Fermentation performance assessment of a genomically integrated xylose-utilizing recombinant of Zymomonas mobilis 39676.

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.

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate.

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.

Comparative energetics of glucose and xylose metabolism in recombinant Zymomonas mobilis.

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 YATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. YATP 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 Yx/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 Yx/s and m were 0.0417 g of DCM/g of xylose (YATP = 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, YATP = 8.71 and m = 2.05 g of glu/(g of cell.h) whereas at D < 0.1/h, YATP = 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.

Continuous fermentation studies with xylos-utilizing recombinant Zymomonas mobilis.

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 cents/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 (Dmax), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted Dmax from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), Dmax 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 Dmax 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 Dmax 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).

Improving fermentation performance of recombinant Zymomonas in acetic acid-containing media.

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.

Conditions that promote production of lactic acid by Zymomonas mobilis in batch and continuous culture.

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.

Continuous culture studies of xylose-fermenting Zymomonas mobilis.

The continuous cofermentation performance of xylose-fermenting Zymomonas mobilis at 30 degrees 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.

Fermentation of biomass-derived glucuronic acid by pet expressing recombinants of E. coli B.

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.

Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinant Zymomonas.

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.

Corn steep liquor as a cost-effective nutrition adjunct in high-performance Zymomonas ethanol fermentations.

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 of >5% (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.

Factors contributing to the loss of ethanologenicity of Escherichia coli B recombinants pL0I297 and KO11.

To be economic and to be compatible with modern continuous bioconversion systems, it is imperative that the process organism exhibits an extremely high degree of stability. In the case of ethanol production from lignocellulosic biomass, functional stability of the potential process biocatalyst can be assessed in terms of the capacity to sustain high-performance fermentation during the continuous fermentation of biomass-derived sugars. This investigation employed glucose- or xylose-limited chemostat culture to examine the functional stability of two patented, genetically engineered E. coli-namely E. coli B (ATCC 11303) carrying the Zymomonas genes for pyruvate decarboxylase and alcohol dehydrogenase II on a multicopy plasmid pLOI297 and a chromosomal pet integrant of strain 11303, designated as strain KO11. Both recombinants carry markers for antibiotic resistance and have been reported to exhibit genetic stability in the absence of antibiotic selection. Chemostats were fed with Luria broth (LB) (with 25 g/L sugar) at a dilution rate of 0.14 and 0.07/h when the feed medium was glucose-LB and xylose-LB, respectively. They pH was controlled at 6.3. With glucose, both recombinants exhibited a rapid loss of ethanologenicity even when selection pressure was imposed by the inclusion of antibiotics in the feed medium. With strain KO11, increasing the concentration of chloramphenicol from 40 to 300 mg/L, resulted in a retardation in the rate of loss of ethanologenicity, but it did not prevent it. Under xylose limitation, the plasmid-bearing recombinant appeared to be stabilized by antibiotics, but this did not reflect genetic stability, since the slower-growing revertant was washed out at a dilution rate of 0.07/h. With both recombinants, interpretation of functional stability with xylose was complicated by the inherent ethanologenicity associated with the host culture. Based on an average cost for large bulk quantities of antibiotics at $55/kg and an amendment level of 40 g/m3, the estimated economic impact regarding the potential requirement for operational stabilization by antibiotics in a plant operating in batch mode varied from a maximum of 29 cents/gal of E95 ethanol for antibiotic amendment of all fermentation media to a minimum of 0.45 cents/gal where antibiotics were used exclusively for the preparation of the inocula for every fourth batch fermentation cycle. The high degree of instability observed in these continuous fermentations does not auger well for the proposed potential industrial utility of these patented, genetically engineered constructs for the production of fuel ethanol from biomass and wastes.

The relationship between growth enhancement and pet expression in Escherichia coli.

The pet operon consists of genes coding for enzymes responsible for ethanol production and consists of pyruvate dehydrogenase and alcohol dehydrogenase II from the high-performance ethanologen Zymomonas mobilis. This article describes the physiological influence of pet expression in Escherichia coli B (ATCC 11303) in terms of growth rate and overall concentrations of cell mass and catabolic end products achieved under well-defined cultivation conditions that included constant pH and carbon (energy) limitation. Glucose, mannose, and xylose were used as substrates, because they represent the principal fermentable components of lignocellulosic biomass and because fermentation of these sugars involves different metabolic pathways. Two different types of ethanologenic recombinants were used-a strain in which pet expression was via a multicopy plasmid (pLO1297) and a chromosomal integrant, strain KO11. Under the condition of sugar substrate limitation, there was no growth enhancement by pet expression with either glucose or mannose. Whereas the host strain produced exclusively lactic acid from glucose and mannose, both recombinants produced mostly ethanol. Both the plasmid-carrying strain and the pet integrant exhibited slower growth compared to the host culture with glucose or mannose as fermentation substrate. With mannose, the plasmid recombinant grew appreciably slower than either the host culture or the recombinant KO11. Use of a magnesium-deficient medium produced different results with glucose since substrate and turbidometric measurements proved to be unreliable in terms of estimating overall biomass levels. At pH 6.3, pet expression improved overall biomass yield; but at pH 7.0, the cell yields exhibited by the plasmid recombinant and the host strain were the same. E. coli B did not grow well on xylose as sole carbon source. With xylose, pet expression increased the growth rate, but had no effect on the overall biomass yield. In comparing our observations with the reports of others, it was concluded that the effect of pet expression on growth of E.coli is dependent on several different biochemical, physiological, genetic, and environmental factors, which largely precludes a statement of generality regarding this phenomenon.

Studies on nutrient requirements and cost-effective supplements for ethanol production by recombinant E. coli.

This article describes a systematic study of the nutritional requirements of a patented recombinant ethanologenic Escherichia coli (11303:pLO1297) and provides cost-effective formulations that are compatible with the production of fuel ethanol in fermentations of lignocellulosic prehydrolysate characterized by high xylose conversion efficiency. A complex and nutrient-rich laboratory medium, Luria broth (LB), provided the benchmark with respect to fermentation performance standard. Xylose fermentation performance was assessed in terms of the target values for operational process parameters established by the US National Renewable Energy Laboratory (NREL)-final ethanol concentration (25 g/L), xylose-to-ethanol conversion efficiency (90%), and volumetric productivity (0.52 g/L.h). Biomass prehydrolysates that are rich in xylose also contain acetic acid, and in anticipation of a need to reduce acetic acid toxicity, the fermentors were operated with a pH control set-point of 7.0 Growth and fermentation in the minimal defined salts (DS) medium was only about 15% compared to the reference medium. Amendment of the minimal medium containing 6 wt% xylose with both vitamins and amino acids resulted in improved growth, but the volume productivity (0.59 g/L.h) was still only about 54% of that with LB (1.1g/L.h). Formulations directed at cost reduction through the use of less expensive commercial complex nutritional supplements were within 90% of the NREL process target with respect to yield and provided a productivity at about 80% of the LB medium, but were not economical. Corn steep liquor (CSL) at about 7-8 g/L was shown to be a complete source of nutritional requirements and supported a fermentation performance approaching that of LB. At a cost of CSL of $50/t(dry wt), the economic impact of using this amount CSL as the sole nutritional supplement in a cellulosic ethanol plant was estimated to be about 4 cents/gal of ethanol.

Comparative energetics of glucose and xylose metabolism in ethanologenic recombinant Escherichia coli B.

This study compared the anaerobic catabolism of glucose and xylose by a patented, recombinant ethanologenic Escherichia 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 gamma ATP 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 (Yx/s, g dry wt cells/g sugar) should be proportional to the net energy yield from sugar metabolism. The value of Yx/s was reduced, on average, by about 50% (from 0.096 g/g glu to 0.51 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.

Effect of oxygen on ethanol production by a recombinant ethanologenic E. coli.

Escherichia coli strain B, bearing the pet plasmid pLO1297, and the wild-type culture, lacking the plasmid, responded to aeration of the complex medium by an approximate three- and fourfold increase in both growth rate and growth yield with glucose and xylose, respectively. At a relatively low oxygen transfer rate (8 mmol O2/L/h), the sugar-to-ethanol conversion efficiency exhibited by the recombinant strain decreased 40% and 30% for glucose and xylose, respectively. At a high aeration efficiency (100 mmol O2/L/h), the ethanol yield with respect to xylose was 0.15 g/g for the recombinant and 0.25 g/g for the culture lacking the plasmid. These observations suggest that oxygen reduces the ethanologenic efficiency of recombinant E. coli by diverting carbon to growth and end products other than ethanol. Previous observations, by others, on the effect of oxygen on ethanologenic recombinant E. coli were made with different strains bearing different plasmids. In addition to the possibility of strain and plasmid specificity, the results of this study suggest that previous conclusions were influenced by the particular environmental conditions imposed on the culture, including poor aeration efficiency and lack of pH control.

Relative rates of sugar utilization by an ethanologenic recombinant Escherichia coli using mixtures of glucose, mannose, and xylose.

The volumetric rates of glucose (G), mannose (M), and xylose (X) utilization by recombinant Escherichia coli B (pLO1297) were compared in pH-stat batch fermentations with Luria broth containing various combinations of two of these sugars at differing mass ratios. Using single substrate media, the rates of glucose, mannose, and xylose utilization were 3.0, 0.8, and 1.5 g/L/h, respectively. With all two substrate media, hexose and pentose sugars were consumed simultaneously. At a mass ratio of 2:1 (M or X:G), the rate of glucose utilization was reduced to 1.7 and 1.2 g/L/h by mannose and xylose, respectively. In media containing glucose and xylose, the rate of xylose utilization was inhibited when the glucose component exceeded about 40% of the total sugar mass in the medium. At a mass ratio of 2M:1X, mannose did not inhibit the rate of xylose utilization. At a mass ratio of 1:2 (G or X:M), the rate of mannose utilization was unaffected by either glucose or xylose. Synthetic media containing a mixture of hexose and pentose sugars were formulated to mimic different biomass hemicellulose hydrolysates. Relative to the rate in a single substrate medium, the respective rates of glucose and xylose utilization were 70% (2.1 g/L/h) and 40% (0.6 g/L/h) in a synthetic softwood prehydrolysate (SW) medium with a total reducing sugar (TRS) content of 45.7 g/L (20 wt% glucose, 30% xylose, and 50% mannose). However, the rate of mannose utilization in the SW medium was not inhibited. The respective rates of glucose and xylose utilization were 30% (0.9 g/L/h) and > 90% (1.4 g/L/h) in a synthetic crop residue prehydrolysate (CR) medium with a TRS content of 46.9 g/L (10 wt% glucose, 73% xylose, and 17% arabinose). Based on the results of this study, we suggest that the apparent "preference" for fermentation of hexose sugars by recombinant E. coli may be owing to the decreased rate of xylose transport caused by hexose sugars. Glucose is a more potent modulator of xylose utilization than mannose, but since xylose affects the rate of glucose utilization, this study also points to the importance of the concentration of the different sugars in terms of the relative rates of utilization by recombinant E. coli.

Effects of pH and acetic acid on glucose and xylose metabolism by a genetically engineered ethanologenic Escherichia coli.

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.

Production of ethanol from pulp mill hardwood and softwood spent sulfite liquors by genetically engineered E. coli.

Although lignocellulosic biomass and wastes are targeted as an attractive alternative fermentation feedstock for the production of fuel ethanol, cellulosic ethanol is not yet an industrial reality because of problems in bioconversion technologies relating both to depolymerization and fermentation. In the production of wood pulp by the sulfite process, about 50% of the wood (hemicellulose and lignin) is dissolved to produce cellulose pulp, and the pulp mill effluent ("spent sulfite liquor" SSL) represents the only lignocellulosic hydrolysate available today in large quantities (about 90 billion liters annually worldwide). Although softwoods have been the traditional feedstock for pulping operations, hardwood pulping is becoming more popular, and the pentose sugars in hardwood SSL (principally xylose) are not fermented by the yeasts currently being used in the production of ethanol from softwood SSL. This study assessed the fermentation performance characteristics of a patented (US Pat. 5,000,000), recombinant Escherichia coli B (ATCC 11303 pLOI297) in anaerobic batch fermentations of both nutrient-supplemented soft and hardwood SSL (30-35 g/L total reducing sugars). The pH was controlled at 7.0 to maximize tolerance to acetic acid. In contrast to the high-performance characteristics exhibited in synthetic media, formulated to mimic the composition of softwood and hardwood SSL (yield approaching theoretical maximum), performance in SSL media was variable with conversion efficiencies in the range of 67-84% for hardwood SSL and 53-76% for softwood SSL. Overlimiting treatment of HSSL, using Ca(OH)2, improved overall volumetric productivity two- to sevenfold to a max of 0.42 g/L/h at an initial cell loading of 0.5 g dry wt/L. A conversion efficiency of 92% (6.1 g/L ethanol) was achieved using diluted Ca(OH)2-treated hardwood SSL. The variable behavior of this particular genetic construct is viewed as a major detractant regarding its candidacy as a biocatalyst for SSL fermentations.

Effect of acetic acid on xylose conversion to ethanol by genetically engineered E. coli.

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.

Ethanol production by recombinant Escherichia coli carrying genes from Zymomonas mobilis.

Efficient utilization of lignocellulosic feedstocks offers an opportunity to reduce the cost of producing fuel ethanol. The fermentation performance characteristics of recombinant Escherichia coli ATCC 11303 carrying the "PET plasmid" (pLOI297) with the lac operon controlling the expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes cloned from Zymomonas mobilis CP4 (Alterthum & Ingram, 1989) were assessed in batch and continuous processes with sugar mixtures designed to mimic process streams from lignocellulosic hydrolysis systems. Growth was pseudoexponential at a rate (generation time) of 1.28 h at pH 6.8 and 1.61 h at pH 6.0. The molar growth yields for glucose and xylose were 17.28 and 7.65 g DW cell/mol, respectively (at pH 6.3 and 30 degrees C), suggesting that the net yield of ATP from xylose metabolism is only 50% compared to glucose. In pH-stat batch fermentations (Luria broth with 6% sugar, pH 6.3), glucose was converted to ethanol 4-6 times faster than xylose, but the glucose conversion rate was much less than can be achieved with comparable cell densities of Zymomonas. Sugar-to-ethanol conversion efficiencies in nutrient-rich, complex LB medium were near theoretical at 98 and 88% for glucose and xylose, respectively. The yield was 10-20% less in a defined-mineral-salts medium. Acetate at a concentration of 0.1M (present in lignocellulosic hydrolysates from thermochemical processing) inhibited glucose utilization (about 50%) much more than xylose, and caused a decrease in product yield of about 30% for both sugars. With phosphate-buffered media (pH 7), glucose was a preferred substrate in mixtures with a ratio of hexose to pentose of 2.3 to 1. Xylose was consumed after glucose, and the product yield was less (0.37 g/g). Under steady-state conditions of continuous culture, the specific productivity ranged from 0.76-1.24 g EtOH/g cell/h, and the maximum volumetric productivity, 2.5 g EtOH/L/h, was achieved with a rich complex LB medium (glucose) at pH 6.0 (30 degrees C) and ethanol at 1.63% (v/v). Growth and fermentation were poor in a buffered-wood (aspen) "hemicellulose hydrolysate" containing 4% xylose and 0.1M acetate with added thiamine and mineral salts.