Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.     

Xylose reductase activity of Candida guilliermondii during xylitol production by fed-batch fermentation

Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 2^4-1 fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S _F ) and in the fermentor (S ₀), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future. The best experimental result was achieved at S _F =45 g/L, S ₀=40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81 U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L·h) and a xylose xylitol yield factor of 0.68 g/g.     

Preliminary kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugarcane bagasse hydrolysate for xylitol production

Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH₄)₂SO₄, 0.1 g/L of CaCl₂·2H₂O, and 20.0 g/L of rice bran at 35°C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K _M for XR and XD against substrates and cofactors were as follows: for XR, 6.4×10⁻² M (xylose) and 9.5×10⁻³ mM (NADPH); for XD, 1.6×10⁻¹ M (xylitol) and 9.9×10⁻² mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the V _max, some interference on the overall xylitol production by the yeast could be expected.     

Enhancement of 1,3-Propanediol production by cofermentation inEscherichia coli expressingKlebsiella pneumoniae dha regulon genes

1,3-Propanediol (1,3-PD) is an intermediate in chemical and polymer synthesis. We have previously expressed the genes of a biochemical pathway responsible for 1,3-PD production, thedha regulon ofKlebsiella pneumoniae, inEscherichia coli. An analysis of the maximum theoretical yield of 1,3-PD from glycerol indicates that the yield can be improved by the cofermentation of sugars, provided that kinetic constraints are overcome. The yield of 1,3-PD from glycerol was improved from 0.46 mol/mol with glycerol alone to 0.63 mol/mol with glucose cofermentation and 0.55 mol/mol with xylose cofermentation. The engineeredE. coli also provides a model system for the study of metabolic pathway engineering.     

Comparative energetics of glucose and xylose metabolism in ethanologenic recombinantEscherichia coli B

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 Y_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 (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.     

Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus

This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus. Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose. The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum. The carbon balance in the medium with glucose and xylose was virtually 100%. The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate. Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate. The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L·h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9–11 mmol/[L·h]). The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate.     

Fermentation of xylose into acetic acid by Clostridium thermoaceticum

For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation, d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities.     

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered saccharomyces yeast capable of cofermenting glucose and xylose

Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.     

Evaluation of recombinant strains of Zymomonas mobilis for ethanol production from glucose/xylose media

The fermentation characteristics of two recombinant strains of Zymomonas mobilis, viz. CP4 (pZB5) and ZM4 (pZB5), capable of converting both glucose and xylose to ethanol, have been characterized in batch and continuous culture studies. The strain ZM4 (pZB5) was found to be capable of converting a mixture of 65 g/L glucose and 65 g/L xylose to 62 g/L ethanol in 48h with a yield of 0.46 g/g. Higher sugar concentrations resulted in incompletexylose utilization (80h) presumably owing to ethanol inhibition of xylose assimilation or metabolism. The fermentation results with ZM4 (pZB5) show a significant improvement over results published previously for recombinant yeasts and other bacteria capable of glucose and xylose utilization.     

Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33)

Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400 (pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.     

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.     

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.     

Production of xylitol byCandida mogii from rice straw hydrolysate

The influence of aeration level, initial pH, initial cell concentration, and fermentation time on the xylitol production from rice straw hemicellulose hydrolysate byCandida mogii was studied. A multifactorial experimental design was adopted to evaluate this influence. A statistical analysis of the results showed that the aeration level and the initial pH had significant effects on yield factor, volumetric productivity, and xylose consumption. For the latter, fermentation time was also a significant variable. Based on the response surface methodology, models for the range investigated were proposed. The maximum values for the yield factor (Y_p/s) and volumetric productivity (Q_p) were, respectively, 0.71 g/g and 0.46 g(Lh).     

Diminished Respirative Growth and Enhanced Assimilative Sugar Uptake Result in Higher Specific Fermentation Rates by the MutantPichia stipitis FPL-061

A mutant strain ofPichia stipitis, FPL-061, was obtained by selecting for growth on L-xylose in the presence of respiratory inhibitors. The specific fermentation rate of FPL-061, was higher than that of the parent,Pichia stipitis CBS 6054, because of its lower cell yield and growth rate and higher specific substrate uptake rate. With a mixture of glucose and xylose, the mutant strain FPL-061 produced 29.4 g ethanol/L with a yield of 0.42 g ethanol/g sugar consumed. By comparison, CBS 6054 produced 25.7 g ethanol/L with a yield of 0.35 gJg. The fermentation was most efficient at an aeration rate of 9.2 mmoles O₂ L^-1 h^-1. At high aeration rates (22 mmoles O₂ L^-1 h^-1), the mutant cell yield was less than that of the parent. At low aeration rates, (1.1 to 2.5 O₂ L^-1 h^-1), cell yields were similar, the ethanol formation rates were low, and xylitol accumulation was observed in both the strains. Both strains respired the ethanol once sugar was exhausted. We infer from the results that the mutant, P.stipitis FPL-061, diverts a larger fraction of its metabolic energy from cell growth into ethanol production.     

Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.     

Activity of xylose reductase from Candida mogii grown in media containing different concentrations of rice straw hydrolysate

Xylose reductase (XR) activity was evaluated in extracts of Candida mogii grown in media containing different concentrations of rice straw hydrolysate. Results of X Ractivity were compared to xylitol production and a similar behavior was observed for these parameters. Highest values of specific production and productivity were found for xylose reductase (35 U/g of cell and 0.97 U/[g of cell·h], respectively) and for xylitol (5.63 g/g of cell and 0.13 g/[g of cell·h]) in fermentation conducted in medium containing 49.2 g of xylose/L. The maximum value of XR:XD ratio (1.82) was also calculated under this initial xylose concentration with 60 h of fermentation.     

An epimerasic activity on galactose induced by xylose inKluyveromyces sp

Production of galactose epimerase by anKluyveromyces sp. isolated from Kefir (dairy product) was investigated in batch culture. The microorganism was cultured in media with 1% galactose, 1% xylose, or 0.5% xylose plus 0.5% galactose, in Erlenmeyer flasks shaken at 200 rpm and maintained at 30°C. After 48 h, the biomass was harvested by centrifugation and permeated with 80% ethanol. Permeated cells were suspended in 0.1M sodium phosphate buffer pH 6.5. A part of this suspension was shaken for 17h at 140 rpm. The supernatant, free of cells, was separated. Partial characterization ofKluyveromyces sp. epimerase was carried out in the cellular suspension and the supernatant solution. Enzymatic activity, using galactose as substrate, was measured. The product of this reaction was measured by the use of glucose oxidase. The results indicated: (1) there was a strong effect of xylose on induction of epimerase activity; (2) the epimerase obtained was independent of the energetic cell activity; (3) the epimerase activity in whole cells was similar to the activity obtained from the supernatant; (4) epimerase showed a typical substrate-inhibition curve and dependence on magnesium; and (5) the best pH range was between 5.5 and 6.5 and the optimal temperature was 30°C.     

Cloning and expression of the gene for xylose isomerase fromThermus flavus AT62 inEscherichia coli

The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn²⁺, Co²⁺, and Mg²⁺ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.     

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that d-xylulokinase limits metabolic flux in recombinant S. cerevisiae.     

Microbial Production of 1,3-Propanediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> XJPD-Li under Different Aeration Strategies

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae XJPD-Li under different aeration strategies were investigated. In batch fermentation, the results showed that the final concentration of 1,3-PD and yield on glycerol were 13.44 g/l and 0.73 mol/mol under the anaerobic condition (N₂, 0.4 vvm), 11.55 g/l and 0.62 mol/mol without aeration, and 8.73 g/l and 0.47 mol/mol under the aerobic condition (air, 0.4 vvm), respectively. Under the aerobic condition, the yield of 1,3-PD on glycerol was the lowest, while the biomass (optical density at 650 nm) was the highest among these three conditions. In the fed-batch culture, the final concentration and the yield of 1,3-PD was 60.82 g/l and 0.61 mol/mol under the anaerobic condition (N₂, 0.4 vvm), 56.43 g/l and 0.53 mol/mol without aeration, and 65.26 g/l and 0.56 mol/mol under the aerobic condition. All these three conditions had good productivities of 1,3-PD, which were 3.35 g/l·h under the anaerobic condition (N₂, 0.4 vvm), 3.13 g/l·h without aeration, and 3.16 g/l·h under the aerobic condition within the initial 12 h.