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.     

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2³ full factorial design with six axial points. Temperatures ranged from 132 to 180 °C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l⁻¹ h⁻¹. The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of β-glucosidase by P. stipitis. During SSF, free extracellular β-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g.     

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     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Effects of culture conditions on the co-fermentation of a glucose and xylose mixture to ethanol by a mutant of Saccharomyces diastaticus associated with Pichia stipitis

Substrates that contain hexose as well as pentose sugars can form an interesting substrate for the production of ethanol. Pichia stipitis and a respiratory-deficient mutant of Saccharomyces diastaticus were used to convert such a substrate into ethanol under continuous culture conditions. With a sugar mixture (glucose 70%/xylose 30%) at 50 g/l, the xylose was entirely consumed when the dilution rate (D) did not exceed 0.006 h^–1 whereas the glucose was entirely consumed whatever the D. The study of influence of initial substrate concentration (S₀) was performed at D = 0.015 h^–1. Under these conditions the substrate was entirely consumed when its initial concentration did not exceed 20 g/l. With S₀ = 80 g/l the residual xylose concentration reached 20.5 g/l. At low D or at low S₀, P. stipitis was the dominant species in the fermentor. Increasing the D or S₀ resulted in the wash-out of P. stipitis mainly because of its low ethanol tolerance. Correspondence to: J. P. Delgenes     

Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis

An enhanced inhibitor-tolerant strain of Pichia stipitis was successfully developed through adaptation to acid-treated rice straw hydrolysate. The ethanol production obtained by fermentation of NaOH-neutralized hydrolysate without detoxification using the adapted P. stipitis was comparable to fermentation of overliming-detoxified hydrolysate. The ethanol yield using the adapted P. stipitis with both types of hydrolysate at pH 5.0 achieved 0.45 g_p g_s⁻¹, which is equivalent to 87% of the maximum possible ethanol conversion. Furthermore, the newly adapted P. stipitis demonstrated significantly enhanced tolerance to sulfate and furfural despite the fact that both inhibitors had not been removed from the hydrolysate by NaOH neutralization. Finally, the ethanol conversion could be maintained at 60% and above when the neutralized hydrolysate contained 3.0% sulfate and 1.3 g L⁻¹ furfural.     

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (g ethanol g^?1 biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82 g g^?1 h^?1). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80 g L^?1) and the RHO2-3RD strain was able to tolerate up to 1.5 g L^?1 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22 %, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.     

The ethanol tolerance of Pichia stipitis Y 7124 grown on a d-xylose,d-glucose and l-arabinose mixture

The tolerance of Pichia stipitis Y 7124 to initial added ethanol was evaluated in anaerobic and microaerobic conditions, during the fermentation of a sugar mixture (d-glucose 20%, d-xylose 75%, l-arabinose 5%). The ethanol tolerance depends on the presence of oxygen. In microaerobiosis, the fermentative capacity of P. stipitis is not inhibited when the initial ethanol concentration does not exceed 20 g/l; in this added ethanol range, the strain produced ethanol with a yield up to 0.40 g/g and a specific rate of 0.1 g/g·h. An increase of the initial ethanol level decreases the rate of ethanol production but the ethanol yield appears to be less sensitive to ethanol inhibition. In anaerobiosis, maximum fermentative performances are obtained in the zero initial ethanol culture. When initial ethanol increases, growth and ethanol production decline gradually. But P. stipitis produces ethanol at an initial ethanol level of 50 g/l, even though this totally inhibits the strain activity in microaerobiosis.     

D-xylose fermentation by Candida shehatae and pichia stipitis at low dissolved oxygen levels in fed-batch cultures

Summary The fermentation of D-xylose byCandida shehatae andPichia stipitis was studied in fed-batch fermentations using dissolved oxygen tension (DOT) control in the range of 0.2 to 1.4% air saturation. The response of these two yeasts to DOT was significantly different. Whereas the ethanol yield withC. shehatae was 0.35 to 0.38 g.g^–1 at all DOT levels, that ofP. stipitis decreased from 0.44 at a zero DOT reading to 0.19 g.g^–1 at 1.4% DOT.     

Influence of metal addition on ethanol production with <Emphasis Type="Italic">Pichia stipitis</Emphasis> ATCC 58784

Trace metals always act as cofactors or coenzymes in many cellular processes. Deficiency or excess of some metals will affect the fermentation of lignocellulosic hydrolysate. In order to make sure the deficient or excessive states of metals in culture medium, metal contents analysis was conducted in Pichia stipitis ATCC 58784 cells, synthetic medium, and diluted acid hydrolysate of rice straw. The results showed that Cu, Ni, and Co were deficient, and Al was a little excessive. So the influences of Cu²⁺, Al³⁺, Ni²⁺, and Co²⁺ additions on the growth and ethanol production of ATCC 58784 were further researched. Low concentration additions of Cu²⁺ and Al³⁺ (<0.24 mM and <0.23 mM, respectively) improved biomass growth of ATCC 58784 by 34 and 13%, respectively; however, higher concentrations decreased biomass growth. On the other hand, addition of Cu²⁺ (0.39 mM) did not affect volumetric ethanol production significantly (P = 0.05) and addition of Al³⁺ (0.38 mM) showed no influence on volumetric ethanol production (P = 0.68). Addition of 0.074 mM Co²⁺ inhibited biomass growth of ATCC 58784 by 13% and volumetric ethanol production by 10%. The biomass growth and volumetric ethanol production of ATCC 58784 was arrested by the addition of 0.33 mM of Ni²⁺ by 53 and 65%, respectively.     

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae

Respiratory and fermentative pathways co-exist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiaeURA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote. Received: 6 January 1998 / Received revision: 9 April 1998 / Accepted: 19 April 1998     

Influence of magnesium concentration on growth,ethanol and xylitol production by Pichia stipitis NRRL Y-7124

Summary The effect of Mg⁺² on Pichia stipitis growth and ethanol production was studied under condition of constant oxygen uptake rate (OUR) . Biomass/xylose and biomass/Mg⁺² yields increased with Mg⁺² concentration with a maximum value at Mg⁺² 4mM, ethanol being the main product obtained. At low Mg⁺² levels (ImM) 49 % of carbon flux to ethanol was redirected to xylitol production, accomplished through NADH intracellular accumulation.     

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g_p/g_s by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO₂ released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Effect of growth rate on ethanol tolerance ofSaccharomyces cerevisiae

Δ^5,7 Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium. The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of Δ^5,7. Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance. Differences between the Δ^5,7 and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.     

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Δxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.     

Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH,temperature and substrate concentration

The effects of temperature, pH and xylose concentration on the fermentation parameters of Candida shehatae and Pichia stipitis were evaluated. The optimum pH was in the region of pH 4–5.5, with an optimum fermentation temperature of 30°C. Maximum fermentation rates were reached at 50 g l⁻¹ xylose. A maximum volumetric ethanol productivity of about 0.9 g (l h)⁻¹ was obtained with both yeast strains. The ethanol yield of C. shehatae decreased considerably when cultivated above 30°C or when the xylose concentration was increased. Xylitol accumulated concomitantly. Xylitol production by P. stipitis was observed only during cultivation at 36°C. Whereas the ethanol yield of C. shehatae was usually about 75% of the theoretical maximum, it was 85–90% with P. stipitis.     

Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

Background  

Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. The availability of NAD⁺ for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.     

Xylose fermentation by yeasts

Summary Utilization and fermentation of xylose by the yeasts Pachysolen tannophilus I fGB 0101 and Pichia stipitis 5773 to 5776 under aerobic and anaerobic conditions are investigated. Pa. tannophilus requires biotin and thiamine for growth, whereas Pi. stipitis does not, and growth of both yeasts is stimulated by yeast extract. Pi. stipitis converts xylose (30 g/l) to ethanol under anaerobic conditions with high yields of 0,40 and it produces only low amounts of xylitol. The yield coefficient is further increased at lower xylose concentrations.Publication Nr. 2 of this series: Eur. J. Appl. Microbiol. Biotechnol. (1983) 17, 287–291.     

Effect of temperature on ethanol tolerance of thermotolerant Isshatchenkia orientalis IPE100

Tolerance to high temperature and ethanol is a major factor in high‐temperature bio‐ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L^?1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (P_m) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L^?1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high‐temperature bio‐ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature‐dependent inhibitory effects of ethanol on yeast growth.