn stover hydrolysate (8% glucose and 3.5% xylose) lacking acetic acid, the xylose-utilizing, tetracycline (Tc)-sensitive, genomically integrated variant of Zymomonas mobilis ATCC 39676 (designated strain C25) exhibited growth and fermentation performance that was inferior to National Renewable Energy Laboratory's first-generation, Tc-resistant, plasmid-bearing Zymomonas recombinants. With C25, xylose fermentation following glucose exhaustion was markedly slower, and the ethanol yield (based on sugars consumed) was lower, owing primarily to an increase in lactic acid formation. There was an apparent increased sensitivity to acetic acid inhibition with C25 compared with recombinants 39676:pZB4L, CP4:pZB5, and ZM4:pZB5. However, strain C25 performed well in continuous fermentation with nutrient-rich synthetic corn stover medium over the dilution range 0.03-0.06/h, with a maximum process ethanol yield at D = 0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L x h). With 0.35% (w/v) acetic acid in the medium, the process yield at D = 0.04/h dropped to 0.32 g/g, and the maximum productivity decreased by 50% to 1.5 g/(L x h). Under the same operating conditions, rec Zm ZM4:pZB5 performed better; however, the medium contained 20 mg/L of Tc to constantly maintain selective pressure. The absence of any need for antibiotics and antibiotic resistance genes makes the chromosomal integrant C25 more compatible with current regulatory specifications for biocatalysts in large-scale commercial operations.

This study documents the similar pH-dependent shift in pyruvate metabolism exhibited by Zymomonas mobilis ATCC 29191 and ATCC 39676 in response to controlled changes in their steady-state growth environments. The usual high degree of ethanol selectivity associated with glucose fermentation by Z. mobilis is associated with conditions that promote rapid and robust growth, with about 95% of the substrate (5% w/v glucose) being converted to ethanol and C)2, and the remaining 5% being used for the synthesis of cell mass. Conditions that promote energetic uncoupling cause the conversion efficiency to increase to 98% as a result of the reduction in growth yield (cell mass production). Under conditions of glucose-limited growth in a chemostat, with the pH controlled at 6.0, the conversion efficiency was observed to decrease from 95% at a specific growth rate of 0.2/h to only 80% at 0.042/h. The decrease in ethanol yield was solely attributable to the pH-dependent shift in pyruvate metabolism, resulting in the production of lactic acid as a fermentation byproduct. At a dilution rate (D) of 0.042/h, decreasing from pH 6.0 to 5.5 resulted in a decrease in lactic acid from 10.8 to 7.5 g/L. Lactic acid synthesis depended on the presence of yeast extract (YE) or tryptone in the 5% (w/v) glucose-mineral salts medium. At D = 0.15/h, reduction in the level of YE from 3 to 1 g/L caused a threefold decrease in the steady-state concentration of lactic acid at pH 6. No lactic acid was produced with the same mineral salts medium, with ammonium chloride as the sole source of assimilable nitrogen. With the defined salts medium, the conversion efficiency was 98% of theoretical maximum. When chemostat cultures were used as seed for pH-stat batch fermentations, the amount of lactic acid produced correlated well with the activity of the chemostat culture; however, the mechanism of this prolonged induction effect is unknown. The levels of lactic acid produced by Z. mobilis in this study have not been previously reported. Zymomonas is Gram-negative, and at no time did microscopic inspection of lactic-acid-producing cultures indicate the presence of Gram-positive organisms. Although these observations are very preliminary in nature, they have implications for the regulation of glycolytic flux in Zymomonas, and demonstrate the possibility of an alternative fate for pyruvate previously presumed not to exits.

In the production of ethanol from lignocellulosic biomass, the hydrolysis of the acetylated pentosans in hemicellulose during pretreatment produces acetic acid in the prehydrolysate. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process that uses a proprietary metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. Acetic acid toxicity represents a major limitation to bioconversion, and cost-effective means of reducing the inhibitory effects of acetic acid represent an opportunity for significant increased productivity and reduced cost of producing fermentation fuel ethanol from biomass. In this study, the fermentation performance of recombinant Z. mobilis 39676:pZB4L, using a synthetic hardwood prehydrolysate containing 1% (w/v) yeast extract, 0.2% KH2PO4, 4% (w/v) xylose, and 0.8% (w/v) glucose, with varying amounts of acetic acid was examine. To minimize the concentration of the inhibitory undissociated form of acetic acid, the pH was controlled at 6.0. The final cell mass concentration decreased linearly with increasing level of acetic acid over the range 0-0.75% (w/v), with a 50% reduction at about 0.5% (w/v) acetic acid. The conversion efficiency was relatively unaffected, decreasing from 98 to 92%. In the absence of acetic acid, batch fermentations were complete at 24 h. In a batch fermentation with 0.75% (w/v) acetic acid, about two-thirds of the xylose was not metabolized after 48 h. In batch fermentations with 0.75% (w/v) acetic acid, increasing the initial glucose concentration did not have an enhancing effect on the rate of xylose fermentation. However, nearly complete xylose fermentation was achieved in 48h when the bioreactor was fed glucose. In the fed-batch system, the rate of glucose feeding (0.5 g/h) was designed to simulate the rate of cellulolytic digestion that had been observed in a modeled SSCF process with recombinant Zymomonas. In the absence of acetic acid, this rate of glucose feeding did not inhibit xylose utilization. It is concluded that the inhibitory effect of acetic acid on xylose utilization in the SSCF biomass-to-ethanol process will be partially ameliorated because of the simultaneous saccharification of the cellulose.

The five-carbon sugar D-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design.Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinant Z. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a "benchmark" yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5-2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.

This study compared the anaerobic catabolism of glucose and xylose by a patented, recombinant ethanologenicEscherichia coli B 11303:pLOI297 in terms of overall yields of cell mass (growth), energy (ATP), and end product (ethanol). Batch cultivations were conducted with pH-controlled stirred-tank bioreactors using both a nutritionally rich, complex medium (Luria broth) and a defined salts minimal medium and growth-limiting concentrations of glucose or xylose. The value of YATP was determined to be 9.28 and 8.19 g dry wt cells/mol ATP in complex and minimal media, respectively. Assuming that the nongrowth-associated energy demand is similar for glucose and xylose, the mass-based growth yield (Y x/s , g dry wt cells/g sugar) should be proportional to the net energy yield from sugar metabolism. The value ofY x/s was reduced, on average, by about 50% (from 0.096 g/g glu to 0.051 g/g xyl) when xylose replaced glucose as the growth-limiting carbon and energy source. It was concluded that this observation is consistent with the theoretical difference in net energy (ATP) yield associated with anaerobic catabolism of glucose and xylose when differences in the mechanisms of energy-coupled transport of each sugar are taken into account. In a defined salts medium, the net ATP yield was determined to be 2.0 and 0.92 for glucose and xylose, respectively.

The two cultures under investigation were genetically engineered for the purpose of producing fuel ethanol from biomass and wastes. In this study, stability was viewed in pragmatic terms whereby the fermentation performance of the different genetic constructs was assessed solely with respect to their capacity to maintain a high efficiency of sugar-to-ethanol conversion. Two serial transfers of test tube batch cultures accounted for about 12 generations of semi-continuous growth in LB medium containing one of 4 different sugars - glucose, galactose, mannose or xylose. Both the plasmid-bearing recombinant E. coli B (ATCC 11303) pLOI297 and the chromosomally-integrated recombinant KO11 exhibited dramatic loss of ethanologenicity during growth in a selective (antibiotic-supplemented) medium with mannose as fermentation substrate. In the absence of antibiotics, both recombinants exhibited instability, with the exception of KO11 with xylose as substrate. These observations with short term cultures call into question previous claims regarding the stability of these genetic constructs.

The anaerobic conversion of xylose to ethanol by a genetically engineredE. coli B (pLOI297) was investigated using anaerobically and aerobically grown cultures as inocula. Using anaerobically grown cells, an increase in the inoculation density from 50 to 340 mg dry wt. cells/L resulted in an increase in the overall volumetric productivity from 0.57 to 0.71 g/L/h. At the higher inoculation density, substitution of the anaerobic inoculum by aerobically grown cells resulted in a 15% reduction in volumetric productivity (0.61 g/L/h) that was caused by the introduction of a lag period during which the aerobic inoculum adapted to the anaerobic environment. In all cases, the ethanol yield from xylose approached the theoretical maximum and seemed unaffected by the physiological history of the inoculum with respect to aeration. It is concluded that aeration should be avoided in the production of high performance starter cultures.

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. The patented, genetically engineered, ethanologen Escherichia coli B (pLOI297) exhibits high-performance characteristics with respect to both yield and productivity in xylose-rich lab media. In addition to producing monomer sugar residues, thermochemical processing of biomass is known to produce substances that are inhibitory to both yeast and bacteria. During prehydrolysis, acetic acid is formed as a consequence of the deacetylation of the acetylated pentosan. Our investigations have shown that the acetic acid content of hemicellulose hydrolysates from a variety of biomass/waste materials was in the range 2-10 g/L (33-166 mM). Increasing the reducing sugar concentration by evaporation did not alter the acetic acid concentration. Acetic acid toxicity is pH dependent. By virtue of its ability to traverse the cell membrane freely, the undissociated (protonated) form of acetic acid (HAc) acts as a membrane protonophore and causes its inhibitory effect by bringing about the acidification of the cytoplasm. With recombinant E. coli B, the pH range for optimal growth with glucose and xylose was 6.4-6.8. With glucose, the pH optimum for ethanol yield and volumetric productivity was 6.5, and for xylose it was 6.0 and 6.5, respectively. However, the decrease in growth and fermentation efficiency at pH 7 is not significant. At pH 7, only 0.56% of acetic acid is undissociated, and at 10 g/L, neither the ethanol yield nor the maximum volumetric productivity, with glucose or xylose, is significantly decreased. The "uncoupling" effect of HAc is more pronounced with xylose and the potency of HAc is potentiated in a minimal salts medium. Controlling the pH at 7 provided an effective means of circumventing acetic acid toxicity without significant loss in fermentation performance of the recombinant biocatalyst.

Recombinant E. coli B (pLOI297) grows in Luria broth with mannose at a rate that is only about one-half of the rate with xylose and about one-quarter of the rate with glucose as carbon source. For a sugar concentration of about 2 % (w/v), the corresponding specific ethanol productivities (qp) are 0.22, 0.45 and 0.70 g ethanol/g cell/h for mannose, xylose and glucose. At higher sugar concentrations (8–11 %), the sp. productivities are 0.12, 0.33 and 0.35 g ethanol/g cell/h for mannose, xylose and glucose. Using a synthetic softwood prehydrolysate medium, in which the mass ratio of mannose:xylose:glucose was approx. 1.0:0.6:0.4 (total sugar conc'n 4.5 %), the sp. productivities associated with glucose and xylose metabolism were decreased by about 50 % and 75 % respectively, whereas mannose metabolism appeared unaffected by the presence of the other sugars. In all cases, the sugar-to-ethanol conversion efficiency was >90 % of theoretical maximum

Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.

Recombinant E. coli B (pLOI297) produced ethanol from a nutrient-supplemented, newsprint prehydrolysate medium, at about a 20% reduction in both yield and productivity compared to a synthetic softwood hemicellulose hydrolysate medium (lacking acetic acid). With pH controlled at 7, the sugar-to-ethanol conversion efficiency with the newsprint prehydrolysate was 74.5% of theoretical maximum. The final ethanol concentration was 14.6 g/L. Reduced ethanol yield was due to by-product formation, principally lactic acid. The specific rates of glucose, mannose and xylose utilization in the synthetic medium were 0.73, 0.42 and 0.22 g/g cell/h respectively. The ethanol yield from the pretreatment processing of newsprint is estimated at 85L per dry metric ton.

The hemicellulose component of corn cobs was completely hydrolyzed by treatment with dilute sulphuric acid. HPLC analysis showed the prehydrolysate to contain 36g/L xylose; 7g/L arabinose; 6g/L glucose and 4.2g/L acetic acid. The patented genetically engineered ethanologenEscherichia coli B (ATCC 11303 pLOI 297) exhibited high performance characteristics with a synthetic model medium in which the pH was controlled at 7 in order to minimize sensitivity to acetic acid. With the synthetic medium, fermentation was completed in 36h and the process yield was 0.49g/g, equivalent to 96% max. theoretical conversion efficiency. However, with the supplemented corn cob prehydrolysate medium, about 20% of the sugar remained unfermented after 8 days, with a conversion efficiency of

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. During prehydrolysis (acid hydrolysis or autohydrolysis of hemicellulose), acetic acid is formed as a consequence of the deacetylation of the acetylated moiety of hemicellulose. Recombinant Escherichia coli B (ATCC 11303), carrying the plasmid pLO1297 with pyruvate decarboxylase and alcohol dehydrogenase II genes from Zymomonas mobilis (CP4), converts xylose to ethanol with a product yield that approaches theoretical maximum. Although other pentose-utilizing microorganisms are inhibited by acetic acid, the recombinant E. coli displays a high tolerance for acetic acid. In xylose fermentations with a synthetic medium (Luria broth), where the pH was controlled at 7, neither yield nor productivity was affected by the addition of 10.7 g/L acetic acid. Nutrient-supplemented, hardwood (aspen) hemicellulose hydrolysate (40.7 g/L xylose) was completely fermented to ethanol (16.3 g/L) in 98 h. When the acetic acid concentration was reduced from 5.6 to 0.8 g/L, the fermentation time decreased to 58 h. Overliming, with Ca(OH)2 to pH 10, followed by neutralization to pH 7 with sulfuric acid and removal of insolubles, resulted in a twofold increase in volumetric productivity. The maximum productivity was 0.93 g/L/h. The xylose-to-ethanol conversion efficiency and productivity in Ca(OH)2-treated hardwood prehydrolysate, fortified with only mineral salts, were 94% and 0.26 g/L/h, respectively. The recombinant E. coli exhibits a xylose-to-ethanol conversion efficiency that is superior to that of other pentose-utilizing yeasts currently being investigated for the production of fuel ethanol from lignocellulosic materials.

Acetic acid acts to promote uncoupling withZymomonas. At pH 5, 36% of acetic acid is in the uncharged and undissociated form (HAc), which is able to permeate the plasma membrane. The transmembrane ΔpH drives the accumulation of acetic acid, which results in the acidification of the cytoplasm. The consequential increase in maintenance metabolism represents a diversion of energy that would otherwise be available for growth. At pH 5, the growth ofZ. mobilis (ATCC 29191) was 50% inhibited with 8.3 g/L acetic acid (50 mM HAc) and completely inhibited by 11 g/L. Addition of 6 g/L acetic acid caused the glucose-to-ethanol conversion efficiency to decrease from 98 to 90% of theoretical maximum. The growth yield coefficient for glucose was 50% decreased by 2.3 g/L acetic acid (13.5 mM HAc) from 0.036 to 0.018 g cell/g glucose. However, the specific (ethanol) productivity of batch cultures was enhanced by

A patent culture ofAlcaligenes faecalis var.myxogenes (ATCC 31749) synthesizes “curdlan” during the stationary phase of an aerobic batch fermentation following the depletion of assimilable nitrogen. Because this β-1,3-homoglucan exopolymer is water insoluble, the fermentation broth is of a relatively low viscosity, and consequently offers little resistance to oxygen transfer from gas to the liquid. However, the layer of insoluble exopolymer surrounding the cell mass offers a resistance to oxygen transfer from the liquid to the cell, thereby necessitating an unexpectedly high dissolved oxygen concentration for maximal productivity. The shear sensitive nature of this fermentation restricts improving oxygen transfer by increasing agitation intensity. The requirement for high volumetric oxygen transfer can be met by low shear designs with axial-flow impellers, providing gas dispersion is assisted by the use of sparging devices consisting of microporous materials. The specific respiration rates for growing and curdlan-producing stationary-phase culture were determined to be 7 and 2.7 mmol2/g cell/h, respectively. At a cell density of 3 g cell/L, the maximal rate of curdlan biosynthesis was about 100 mg/g cell/h, with a requirement for dissolved oxygen (DO) of 6.5 mg O2/L (86% air saturation at 30°C and 1 atm.). Whereas, at constant impeller speed, the volumetric oxygen transfer was improved both by increasing the air sparging rate and by using2-enriched air (30% O2), productivity was not consistently improved by operating at a twofold higher cell density and at aDO>6.5 mg O2/L. At higher cell densities, it would appear that shear must be increased to minimize culture clumping and assist in oxygen transfer to the cell.

The first objective of this study was the measurement of physical properties of P(3HB-co-3HV) copolymers with different (hydroxybutyrate) HB to (hydroxyvalerate) HV ratios produced by Bacillus cereus (TRY2) isolated from activated sludge. The 3HV PHBV copolymers were 0.05, 22.6, 39.2, 54.1, and 69.1 mol%, respectively. The second objective was to study possible waste-water treatment and production of PHAs at the same time by B. cereus (TRY2) and Pseudotnonas spp. (TOB17) (both were isolated from activated sludge), recombinant Bacillus DH5a, and a combination of the above three bacteria. The results were satisfactory; the maximum COD and TOC of the sewage sludge reduced were 53.5% and 67.5%, respectively.

An examination into the effect of bioreactor design on the production of β,1,3-glucan exopolysaccharide (“curdlan”) by selected patent cultures ofAlcaligenes faecalis andAgrobacterium radiobacter revealed that low shear mixing achieved through the replacement of the radial-flow flat-blade impellers that are commonly supplied in “standard” commercial bioreactors, by low shear (high-pumping) axialflow impellers, leads to an increase in thequality of the exopolymer recovered during the stationary-phase of batch fermentations. Whereas “Rushton turbine” impellers were effective in providing high rates of oxygen transfer necessary for high cell density fermentations, the high shear-to-flow ratio characteristic of this design produced a product of inferior quality, but with characteristics very similar to that of the commercially available “curdlan standard.” Curdlan is water insoluble, and consequently, the fermentation broth is of a relative low viscosity compared to other soluble microbial polysaccharides. Whereas curdlan does not constrain mass transfer from gas to liquid, it nevertheless offers a resistance to oxygen transfer from the liquid to the cell by virtue of the layer of insoluble exopolymer surrounding the cell mass, thereby necessitating an unexpectedly high dissolved oxygen concentration for maximal productivity. The requirement for high volumetric oxygen transfer can be met by low shear designs with axial-flow impellers, providing gas dispersion is assisted by the use of sparging devices consisting of microporous materials.

Escherichia coli B (ATCC 11303) carrying the PET operon on plasmid pLOI 297 converted hemicellulose hydrolysate to ethanol at an efficiency of 94% theoretical maximum, which is 15% better than the highest efficiency reported for pentose utilizing yeasts in a comparable system. Aspen prehydrolysate (APH), that had been produced by theBio-Hol Process using a Wenger extruder with SO2 as catalyst, was used as feedstock. The fermentation medium contained predominantly xylose (35g/L) with acetic acid present at about 6g/L. With the pH controlled at 7.0, this concentration of acetic acid was not inhibitory for growth or xylose fermentation. When the APH was fortified with nutrients (tryptone and yeast extract), the recombinant (inoculated at 0.5 g dry wt/L) converted 100% of the xylose to ethanol with a volumetric productivity of 0.29 g/L/hr. Overliming the APH with Ca(OH)2, followed by neutralization to pH 7 with sulphuric acid and removal of the insolubles, resulted in a 2-fold increase in productivity. The max. productivity was 0.76 g/L/hr. The productivity in Ca(OH)2-treated APH, fortified with only mineral salts, was 0.26 g/L/hr.

The performance of Thermoanaerobacter ethanolicus was evaluated in continuous culture with media containing concentrations of xylose (8 to 20 g/liter) greater than those previously reported. The ethanol yield declined from to 0.42 to 0.29 g of ethanol per g of xylose consumed when input xylose was increased from 4 to 20 g/liter. Yields of both total C 2 and C 3 products from consumed xylose and of cell biomass from ATP produced declined as the input xylose concentration was increased, which was not the case when glucose was the substrate. This suggested that yeast extract functioned as a significant energy and carbon source for cells in fermentations of xylose but not of glucose. The feasibility of this interpretation was confirmed by (i) the calculation of the products theoretically obtainable from yeast extract and (ii) the observation of significant quantities of fermentation products in inoculated sugar-free media. Markedly different patterns of metabolism for the two sugar substrates were also evidenced by the cell yield for glucose being twice that of xylose at elevated sugar concentrations. It was noted that caution must be exerted when results obtained at low xylose concentrations are extrapolated to predict those which can be obtained at higher concentrations.

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most impor tant characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC19 /PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. col i HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor cul ture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatogra phy, proton nuclear magnetic resonance spectroscopy, and differential scan ning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applica tions in industry and medicine.

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. logen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion, Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The “Iogen Process” for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wild-type Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.

Polyhydroxyalkanoates, biodegradable plastics with the desired physical and chemical properties of conventional synthetic plastics, are extensively investigated. In this study, specific bacterial strains produced specific copolymers from food waste. Copolymers of HB and HV (poly[3-hydroxybutyrate-co-3-hydroxyvalerate]) were obtained using various ratios of butyric acid (C4) and valeric acid (C5) as carbon sources. The C4 to C5 ratio affected the melting points of the copolymers. Melting and glass transition temperatures and many other thermal properties are important parameters relative to in-service polymer applications. Higher ratios of butyrate to valerate gave higher melting points. When a mixed culture of activated sludge was employed to produce copolymers using food wastes as nutrients, the obtained copolymers showed various monomer compositions. Copolymers with a higher portion of HV were obtained using soy waste; copolymers with less HV were obtained using malt wastes. Pure strains, (i.e., Alcaligenes latus DSM 1122, and DSM 1124, Staphylococcus spp., Klebsiella spp.) produced specific copolymers from food waste. Only Klebsiella spp. produced different copolymers; the ratios of HB:HV were 93:7 and 79:21 from malt waste and soy waste, respectively. The other strains produced polymers of 100% HB. Selecting industrial food wastes as carbon sources can further reduce the cost of producing copolymers.

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN's biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200-250 degrees C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4cents/gal of ethanol. This work examined the growth and fermentation performance of xylose-utilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated as the "adapted" strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30 h, followed by CP4:pZB5 at 48 h, with corresponding volumetric productivities of 1.4 and 0.89 g/ (L x h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to ferment arabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of D - and L-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of DL-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, D-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. D-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.

Recombinant Zymomonas mobilis CP4:pZB5 was grown with pH control in batch and continuous modes with either glucose or xylose as the sole carbon and energy source. In batch cultures in which the ratio of the final cell mass concentration to the amount of sugar in the medium was constant (i.e., under conditions that promote “coupled growth”), maximum specific rates of glucose and xylose consumption were 8.5 and 2.1 g/(g of cell.h), respectively; maximum specific rates of ethanol production for glucose and xylose were 4.1 and 1.0 g/(g of cell.h), respectively; and average growth yields from glucose and xylose were 0.055 and 0.034 g of dry cell mass (DCM)/g of sugar, respectively. The corresponding value of γATp for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. γATp for the wild-type culture CP4 with glucose was 10.4 g of DCM/mol of ATP. For single substrate chemostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or “true” growth yield (max γ x/s) was calculated from Pirt plots as the inverse of the slope of the best-fit linear regression for the specific sugar utilization rate as a function of D, and the “maintenance coefficient” (m) was determined as the y-axis intercept. For xylose, values of max γ and m were 0.0417 g of DCM/g of xylose (γATp = 6.25) and 0.04 g of xylose/(g of cell.h), respectively. However, with glucose there was an observed deviation from linearity, and the data in the Pirt plot was best fit with a second-order polynomial in D. At D > 0.1/h, γATP = 8.71 and m = 2.05 g of glu/(g of cell.h) whereas at D < 0.1/h, γ ATP, = 4.9 g of DCM/mol of ATP and m = 0.04 g of glu / (g of cell.h). This observation provides evidence to question the validity of the unstructured growth model and the assumption that Pirt’s maintenance coefficient is a constant that is independent of the growth rate. Collectively, these observations with individual sugars and the values assigned to various growth and fermentation parameters will be useful in the development of models to predict the behavior of rec Zm in mixed substrate fermentations of the type associated with biomass-to-ethanol processes

This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the cofermentation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5¢/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L.h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell.h).

Xylose-fermenting recombinant Zymomonas mobilis has been proposed as a candidate biocatalyst for the production of fuel ethanol from cellulosic biomass and wastes. This study documents the effect of glucose on xylose utilization by recombinant Z. mobilis CP4:pZB5 using a nutrient-rich synthetic (pure sugar) hardwood dilute-acid prehydrolyzate medium containing 0.8% (w/v) glucose and 4% (w/v) xylose that was enriched with respect to xylose concentration within the range 6-10% (w/v) xylose. Supplementation with glucose to a final concentration of 2% (w/v) resulted in faster xylose utilization of both 6% and 8% xylose; however, higher levels of glucose supplementation (>2%) did not result in a decrease in the time required for fermentation of either 6% or 8% xylose. An improvement in the rate of 8% xylose utilization was also achieved through continuous glucose feeding in which the total glucose concentration was about 1.3% (w/v). This fed-batch experiment was designed to mimic the continuous supply of glucose provided by the cellulose saccharifying enzymes in a simultaneous saccharifying and cofermentation process. The upper limit ethanol concentration at which xylose utilization by recombinant Z. mobilis CP4:pZB5 is completely inhibited is about 5.5% (w/v) at pH 5 and >6% at pH 5.75. At pH 5.75, this level of ethanol was achieved with the following media of pure sugar mixtures (each containing the same sugar loading of 12% (w/v): 1. 6% xylose + 6% glucose; 2. 8% xylose + 4% glucose; and 3. 4% xylose + 8% glucose.

The continuous cofermentation performance of xylose-fermenting Zymomonas mobilis at 30°C and pH 5.5 was characterized using a pure-sugar feed solution that contained 8 g/L glucose and 40 g/L xylose. Successful chemostat start up resulted in complete utilization of glucose and greater than 85% utilization of xylose, but was only reproducibly achieved using initial dilution rates at or less than 0.04/h; once initiated, cofermentation could be maintained at dilution rates of 0.04 to 0.10/h. Whereas xylose and cell-mass concentrations increased gradually with increasing dilution rate, ethanol concentrations and ethanol yields on available sugars remained approximately constant at 20-22 g/L and 80-90% of theoretical, respectively. Volumetric and specific ethanol productivities increased linearly with increasing dilution rate, rising from approx 1.0 each (g/L/h or g/g/h) at a dilution rate of 0.04/h to approx 2.0 each (g/L/h or g/g/h) at a dilution rate of 0.10/h. Similarly, specific sugar-utilization rates increased from approx 2.0 g/g/h at dilution rate 0.04/h to approx 3.5 g/g/h at dilution rate of 0.10/h. The estimated values of 0.042 g/g for the maximum Z. mobilis cell-mass yield on substrate and 1.13 g/g/h for the minimum specific substrate utilization rate required for cellular maintenance energy are within the range of values reported in the literature. Results are also presented which suggest that long-term adaptation in continuous culture is a powerful technique for developing strains with higher tolerance to inhibitory hemicellulose hydrolyzates.

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-O-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) by Escherichia coli B (ATCC 11303) and two different ethanologenic recombinants--a strain in which pet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-type E. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.

The ethanologenic bacterium Zymomonas mobilis has been demonstrated to possess several fermentation performance characteristics that are superior to yeast. In a recent survey conducted by the National Renewable Energy Laboratory (NREL), Zymomonas was selected as the most promising host for improvement by genetic engineering directed to pentose metabolism for the production of ethanol from lignocellulosic biomass and wastes. Minimization of costs associated with nutritional supplements and seed production is essential for economic large-scale production of fuel ethanol. Corn steep liquor (CSL) is a byproduct of corn wet-milling and has been used as a fermentation nutrient supplement in several different fermentations. This study employed pH-controlled batch fermenters to compare the growth and fermentation performance of Z. mobilis in glucose media with whole and clarified corn steep liquor as sole nutrient source, and to determine minimal amounts of CSL required to sustain high-performance fermentation. It was concluded that CSL can be used as a cost-effective single-source nutrition adjunct for Zymomonas fermentations. Supplementation with inorganic nitrogen significantly reduced the requirement for CSL. Depending on the type of process and mode of operation, there can be a significant contribution of nutrients from the seed culture, and this would also reduce the requirement for CSL. Removal of the insolubles (40% of the total solids) from CSL did not detract significantly from its nutritional effectiveness. On an equal-volume basis, clarified CSL was 1.33 times more "effective" (in terms of cell mass yield and fermentation time) than whole CSL. For fermentations at sugar loading of5% (w/v), the recommended level of supplementation with clarified CSL is 1.0% (v/v). Based on CSL at US $50/t, the cost associated with using clarified CSL at 1.0% (v/v) is 88 cents/1000 L of medium and 5.3 cents/gal of undenatured ethanol for fermentation of 10% (w/v) glucose. This cost compares favorably to estimates for using inorganic nutrients. The cost impact is reduced to 3.1 cents/gal if there is a byproduct credit for selling the insolubles as animal feed at a price of about US $100/t. Therefore, the disposition of the CSL insolubles can significantly impact the calculations of cost associated with the use of CSL as a nutritional adjunct in large-scale fermentations.