Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

Summary Three strains ofPichia stipitis and three ofCandida shehatae were compared withPachysolen tannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

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.     

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.     

Simultaneous saccharification and fermentation by mixed cultures ofBrettanomyces clausenii andPichia spipitis R of SO2-prehydrolysed wood

Summary Using pilot scale Wenger and Stake II reactors for prehydrolysing aspen and coniferous wood chips in the presence of SO₂ catalyst, highly digestible lignocellulosic substrates were generated from which about 90% yields of hemicellulose mostly in monomeric form could be recovered. Simultaneous saccharification and fermentation (SSF) of these SO₂ feedstocks by a mixed culture ofBrettanomyces clausenii andPichia stipitis R resulted in rapid and efficient fermentation giving a final yield of 369 and 360 L ethanol/tonne of the prehydrolysed woods, respectively. BecauseB. clausenii is an excellent cellobiose fermenter, no -glucosidase was needed during SSF.     

Evaluation of hardboard manufacturing process wastewater as a feedstream for ethanol production

Waste streams from the wood processing industry can serve as feedstream for ethanol production from biomass residues. Hardboard manufacturing process wastewater (HPW) was evaluated on the basis of monomeric sugar recovery and fermentability as a novel feedstream for ethanol production. Dilute acid hydrolysis, coupled with concentration of the wastewater resulted in a hydrolysate with 66 g/l total fermentable sugars. As xylose accounted for 53 % of the total sugars, native xylose-fermenting yeasts were evaluated for their ability to produce ethanol from the hydrolysate. The strains selected were, in decreasing order by ethanol yields from xylose (Y _p/s, based on consumed sugars), Scheffersomyces stipitis ATCC 58785 (CBS 6054), Pachysolen tannophilus ATCC 60393, and Kluyveromyces marxianus ATCC 46537. The yeasts were compared on the basis of substrate utilization and ethanol yield during fermentations of the hydrolysate, measured using an HPLC. S. stipitis, P. tannophilus, and K. marxianus produced 0.34, 0.31, and 0.36 g/g, respectively. The yeasts were able to utilize between 58 and 75 % of the available substrate. S. stipitis outperformed the other yeast during the fermentation of the hydrolysate; consuming the highest concentration of available substrate and producing the highest ethanol concentration in 72 h. Due to its high sugar content and low inhibitor levels after hydrolysis, it was concluded that HPW is a suitable feedstream for ethanol production by S. stipitis.     

Production of poly(β-l-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans

For the first time the production of poly(β-l -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H₂O₂-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO₃ plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO₃ and supplementary enzymes.     

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)     

Influence of oxygen transfer rate and media composition on fermentation ofd-xylose byPichia stipitis NRRL Y-7124

Summary The optimization of ethanol production byPichia stipitis NRRL Y-7124 was analysed by ATP balance. Ethanol volumetric productivity was maximal (0.5–0.6 g/l h) only over a narrow range of oxygen transfer rates (3–5 mmol O₂/l h). Trace element supplements increased ethanol volumetric productivity 20%. Biotin and thiamine did not significantly affect ethanol yield. Vitamins and trace elements were not synergistic. Organic nitrogen source from yeast extract was used for growth simultaneously to ammonia.     

NADPH-specific and NADH-specific xylose reduction is catalyzed by two separate enzymes in Pachysolen tannophilus

Summary Pachysolen tannophilus contains — in addition to an NADPH-linked xylose reductase — a separate NADH-linked one, in this respect differing from the yeast Pichia stipitis. Both enzyme proteins can conveniently be separated from each other by either ion exchange chromatography or chromatofocusing.     

Effect of intermittent feeding of cellulose hydrolyzate to hemicellulose hydrolyzate on ethanol yield byPachysolen tannophilus

Summary Intermittent-feeding of cellulose hydrolyzate to hemicellulose hydrolyzate of hardwood resulted in greater yields of ethanol usingPachysolen tannophilus than batch fermentations of either hydrolyzate alone or as a mix. Conversion efficiencies as great as 0.40 g ethanol/g sugar fed were achieved.     

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     

Citric acid production by Yarrowia lipolytica: Effect of nitrogen and biomass concentration on yield and productivity

Summary Citric acid yields of Yarrowia lipolytica (NRRL Y-1095) grown on glucose ranged from 0.38–0.77 g/g and were dependent on both biomass and nitrogen concentration (as NH₄Cl and yeast extract). Increasing the biomass concentration by 3% (w/v) increased fermentor productivities from 0.6 to 1.22 g citric acid/L h.     

Acetone-butanol fermentation of xylose and sugar mixtures

Summary Three strains ofCl. acetobutylicum and one ofCl. butyricum have been tested for their ability to ferment xylose to butanol. ATCC 824 and NRRL 527 produced 0.28 g solvents/g xylose, while ATCC 8260 and NRRL 594 produced much butyric acid. In 2-stage fermentations in which ATCC 8260 or NRRL 594 acted upon xylose for 12 to 20 h, followed by NRRL 527 for a total of 3 days, yields of solvent were better, 0.32 g/g xylose. Upon fermenting a mixture of sugars simulating sulphite waste liquor 0.36 g solvents/g sugar were obtained. Sugar consumption in both cases was about 96%.     

Fermentation of cellobiose and wood sugars to ethanol byCandida shehatae andPichia stipitis

Summary Three pentose fermenting yeast strains ofCandida shehatae and three ofPichia stipitis were examined for their ability to produce ethanol from cellobiose and from sugars liberated by hydrolysis of lignocellulosic biomass. All of thePichia strains tested produced some ethanol;P. stipitis CBS 5776 gave the highest yield: 10.3 g/L on complete fermentation of 25 g/L cellobiose within 48 hours. This yeast also produced considerably more ethanol from the wood sugar mixture.     

Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

Genetic Engineering of Inhibitor-Tolerant Saccharomyces cerevisiae for Improved Xylose Utilization in Ethanol Production

For economical lignocellulose-to-ethanol production, a desirable biocatalyst should tolerate inhibitors derived from preteatment of lignocellulose and be able to utilize heterogeneous biomass sugars of hexoses and pentoses. Previously, we developed an inhibitor-tolerant Saccharomyces cerevisiae strain NRRL Y-50049 that is able to in situ detoxify common aldehyde inhibitors such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF). In this study, we genetically engineered Y-50049 to enable and enhance its xylose utilization capability. A codon-optimized xylose isomerase gene for yeast (YXI) was synthesized and introduced into a defined chromosomal locus of Y-50049. Two newly identified xylose transport related genes XUT4 and XUT6, and previously reported xylulokinase gene (XKS1), and xylitol dehydrogenase gene (XYL2) from Scheffersomyces stipitis were also engineered into the yeast resulting in strain NRRL Y-50463. The engineered strain was able to grow on xylose as sole carbon source and a minimum ethanol production of 38.6?g?l^?1 was obtained in an anaerobic fermentation on mixed sugars of glucose and xylose in the presence of furfural and HMF.     

Callus formation from leaf mesophyll protoplasts of Malus Xdomestica Borkh. cv. Greensleeves

Large yields (1.85 × 10⁷/g.f.wt.) of viable protoplasts were obtained from leaves of axenic shoot cultures of Malus Xdomestica Borkh. cv. Greensleeves. Protoplasts cultured in liquid or agarose semi-solidified KM8P medium underwent cell wall regeneration and colony formation.Protoplast-derived cell colonies developed to callus on semi-solid KM8 medium. This is the first report of callus formation from mesophyll protoplasts of apple.Abbreviations BAP 6-benzylaminopurine - K kinetin - Z zeatin - GA₃ gibberellic acid - IBA 3-indole butyric acid - NAA 1-naphthalene acetic acid - IAA 3-indole acetic acid - ABA abscisic acid - f.wt. fresh weight - MS Murashige and Skoog (1962)     

Comparison of the ability of three Aspergillus strains to form aflatoxins on bakery products and on nutrient agar

The growth of Aspergillus parasiticus NRRL 2999, A. parasiticus NRRL 3000 and A. flavus NRRL 3251 on whole wheat bread and on cake (Rührkuchen) was compared and the formation of the aflatoxins B₁, B₂, G₁, G₂ and M₁ on these substrates and, for purpose of comparison, on malt extract agar was determined. On cake the moulds grew better than on bread and formed the highest yields of aflatoxins. Malt extract agar was the most unfavourable substrate for toxin production. The ratio M₁/B₁ on bread and cake was in the order of 0.1–0.4 and was higher than the data reported for grains. The highest yields of aflatoxin B₁ (1.0 g/g) were produced by A. flavus NRRL 3251 on cake.     

NADH-linked aldose reductase: the key to anaerobic alcoholic fermentation of xylose by yeasts

Summary The kinetics and enzymology of d-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD⁺-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD⁺/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase.     

Strain improvement of the xylose-fermenting yeastPachysolen tannophilus by hybridisation of two mutant strains

Two previously reported mutants ofPachysolen tannophilus, which accumulate ethanol more rapidly and in greater yield than the wild-type NRRL Y2460, have been cross-mated. Aneth 2-1 mutant which is unable to grow on ethanol, was mated with the mutant NO₃–NO₃-4 which possesses increased levels of pentose phosphate pathway enzymes. The new hybrid strain combines the properties of both parents and possesses improved characteristics for xylose fermentation to ethanol.