Enhanced rate of ethanol production from D-xylose using recycled or immobilized cells ofPachysolen tannophilus

Summary Enhanced rates of ethanol production byPachysolen tannophilus from D-xylose were obtained by performing the fermentation with recycled cells in suspension culture or immobilized in a Ca-alginate gel. Fermentation under these conditions did not require aeration. Increasing temperature from 30 to 37°C enhanced the amount of ethanol produced in 24 hours from the recycled or the immobilized cells.Issued as National Research Council of Canada Publication Number 19475.     

Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus

The yeast Pachysolen tannophilus was found to be capable of converting D-xylose to ethanol. Batch cultures initially containing 50 g/L D-xylose yielded 0.34 g of ethanol per gram of pentose consumed. Aerobic conditions were required for cell growth but not for ethanol production. Both alcohol formation and growth were optimum when incubation temperature was 32 degrees C, when pH was near 2.5, and when D-xylose and ethanol concentrations did not exceed 50 and 20 g/L, respectively.     

Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus

Summary The yeast Pachysolen tannophilus has been identified as being able to convert an aldopentose, D-xylose, into ethanol. A feature of the conversion is that it can take place under aerobic conditions.Issued as N.R.C.C. Publication No. 19095.     

Continuous conversion of D-xylose to ethanol by immobilized pachysolen tannophilus

The yeast Pachysolen tannophilus was entrapped in calcium alginate beads to ferment D-xylose on a continous basis in the presence of high cell densities. Experimental operating variables included the feed D-xylose concentration, the dilution rate, and the fermentor biomass concentration. Under favorable operating conditions, cultures retained at least 50% of their initial productivity after 26 days of operation. The specific ehanol production rate was dependent on the substrate level in the fermentor, passing through an optimum when the D-xylose concentration was between 28 and 35 g/L. Consequently, reactor productivity increased with dilution rate and feed D-xylose concentration until a maximum was reached. The ethanol content of the effluent always decreased with increasing dilution rate, but excessive dilution rates diminished the ethanol content without increasing productivity. Unlike production rate, ethanol yield declined monotonically from 0.35 g/g as the fermentor substrate concentration increased. The yield was 69% of that theoretically possible when the D-xylose concentration was near zero, as opposed to 42% when it was in the range supporting the optimum specific rate of ethanol production. As long as D-xylose was supplied to cells faster than they could consume it, productivity increased with the mass of cells immobilized. The effectiveness factor associated with the calcium alginte beads used in this system was 0.4, indicating that only 40% of the entrapped biomass was effective in converting D-xylose to ethanol because of diffusion limitations.     

Ethanol production from D-xylose and other sugars by the yeastPachysolen tannophilus

Summary D-Xylose was fermented to ethanol by a strain ofPachysolen tannophilus in yields greater than 0.3g ethanol per g xylose consumed. Ethanol production was influenced by xylose concentration and was at a maximum at 10%, w/v. Ethanol formation occurred at pH 2.75-2.50 but the yeast would not grow at this pH when the initial pH of the medium was less than 3.0. Ethanol was consumed by the yeast when the xylose concentration became limiting. L-Arabinose, D-glucose, D-fructose, cellobiose, D-glucuronic acid, but not sucrose,were also fermented to ethanol byPachysolen tannophilus. Kinetic studies on xylose fermentation established various parameters involved in growth, substrate utilization and ethanol formation when the yeast was fermenter grown.     

Mutants of Pachysolen tannophilus showing enhanced rates of growth and ethanol formation from d-xylose

Mutants of Pachysolen tannophilus NRRL Y-2460 have been sought that show enhanced rates of d-xylose fermentation. Mutagenesis followed by enrichment in urea-xylitol broth generally resulted in a lower frequency of good ethanol producers than enrichment in nitrate-xylitol broth. Under aerobic conditions, the best xylose-fermenting strains (which were obtained from nitrate-xylitol broth) produced ethanol from xylose twice as fast and in 32% better yield than the parent strain. Under anaerobic conditions, these strains produced ethanol from xylose 50% faster than (but in the same yield as) the parent strain. These findings show that enrichment in nitrate-xylitol broth is a promising method for obtaining mutants of Pachysolen having enhanced fermentation rates.     

The induction of D-xylose catabolizing enzymes inPachysolen tannophilus and the relationship to anaerobic D-xylose fermentation

Summary Aerobic cultures harvested from the lag and early exponential growth phases fermented D-xylose poorly under anaerobic conditions whereas fermentation by late exponential and stationary phase cultures was rapid. These differences could be related to the ratios of NADH- to NADPH-linked xylose reductase (XR) and the levels of NADH-linked XR and NAD-linked xylitol dehydrogenase (XD) present. Under aerobic conditions, induction of NADPH-linked XR preceded NADH-linked XR which suggested the presence of two separate XR't's. Induction of XR and XD was more rapid under aerobic than anaerobic conditions.     

Mutants of Pachysolen tannophilus that produce enhanced amounts of acetic acid from D-xylose and other sugars

Summary Two mutants of Pachysolen tannophilus were isolated which produced considerably more acetic acid from several sugars than a wild type strain. Such mutants are of potential interest for the production of acetic acid rather than ethanol from lignocellulosic hydrolysates.Issued as NRCC No. 20810.     

Demonstration of D-xylose reductase and D-xylitol dehydrogenase in Pachysolen tannophilus

Summary The yeast, Pachysolen tannophilus, can utilize the pentose D-xylose with accumulation of significant quantities of ethanol. Cell extracts of the organism contain NADPH-linked D-xylose reductase (aldose reductase EC 1.1.1.21) and NAD-dependent D-xylitol dehydrogenase (D-xylulose reductase EC 1.1.1.9). D-Xylose was required for induction of both the D-xylitol dehydrogenase and the D-xylose reductase. Neither enzyme was found in glucose grown cell-free extracts.     

Transformation of a glucose negative mutant ofPachysolen tannophilus with a plasmid carrying the cloned hexokinase PII gene fromSaccharomyces cerevisiae

Lithium treated cells of the yeastPachysolen tannophilus have been transformed with a plasmid carrying the gene encoding for the hexokinase PII enzyme fromSaccharomyces cerevisiae. The gene was expressed and the presence of the enzyme within the cell was demonstrated by DEAE-cellulose chromatography of cell-free extracts. Plasmid DNA from the transformants was used to transformE. coli HB101. Plasmid DNA from the bacterial transformants had the same mobility on an agarose gel as the original plasmid.     

The effect of pH on kinetic and yield parameters during the ethanolic fermentation of D-xylose with Pachysolen tannophilus

We have studied the ethanolic fermentation of D-xylose with Pachysolen tannophilus in batch cultures. We propose a model to predict variations in D-xylose consumed, and biomass and ethanol produced, in which we include parameters for the specific growth rate, for the consumption of D-xylose and production of ethanol either related or not to growth.The ideal initial pH for ethanol production turned out to be 4.5. At this pH value the net specific growth rate was 0.26 h^–1, biomass yield was 0.16 g.g^–1, the cell-maintenance coefficient was 0.073 g.g^–1.h^–1, the parameter for ethanol production non-related to growth was 0.064 g.g^–1,h^–1 and the maximum ethanol yield was 0.32 g.g^–1.List of Symbols A _c Carbon atomic weight - a _d1/h Specific cell-maintenance rate defined in Eq. (8) - c Mass fraction of carbon in the biomass - E g/l Ethanol concentration - f _x Correction factor defined in Eq. (13) - f _x Correction factor defined in Eq. (13) - f _xi Correction factor defined in Eq. (14) - k _d1/h Death constant - M _E Ethanol molecular weight - M _s Xylose molecular weight - M _xi Xylitol molecular weight - m g xylose/g biomass Maintenance coefficient for substrate - m _dg xylose/g biomass Maintenance coefficient when k _d - q _Eg ethanol/g biomass. Specific ethanol production rate - s g/l Residual xylose concentration - s ₀ g/l Initial xylose concentration - t h Time - x g/l Biomass concentration - x ₀ g/l Initial biomass concentration - Y _E/sg ethanol/g xylose Instantaneous ethanol yield - ¯Y _E/sg ethanol/g xylose Mean ethanol yield - Y _E ^s/T g ethanol/g xylose Theoretical ethanol yield - Y _E ^s/* g ethanol/g xylose Corrected instantaneous ethanol yield - ¯Y _E ^s/* g ethanol/g xylose Corrected mean ethanol yield - Y _x/sg biomass/g xylose Biomass yield - ¯Y _xi/sg xylitol/g xylose Mean xylitol yield Greek Letters g ethanol/g biomass Growth-associated product formation parameter - g ethanol/g biomass.h Non-growth-associated product formation parameter - _dg ethanol/g biomass.h Non-growth-associated product formation parameter when k _d0 - h Variable defined in Eq. (6) or Eq. (7) - 1/h Specific growth rate - _m1/h Maximum specific growth rate     

Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30v°C, and a culture medium with an initial pH of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum specific growth rate, biomass productivity, specific rate of substrate consumption (q_s) and of ethanol production (q_E), and overall ethanol and xylitol yields. For the calculation of the specific rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, first D-glucose and then D-xylose. In both yeasts, the specific substrate-consumption rate diminished over each culture. The values q_s and q_E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg^у.     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

Effect of hydrogen acceptors on D-xylose fermentation by anaerobic culture of immobilized Pachysolen tannophilus cells

The effect of hydrogen acceptors on the kinetic parameters of D-xylose fermentation under anaerobic conditions was studied in a transient culture of immobilized Pachysolen tannophilus cells. Addition of oxygen to a steady-state culture resulted in a rapid increase (up to fivefold) in the rates of ethanol production and D-xylose uptake, but the rate of xylitol production was unaffected. Furthermore, the molar ethanol yield increased from 0.97 to 1.43 in the presence of oxygen. The moles of ethanol produced per moles of oxygen utilized were considerably greater than would be predicted from the stoichiometry of D-xylose fermentation, which suggests that the organism required oxygen for other functions in addition to its role as a hydrogen acceptor in D-xylose metabolism. When the artificial hydrogen acceptors acetone, acetaldehyde, and acetoin were added to the culture, the rate of ethanol production increased while the xylitol production rate decreased but the rate of xylose uptake was unaffected. The molar ethanol yields increased from 1.03 to 1.63, 1.43, and 1.24 upon addition of acetaldehyde, acetone, and acetoin, respectively, at the expense of the molar xylitol yields. The hydrogen acceptors sodium acetate, methylene blue, benzyl viologen, phenazine methosulfate, indigo carmine, and tetrazolium chloride had no effect on ethanol production.     

Viability of Candida shehatae in D-xylose fermentations with added ethanol

Ethanol was added at concentrations of 25 and 50 g/L to active cultures of Canida shehatae under oxygen-limited (fermentative) conditions. Added ethanol completely inhibited grwoth and fermentation of D-xylose by C. shehatae. Cultures with added ethanol rapidly declined in cell viability as measured by plate counts and methylene blue staining. The rate of decline in cell viability was dependent on the amount of added ethanol. Over the course of the fermentation, cell viability, as measured by plate counts, was significantly lower in all experiments (with or without ethanol addition) compared with the viability measurements by methylene blue staining. Thus, data from the plate counts provided a more sensitive measure of the toxic effects of added ethanol and long-term anaerobiosis on C. shehatae growth/fermentation. Mean cell volume and total cell volume declined in fermentations with added ethanol. (c) 1992 John Wiley & Sons, Inc.     

Effect of slow feeding of xylose on ethanol yield byPachysolen tannophilus

Summary With slow feeding of xylose to a batch fermentation byPachysolen tannophilus, the yield of ethanol from xylose was improved to 0.41 g/g (80% of theoretical) with a maximum ethanol concentration of 26.5 g/L at 120 h. This is a 41% improvement on the ethanol yield observed for batch fermentations without slow feeding. The optimum level of xylose in the medium was determined to be between 5 and 8g/L; xylose at greater than 10 g/L leads to xylitol accumulation, whereas xylose below 3 g/L permits ethanol to be oxidized to acetate. This latter effect is exacerbated by increased aeration.     

The effect of aeration on D-xylose fermentation byPachysolen tannophilus,Pichia stipitis,Kluyveromyces marxianus andCandida shehatae

Summary The fermentation of D-xylose byPachysolen tannophilus Y2460,Pichia stipitis Y7124,Kluyveromyces marxianus Y2415 andCandida shehatae Y12878 was investigated in aerobic, anaerobic and microaerophilic batch cultures. The aeration rate greatly influenced the fermentations; growth, rate of ethanol production and oxidation of ethanol are affected. Of the strains tested,Pichia stipitis appears superior; under anaerobic conditions it converts D-xylose (20 g/l) to ethanol with a yield of 0.40 g/l and it exhibits the highest ethanol specific productivity (3.5 g of ethanol per g dry cell per day) under microaerophilic conditions.     

Influence of the concentrations of d-xylose and yeast extract on ethanol production by Pachysolen tannophilus

To determine the most favorable conditions for the production of ethanol by Pachysolen tannophilus, this yeast was grown in batch cultures with various initial concentrations of two of the constituents of the culture medium: d-xylose (s_o), ranging from 1 g·l⁻¹ to 200 g·l⁻¹, and yeast extract (l_o), ranging from 0 g·l⁻¹ to 8 g·l⁻¹. The most favorable conditions proved to be initial concentrations of S_o=25 g·l⁻¹ and l_o=4 g·l⁻¹, which gave a maximum specific growth rate of 0.26 h⁻¹, biomass productivity of 0.023 g·l⁻¹·h⁻¹, overall biomass yield of 0.094 g·g⁻¹, specific xylose-uptake rate (q_s) of 0.3 g·g⁻¹·h⁻¹ (for t=50 h), specific ethanol-production rate (q_E) of 0.065 g·g⁻¹·h⁻¹ and overall ethanol yield of 0.34 g·g⁻¹; q_s values decreased after the exponential growth phase while q_E remained practically constant.     

Fluorescence measurements under aerobic and anaerobic conditions on Pichia stipitis,Pachysolen tannophilus and Candida utilis grown on D-xylose

Batch growth of the yeasts Candida utilis, Pachysolen tannophilus and Pichia stipitis on 1% D-xylose was monitored using a commercial fluorosensor with an excitation wavelength of 340 nm and a detection wavelength of 460 nm. Step changes in oxygen concentration were made and in the presence of 0.3 g/l of xylose, step changes from aerobic to anaerobic conditions resulted in an increase of the fluorescence level by about 40% for the non-fermentative yeast C. utilis. However, the increases of the fluorescence levels for P. tannophilus and P. stipitis stayed below 10%. These measurements indicate better control of (or better redox balance for) intracellular NADH concentration in P. tannophilus and P. stipitis than in C. utilis.List of Symbols F NFU fluorescence - F ₀ NFU initial fluorescence - F NFU final fluorescence difference - t s time - s time constant