The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by¹³C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-¹³C]xylose or [2-¹³C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-¹³C]xylose or [1-¹³C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-¹³C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.     

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603^T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603^T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.     

The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus

Summary The ability of a Candida shehatae and a Pachysolen tannophilus strain to ferment D-xylose to ethanol was evaluated in defined and complex media under different levels of aeration. Aeration enhanced the ethanol productivity of both yeasts considerably. C. shehatae maintained a higher fermentation rate and ethanol yield than P. tannophilus over a wide range of aeration levels. Ethanol production by C. shehatae commenced during the early stage of the fermentation, whereas with P. tannophilus there was a considerable lag between the initiation of growth and ethanol production. Both yeasts produced appreciable quantities of xylitol late in the fermentation. P. tannophilus failed to grow under anoxic conditions, producing a maximum of only 0.5 g · l^-1 ethanol. In comparison, C. shehatae exhibited limited growth in anoxic cultures, and produced ethanol much more rapidly. Under the condition of aeration where C. shehatae exhibited the highest ethanol productivity, the fermentation parameters were: maximum specific growth rate, 0.15 h^-1; maximum volumetric and specific rates of ethanol production, 0.7 g (l · h)^-1 and 0.34 g ethanol (g cells · h)^-1 respectively; ethanol yield, 0.36 g (g xylose)^-1. The best values obtained with P. tannophilus were: maximum specific growth rate, 0.14 h^-1; maximum volumetric and specific rates of ethanol production, 0.22 g (l · h)^-1 and 0.07 h^-1 respectively; ethanol yield coefficient, 0.28. Because of its higher ethanol productivity at various levels of aeration, C. shehatae has a greater potential for ethanol production from xylose than P. tannophilus.     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Xylitol production from xylose by two yeast strains: Sugar tolerance

The kinetics and enzymology ofd-xylose utilization are studied in micro-, semi-, and aerobic batch cultures during growth ofCandida guilliermondii andCandida parapsilosis in the presence of several initial xylose concentrations. The abilities of xylitol accumulation by these two yeast strains are high and similar, although observed under various growth conditions. WithCandida parapsilosis, optimal xylitol production yield (0.74 g/g) was obtained in microaerobiosis with 100 g/L of xylose, whereas optimal conditions to produce xylitol byCandida guilliermondii (0.69 g/g) arose from aerobiosis with 300 g/L of sugar. The different behavior of these yeasts is most probably explained by differences in the nature of the initial step of xylose metabolism: a NADPH-linked xylose reductase activity is measured with a weaker NADH-linked activity. These activities seem to be dependent on the degree of aerobiosis and on the initial xylose concentration and correlate with xylitol accumulation.     

Enzymatic and physiological study of d-xylose metabolism by Candida shehatae

Summary Candida shehatae carbon metabolic pathways were correlated with fermentative activity under different growth conditions. Reduced nicotine adenine dinucleotide (NADPH) is the coenzyme preferred for xylose reductase by C. shehatae under in vitro anoxic cell culture conditions. To prevent a redox imbalance derived from intracellular accumulation of NADH in the second enzymatic step of xylose metabolism, the operation of phosphoketolase via in addition the classic pentose phosphate pathway essential for NADH dissimilation is suggested. Variation in cultivation conditions showed a different NADH/NADPH ratio coupled to xylose reductase activity. The existence of two xylose reductases is discussed. Like ethanol, xylitol accumulates only under oxygen-limited or anaerobic conditions. Xylitol accumulaiton under unaerobic conditions was higher when using respiring cells than respirofermentative cells. This fact suggests that cells pregrown under oxygen limitation are better adapted to starting alcoholic fermentation than cells previously grown under aerobic conditions.Offprint requests to: M. T. Amaral-Collaço     

Ethanol tolerance of Pichia stipitis and Candida shehatae strains in fed-batch cultures at controlled low dissolved oxygen levels

Summary Fed-batch cultivations of Pichia stipitis and strains of Candida shehatae with d-xylose or d-glucose were conducted at controlled low dissolved oxygen tension (DOT) levels. There were some marked differences between the strains. In general growth was inhibited at lower ethanol concentrations than fermentation, and ethanol levels of up to 47 g·l^-1 were produced at 30°C. Ethanol production was mainly growth associated. The yeast strains formed small amounts of monocarboxylic acids and higher alcohols, which apparently did not enhance the ethanol toxicity. The maximum ethanol concentration obtained on d-xylose could not be increased by using a high cell density culture, nor by using d-glucose as substrate. The latter observation suggested that the low ethanol tolerance of these xylose-fermenting yeast strains was not a consequence of the metabolic pathway used during pentose fermentation. In contrast with the C. shehatae strains, it was apparent with P. stipitis CSIR-Y633 that when the ethanol concentration reached about 28 g·l^-1, ethanol assimilation exceeded ethanol production, despite cultivation at a low DOT of 0.2% of air saturation. Discontinuing the aeration enabled ethanol accumulation to proceed, but with concomitant xylitol production and cessation of growth.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Ethanol Production from Xylose by a Recombinant Candida utilis Strain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD⁺-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD⁺-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.     

The effects of the oxygen transfer coefficient and substrate concentration on the xylose fermentation by Debaryomyces hansenii

Relevant production of xylitol by Debaryomyces hansenii requires semiaerobic conditions since in aerobic conditions the accumulated reduced adenine dinucleotide coenzyme is fully reoxidized leading to the conversion of xylitol into xylulose. For oxygen transfer coefficient values from 0.24 to 1.88 min^-1, in shake flasks experiments, biomass formation increased proportionally to the aeration rate as shown in the oxygen transfer coefficient and xylose concentration isoresponse contours. The metabolic products under study, xylitol and ethanol were mainly growth associated. However, for oxygen transfer coefficient above 0.5 min^-1 higher initial xylose concentration stimulated the rate of production of xylitol. This fact was less evident for ethanol production. The direct relationship between increased biomass and products formation rate, indicated that the experimental domain in respect to the aeration rate was below the threshold level before the decreasing in metabolic production rates reported in literature for xylose-fermenting yeasts. The fact that ethanol was produced, albeit in low levels, throughout the experimental design indicated that the semiaerobic conditions were always attained. Debaryomyces hansenii showed to be an important xylitol producer exhibiting a xylitol/ethanol ratio above four and a carbon conversion of 54% for xylitol.Abbreviations K_La oxygen transfer coefficient - DO(T) dissolved oxygen (tension) - OUR oxygen uptake rate - NAD(H) oxidised (reduced) nicotinamide adenine dinucleotide - NADP(H) oxidised (reduced) nicotinamide adenine dinucleotide phosphate - CRC catabolic reduction charge - C oxygen concentration in the culture medium - C^* oxygen concentration at saturation conditions - Y_i response from experiment i - parameters of the polynomial model - x experimental factor level (coded units) - R² coefficient of multiple determination - t time     

Effects of environmental conditions on production of xylitol byCandida boidinii

Candida boidinii NRRL Y-17213 produced more xylitol thanC. magnolia (NRRL Y-4226 and NRRL Y-7621),Debaryomyces hansenii (C-98 M-21, C-56 M-9 and NRRL Y-7425), orPichia (Hansenula) anomala (NRRL Y-366). WithC. boidinii, highest xylitol productivity was at pH 7 but highest yield was at pH 8, using 5 g urea and 5 g Casamino acids/I. Decreasing the aeration rate decreased xylose consumption and cell growth but increased the xylitol yield. When an initial cell density of 5.1 g/l was used instead of 1.3 g/l, xylitol yield and the specific xylitol production rate doubled. Substrate concentration had the greatest effect on xylitol production; increasing xylose concentration 7.5-fold (to 150 g/l) gave a 71-fold increase in xylitol production (53 g/l) and a 10-fold increase in xylitol/ethanol ratio. The highest xylitol yield (0.47 g/g), corresponding to 52% of the theoretical yield, was obtained with 150 g xylose/l after 14 days. Xylose at 200 g/l inhibited xylitol production.E. Vandeska and S. Kuzmanova were and S. Amartey and T. Jeffries are with the Forest Products Laboratory, Institute for Microbial and Biochemical Technology, 1 Gifford Pinchot Drive, Madison, WI 53703, USA. E. Vandeska and S. Kuzmanova are now with the Faculty of Technology and Metallurgy, Rudjer Boskovic 16, 91000 Skopje, Macedonia     

Temperature profiles of growth and ethanol tolerance of the xylose-fermenting yeasts Candida shehatae and Pichia stipitis

Summary The effect of different ethanol concentrations on the growth of Candida shehatae and Pichia stipitis with xylose as substrate was evaluated in a temperature gradient incubator. The upper limit of the temperature profiles of ethanol tolerance of both yeast strains were similar, although P. stipitis appeared to have a slightly higher ethanol tolerance in the higher temperature range. An increase in the ethanol concentration severely depressed the maximum growth temperature, and also increased the minimum growth temperature slightly. The ethanol tolerance limit of 46–48 g·l^-1 occurred within a narrow temperature plateau of 11 to 22° C. The low ethanol tolerance of these pentose fermenting yeasts is detrimental for commercial ethanol production from hemicellulose hydrolysates.     

A quantitative screening of some xylose-fermenting yeast isolates

Summary A quantitative screening procedure for xylose fermentation was conducted on 56 yeast isolates. Several of the isolates were found to be markedly superior toC. shehatae CSIR-Y492, one of the better xylose-fermenting yeasts identified thus far. The outstanding isolate was a strain ofPichia stipitis which had an ethanol yield coefficient of 0.45 from xylose and which produced no detectable amounts of xylitol.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

The oxygen requirements of yeasts for the fermentation of d-xylose and d-glucose to ethanol

Summary The effect of oxygen availability on d-xylose and D-glucose metabolism by Pichia stipitis, Candida shehatae and Pachysolen tannophilus was investigated. Oxygen was not required for fermentation of d-xylose or d-glucose, but stimulated the ethanol production rate from both sugars. Under oxygen-limited conditions, the highest ethanol yield coefficient (Y_e/s) of 0.47 was obtained on d-xylose with. P. stipitis, while under similar conditions C. shehatae fermented d-xylose most rapidly with a specific productivity (q_pmax) of 0.32 h^-1. Both of these yeasts fermented d-xylose better and produced less xylitol than. P. tannophilus. Synthesis of polyols such as xylitol, arabitol, glycerol and ribitol reduced the ethanol yield in some instances and was related to the yeast strain, carbon source and oxygen availability. In general, these yeasts fermented d-glucose more rapidly than d-xylose. By contrast Saccharomyces cerevisiae fermented d-glucose at least three-fold faster under similar conditions.Nomenclature q_pmax maximum specific rate of ethanol production (g ethanol per g dry biomass per hour) - Y_e/s ethanol yield (g ethanol per g substrate utilized) - Y_p/s polyol yield (g polyol per g substrate utilized) - Y_x/s biomass yield (g dry biomass per g substrate utilized) - _max maximum specific growth rate (per hour)     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

The effect of respiratory inhibitors on the fermentative ability of Pichia stipitis,Pachysolen tannophilus and Saccharomyces cerevisiae under various conditions of aerobiosis

Summary The growth and ethanol production by the d-xylose-fermenting yeasts Pichia stipitis and Pachysolen tannophilus under various conditions of aerobiosis responded similarly to the addition of the respiratory inhibitors potassium cyanide (KCN), antimycin A (AA), sodium azide and rotenone. However, the d-glucose-fermenting yeast Saccharomyces cerevisiae differed markedly from these yeasts in response to the inhibitors. In general the growth of the d-xylose-fermenting yeasts was inhibited by the respiratory inhibitors while ethanol production was either stimulated (especially when oxygen was available) or unaffected or inhibited by rotenone or AA or KCN and sodium azide, respectively. However, by exception KCN and AA stimulated ethanol production under aerobic conditions by Pichia stipitis and Pachysolen tannophilus respectively. Stimulatory or inhibitory effects by respiratory inhibitors were less marked in S. cerevisiae. These data suggest that unimpaired mitochondrial function is necessary for growth on d-xylose and optimal d-xylose fermentation. A requirement for membrane generated energy during d-xylose utilisation is indicated by 2,4-dinitrophenol inhibition of growth and fermentation.     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

An investigation of d-{1-13C} xylose metabolism in Pichia stipitis under aerobic and anaerobic conditions

Summary The uptake of d-{1-¹³C} xylose, the accumulation of intermediates and the distribution of the label in ethanol in Pichia stipitis under aerobic and anaerobic conditions were investigated by nuclear magnetic resonance spectroscopy. The rate-limiting step of d-xylose metabolism under aerobic conditions appeared to be uptake, whereas under anaerobic conditions it was the conversion of xylitol to xylulose. The yeast showed no preference to either the alpha-or beta-forms of d-xylose. Under anaerobic conditions only {2-¹³C{ ethanol was detected and this suggests that NADH but not NADPH was used as cofactor in the conversion of xylose to xylitol. d-Xylose is most likely metabolised by the pentose phosphate pathway in this yeast.