Cellulosic fuel ethanol

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.     

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 xyloseutilizing, 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 wasmarkellyslower, 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 continous ferm entation with nutrient-rich synthetic corn stover medium over the dilution range 0.03–0.06/h, with a maximum provess ethanol yield at D=0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L·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·h). Under the same operating conditions, rec Zm Zm 4: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 antiboitic resistance genes makes the chromosomal integrant C25 more com patible with current regulatory specifications for biocatalysts in large-scale commercial operations.     

Ethanol production usingZymomonas mobilis in a cross-linked immobilized cell reactor

The bacteriumZymomonas mobilis may be utilized to produce ethanol from glucose in a cross-linked immobilized cell reactor. Reactor startup is much more rapid with cross-linkedZymomonas than with the yeastSaccharomyces cerevisiae. Volumetric ethanol productivities (based on liquid holdup) three times those obtained with cross-linked yeast, and comparable to those obtained withZymomonas immobilized by other methods, are possible.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Ethanol fermentation in a tower fermenter using self-aggregatingSaccharomyces uvarum

A self-aggregating strain ofSaccharomyces uvarum (U4) was used as a biocatalyst to carry out continuous ethanol fermentation in a tower fermentor equipped with a cell separator. Cell aggregates (2–3 mm) formed a stable packed bed in the fermentor, and the cell separator retained yeast cells effectively. Corn steep liquor was used as a nitrogen source for the fermentation of corn syrup and black strap molasses. An ethanol productivity of 54 g/L/h was reached using corn syrup at a dilution rate of 0.7/h, and sugar concentration in the feed was 15% (w/v). For molasses fermentation, an ethanol productivity of 22 g/L/h was obtained at a dilution rate of 0.7/h, and sugar concentration in the feed was 12.5% (w/v). Ethanol yields obtained from tower fermentation are higher than those obtained from flask fermentation (96% for corn syrup fermentation and 92% for molasses fermentation). No significant loss in fermentation activity was observed after 3 mo of operation.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.     

Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol

Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C, using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 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 ofZymomonas 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 48 h 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.

     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Continuous fermentation studies with xylose-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 coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/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).     

A simultaneous sucrose bioconversion into ethanol and levan byZymomonas mobilis

Two biotechnological systems were developed for sucrose conversion into levan and ethanol withZymomonas mobilis, ensuring a 66.7% transfer of substrate carbon in a batch and 61% carbon transfer in a continuous culture. The effect of glucose, ethanol, and medium pH on sucrose conversion byZ. mobilis was studied. The addition of ethanol to the fermentation medium, in the final conc. of 100 g/L, uncoupled levan synthesis from ethanol fermentation. For a continuous culture, the most efficient conversion of substrate carbon into levan was reached at pH 4.8, giving 64.2 g/L levan, with the levan yield of 0.22 g/g and the productivity of 3.2 g/L/h.     

Ethanol production from jerusalem artichoke by inulinase and zymomonas mobilis

Ethanol production from Jerusalem artichoke was studied using inulinase and Z.mobilis by simultaneous saccharification and fermentation (SSF) process. The SSF process showed higher ethanol yield and productivity than the acid or enzymatic prehydrolyzed two-step process. The optimum temperature and inulinase concentration for SSF were 35°C and 0.25% (v/w, 4.4 units/g of sugar), respectively. In order to operate the SSF process in a continuous mode, inulinase and Z.mobilis cells were coimmobilized in alginate beads, using chitin as a matrix for enzyme immobilization. The maximum ethanol productivity of the continuous SSF process was 55.1 g/L/h, with 55% conversion yield. At the conversion yield of 90%, the productivity was 32.7 g/L/h. The continuous SSF system could be operated stably over 2 wk with an ethanol concentration of 48.6 g/L (95% of theoretical yield).     

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°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 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, 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 asthe “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 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·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 fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Enhancing Clostridial Acetone-Butanol-Ethanol (ABE) Production and Improving Fuel Properties of ABE-enriched Biodiesel by Extractive Fermentation with Biodiesel

The extractive acetone–butanol–ethanol (ABE) fermentations of Clostridium acetobutylicum were evaluated using biodiesel as the in situ extractant. The biodiesel preferentially extracted butanol, minimized product inhibition, and increased production of butanol (from 11.6 to 16.5 gL⁻¹) and total solvents (from 20.0 to 29.9 gL⁻¹) by 42% and 50%, respectively. The fuel properties of the ABE-enriched biodiesel obtained from the extractive fermentations were analyzed. The key quality indicators of diesel fuel, such as the cetane number (increased from 48 to 54) and the cold filter plugging point (decreased from 5.8 to 0.2 °C), were significantly improved for the ABE-enriched biodiesel. Thus, the application of biodiesel as the extractant for ABE fermentation would increase ABE production, bypass the energy intensive butanol recovery process, and result in an ABE-enriched biodiesel with improved fuel properties.     

Influence of oxygen availability on cell growth and xylitol production by Candida guilliermondii

Oxygen availability is the most important environmental parameter in the production of xylitol by yeasts, directly affecting yields and volumetric productivity. This work evaluated the cell behavior in fermentations carried out with different dissolved oxygen concentrations (0.5–30.0% of saturation), as well as a limited oxygen restriction (0% of saturation), at several oxygen volumetric transfer coefficients (12 ≤ k _L a ≤ 70 h⁻¹). These experiments allowed us to establish the specific oxygen uptake rate limits to ensure high yields and volumetric productivity. When oxygen availability was limited, the specific oxygen uptake rate values were between 12 and 26 mg of O₂/of g cell·h, resulting in a yield of 0.71 g of xylitol/xylose consumed, and 0.85 g/[L·h] for the volumetric productivity. According to the results, the effective control of the specific oxygen uptake rate makes it possible to establish complete control over this fermentative process, for both cell growth and xylitol production.     

Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass

Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLO1297, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but noselective antibiotic. An average of 93 and 95% of the FBR4 and FBR5 cells, respectively, maintained pLO1297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from stock in pH-controlled batch fermentations with 10% (w/v) xylose. Fermentation results were similar for all the cultures. Fermentations were completed within 60 h and ethanol yields were 86–92% of theoretical. Maximal ethanol concentrations were 3.9–4.2% (w/v). The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% (w/v) total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, 90% of the maximum obtainable. Author to whom all correspondence and reprint requests should be addressed. Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA im plies no approval of the product to the exclusion of others that may also be suitable.     

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 Y_ATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. Y_ATP for the wild-type culture CP4 with glucose was 10.4g of DCM/mol of ATP. For single substratechem ostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or “true” growth yield (max Y_a/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 Y _s/s and m were 0.0417g of DCM/g of xylose (Y_ATP=6.25) and 0.04g 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, Y_ATP=8.71 and m=2.05g of glu/(g of cell…h) whereas at D<0.1/h, Y_ATP=4.9g of DCM/mol of ATP and m=0.04g 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 in dependent of the growth rate. Collectively, these observations with individual sugars and the values assign ed 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.     

Fermentation performance characteristics of a prehydrolyzate-adapted xylose-fermenting recombinant Zymomonas in batch and continuous fermentations

Long-term (149 d) continuous fermentation was used to adapt a xylose-fermenting recombinant Zymomonas mobilis, strain 39676:pZB 4L, to conditioned (overlimed) dilute-acid yellow poplar hemicellulose hydrolyzate (“prehydrolyzate”). An “adapted” variant was isolated from a chemostat operating at a dilution rate of 0.03/h with a 50% (v/v) prehydrolyzate, corn steep liquor, and sugar-supplemented medium, at pH 5.75. The level of xylose and glucose in the medium was kept constant at 4% (w/v) and 0.8% (w/v), respectively. These sugar concentrations reflect the composition of the undiluted hardwood prehydrolyzate. The level of conditioned hardwood prehydrolyzate added to the medium was increased in 5% increments startingata level of 10%. At the upper level of 50% prehydrolyzate, the acetic-acid concentration was about 0.75% (w/v). The adapted variant exhibited improved xylose-fermentation performance in a pure-sugar, synthetic hardwood prehydrolyzate medium containing 4% xylose (w/v), 0.8% (w/v) glucose, and acetic acid in the range 0.4–1.0% (w/v). The ethanol yield was 0.48–0.50 g/g; equivalent to a sugar-to-ethanol conversion efficiency of 94–96% of theoretical maximum. The maximum growth yield and maintenance energy coefficients were 0.033 g dry cell mass (DCM)/g sugars and 0.41 g sugars/g DCM/h, respectively. The results confirm that long-term continuous adaptation is a useful technique for effecting strain improvement with respect to the fermentation of recalcitrant feedstocks.