Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugard-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 ofZymomonas 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 recombinantZ. 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.     

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.     

The effect of glucose on high-level xylose fermentations by recombinant Zymomonas in batch and fed-batch fermentations

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

1. 6% xylose+6% glucose;
2. 8% xylose+4% glucose; and
3. 4% xylose+8% glucose.

At the level of inoculum used in this study, complete fermentation of the 12% sugar mixtures required 2–3 d (equivalent to a volumetric ethanol productivity of 0.83–1.25 g ethanol/L.h). The sugar-to-ethanol conversion efficiency was 94–96% of theoretical maximum.     

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 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.     

Fractionation of Cynara cardunculus (cardoon) biomass by dilute-acid pretreatment

Cynara cardunculus L. (cardoon) is a Mediterranean perennial herb offering good potential as substrate for sustainable production of bioethanol. In this work the first approach to the study of dilute-acid pretreatment of cardoon biomass for biological conversion was made. The influence of temperature (160-200 degrees C), acid concentration (0-0.2% [w/w]), and solid concentration (5-10% [w/v]) in the formation of free sugars and sugar decomposition products in the prehydrolyzate was studied using a response surface methodology. Results show a negative interaction effect between acid concentration and temperature in xylose recovery yield in prehydrolyzate, whereas dry matter concentration does not exert a significant effect. Xylose recovery yield reaches a maximum of about 80% of the content in dry untreated raw material at 180 degrees C and 0.1 or 0.2% acid addition. At these conditions the ratio of monomers found in prehydrolyzate in relation to total sugar yield for xylose is close to 100%. Furfural concentration, the major furan determined in the prehydrolyzate, increases as pretreatment severity rises. Maximum furfural yield of 4.2 g/100 g dry untreated raw material was found at 200 degrees C and 0.2% acid concentration. The yield of furfural at the conditions in which maximum xylose recovery is attained is substantially lower, less than 2 g/100 g dry untreated raw material. This fact supports the idea of using moderate temperatures in dilute-acid processes, which at the same time provides reasonably high sugar recovery yield and avoids high inhibitory products formation.     

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.     

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).     

Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5)

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in κ-carrageenan beads having a diameter of 1.5–2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2–4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5–15.3 g/L·h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars. This article has been authored by a contractor of the US go vernment under contract DE-AC05-96OR22464. Accordingly, the US government retains a nonexclusive, royaltyfree license to publish or reproduce the published form of the contribution, or allow others to do so, for US government purposes.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

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.

     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Influence of inhibitory compounds and minor sugars on xylitol production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis>

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3–6.5 g/L), xylose (60.1–92.1 g/L), or arabinose (5.9–9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0–5.0 g/L), hydroxymethylfurfural (0.01–0.30 g/L), acetic acid (0.5–3.0 g/L), and vanillin (0.5–3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto-xylitol bioconversion from complex xylose media from lignocellulosic materials.     

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.     

Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon material balances

Xylitol production by Debaryomyces hansenii NRRL Y-7426 was performed on synthetic medium varying the initial xylose concentration between 50 and 300 g/L. The experimental results of these tests were used to investigate the effect of substrate level on xylose consumption by this yeast. Satisfactory values of product yield on substrate (0.74–0.83 g/g) as well as volumetric productivity (0.481–0.694 g/L·h) were obtained over a wide range of xylose levels (90–200 g/L), while a worsening of kinetic parameters took place at higher concentration, likely due to a substrate inhibition phenomenon. The metabolic behavior of D. hansenii was studied, under these conditions, through a carbon material balance to estimate the fractions of xylose consumed by the cell for different activities (xylitol production, biomass growth, and respiration) during the lag, exponential, and stationary phases.     

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.     

Fermentation Kinetics for Xylitol Production by a Pichia stipitis d-Xylulokinase Mutant Previously Grown in Spent Sulfite Liquor

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3Δ) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h⁻¹). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g_xylose/g_cel h) and xylitol production (0.059 g_xylitol/g_cel h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized. Prepared for 29th Symposium on Biotechnology for Fuels and Chemicals.     

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.     

High-yield fermentation of pentoses into lactic acid

Lactobacillus species capable of fermenting glucose are generally incapable of utilizing xylose for growth or fermentation. In this study, a novel aspect of a well-known Lactobacillus strain, L. casei subsp. rhamnous (ATCC 10863), was uncovered: it can ferment xylose as efficiently as glucose. This strain is a registered organism, extremely stable on long-term operation. Fermentation by this strain is characterized by an initial lag phase lasting 24–72 h before xylose consumption takes place. The yield (grams/gram) of lactic acid from xylose is in excess of 80% with initial volumetric productivity of 0.38 g/(L-h). Acetic acid is the primary byproduct formed at the level of about 10% of the lactic acid. In addition to xylose, it can ferment all other minor sugars in hemicellulose except arabinose. Subjected to mixed sugar fermentation, this strain consumes glucose first, then mannose, followed by almost simultaneous utilization of xylose and galactose. It shows high tolerance for lactic acid as well as extraneous toxins. It can ferment the mixed sugars present in acid-treated hydrolysate of softwood, giving yields similar to that of pure sugar but at a slower rate. Author to whom all correspondence and reprint requests should be addressed.     

Minerals and Organic Nitrogen Present in Grape Marc Hydrolyzates Enhance Xylose Consumption by <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis>

This work deals with the nutritional evaluation of grape marc hydrolyzates as fermentation medium for Lactobacillus pentosus. Usually, the fermentation of xylose and arabinose in the presence of glucose remains a primary obstacle for economical biomass conversion. The few microorganisms that can grow simultaneously on both pentose and hexose sugars contained in lignocellulosic feedstocks typically grow slowly and demonstrate marginal yields and productivities. Moreover, lignocellulosic hydrolyzates contain phenolic compounds and other components originated by the degradation of sugars that can inhibit lactic acid fermentation. However, in this case, grape marc hydrolyzates not only did not need a detoxification stage, but it also improved the xylose consumption by Lactobacillus pentosus with a faster and more efficient conversion of hemicellulosic sugars compared with synthetic media. After analysis of grape marc hydrolyzates, it was observed that minerals such as K (2,707 mg/L), Ca (3,681 mg/L), and Mg (198.5 mg/L) are present in higher concentration than those found in the general medium of Lactobacillus (1,705 mg/L of K, 58.3 mg/L of Ca, and 27.0 mg/L of Mg). Moreover, grape marc hydrolyzates contain an additional source of nitrogen (9.2 g/L) which, together with their elevated mineral concentration, improved lactic acid fermentation compared with synthetic media.