M. T. Amaral-Collaço

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.

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

Abstract The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O2 l−1 min−1 resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O2 l−1 min−1, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O2 l−1 min−1), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg−1 when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O2 l−1 min−1), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O2 l−1 min−1), no significant fermentative activity is reported.

Polyols production during single and mixed substrate fermentations in Debaryomyces hansenii

Abstract The kinetics of polyols production by Debaryomyces hansenii was studied both on single substrate and mixed substrate-containing media. From the single substrate experiments, polyols (xylitol and arabitol) and ethanol were produced from pentose sugars while ethanol was produced in significant amounts only from glucose-grown D. hansenii. The maximal xylitol volumetric productivity (Qxylitol), 0.28 g l−1 h−1 was obtained from D -xylose, whereas the maximal arabitol volumetric productivity (Qarabitol), 0.04 g l−1 h−1 was observed with arabinose. When D. hansenii was cultivated with mixed substrates, a simultaneous sugar consumption pattern occurred both for glucose/arabinose and xylose/arabinose mixtures. The addition of low amounts of xylose to an arabinose medium led to a fourfold increase in the arabitol volumetric productivity: 0.17 g l−1 h−1. Conversely, glucose addition had no effect on arabitol production. Xylitol was the main polyol produced for all tested cultivation conditions by D. hansenii. An enzymatic study of the first two xylose-catabolic enzymes in glucose and xylose-grown D. hansenii revealed that both enzymes were induced by D -xylose. Glucose caused total inhibition of xylitol dehydrogenase, whereas xylose reductase was only partially repressed.

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

SummaryCandida 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.

The influence of hexoses addition on the fermentation of d-xylose in Debaryomyces hansenii under continuous cultivation

The effect of hexoses (glucose and galactose) addition to the feed xylose mineral medium of Debaryomyces hansenii chemostat cultures grown at a constant dilution rate of 0.055 h(-1) was studied. Xylitol was the major product detected amongst all tested conditions. The maximal values for xylitol yield and volumetric productivity (0.56 gg(-1) xylose and 0.21 gl(-1)h(-1), respectively) were obtained for a glucose/xylose feeding ratio of 10%, showing that the addition of small amounts of glucose, but not galactose, enhanced the xylitol production. A xylitol yield increase of 30%, compared with the sole xylose-containing feed medium, was observed. It was found that the oxygen requirement for D. hansenii growth is lower under glucose compared with xylose. Ethanol and glycerol were only produced for glucose/xylose feeding ratio above 30%. The byproducts accumulation was correlated with glucose metabolism, because a direct relationship between the increase of ethanol (and glycerol) concentration and the increase of glucose in the feed medium was found.

Characterization of xylitol dehydrogenase from Debaryomyces hansenii

The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells ofDebaryomyces hansenii was partially purified in two Chromatographic steps, and characterization studies were carried out in order to inves tigate the role of the xylitol dehydrogenase-catalyzed step in the regu lation of D-xylose metabolism. The enzyme was most active at pH 9.0–9.5, and exhibited a broad polyol specificity. The Michaelis con stants for xylitol and NAD+ were 16.5 and 0.55 mM, respectively. Ca2+, Mg2+, and Mn2+ did not affect the enzyme activity. Conversely, Zn2+, Cd2+, and Co2+ strongly inhibited the enzyme activity. It was concluded that NAD+-xylitol dehydrogenase from D.hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and Km value for xylitol, and therefore should be named L-iditol:NAD+-5-oxidoreductase (EC 1.1.1.14). The reason D.hansenii is a good xylitol producer is not because of its value of Km for xylitol, which is low enough to assure its fast oxidation by NAD+ xylitol dehydrogenase. However, a higher Km value of xylitol dehydro genase for NAD+ compared to theKm values of other xylose-ferment ing yeasts may be responsible for the higher xylitol yields.

Characterisation and application of glycanases secreted by Aspergillus terreus CCMI 498 and Trichoderma viride CCMI 84 for enzymatic deinking of mixed office wastepaper

Two enzymatic extracts obtained from xylan-grown Aspergillus terreus CCMI 498 and cellulose-grown Trichoderma viride CCMI 84 were characterised for different glycanase activities. Both strains produce extracellular endoxylanase and endoglucanase enzymes. The enzymes optimal activity was found in the temperature range of 45-60 degrees C. Endoglucanase systems show identical activity profiles towards temperature, regardless of the strain and inducing substrate. Conversely, the endoxylanases produced by both strains showed maximal activity at different pH values (from 4.5 to 5.5), being the more acidic xylanase produced by T. viride grown on cellulose. The endoglucanase activities have an optimum pH at 4.5-5.0. The endoxylanase and endoglucanase activities exhibited high stability at 50 degrees C and pH 5.0. Mannanase, beta-xylosidase, and amylase activities were also found, being the first two activities only present for T. viride extract. These two enzymatic extracts were used for mixed office wastepaper (MOW) deinking. When the enzymatic extract from T. viride was used, a further increase of 24% in ink removal was obtained by comparison with the control. Both enzymes contributed to the improvement of the paper strength properties and the obtained results clearly indicate that the effective use of enzymes for deinking can also contribute to the pulp and paper properties improvement.

Diversity of microfungi in the phylloplane of plants growing in a Mediterranean ecosystem

Mediterranean ecosystems have not been investigated as natural habitats for microorganisms in general, and microfungi in particular. Plants harvested in “Serra da Arrábida” (38°27′ N, 9°02′ W), a Mediterranean ecosystem in Portugal, were analyzed for the filamentous microfungi inhabiting their surface. Two field locations with distinct climatic characteristics were studied: ‘Fonte do Veado’ (38°28′50″ N, 9°0′17″ W; 300 m elevation) located on the northern slope, and ‘Mata do Solitário’ (38°27′55″ N, 8°59′35″ W; 50 m elevation), on the southern slope. From Veado zone, leaf samples yielded a total of 3049 isolates, ranging from 317 to 1328/sample (mean = 762). The number of species/sample ranged from 12 to 24. From Solitario zone, leaf samples yielded a total of 1337 isolates, ranging from 189 to 528/sample (mean = 334). The number of species/sample, in this case, ranged from 10 to 17. Veado zone showed a wider range of species. The fungal species more frequently isolated from both zones (Aureobasidium pullulans (De Bary) Arnaud, Cladosporium cladosporioides (Fresen.) De Vries, C. sphaerospermum Penzig and Alternaria alternata (Fr.) Keissler) were found in all plant samples and represents 80% (Veado) and 85% (Solitario) of the total isolates.

Characterization of a thermotolerant and alkalotolerant xylanase from a Bacillus sp.

In a recent screening for thermophilic bacteria from Azores hot springs, a Bacillus sp strain 3M, exhibiting cellulase-free extracellular xylanolitic activity, was isolated. Further enzyme characterization from liquid cultures grown on birchwood xylan revealed that the endo-l,4-βxylanase retains 100% of activity for at least 3 d at 55°C. At 80°C, it retains 47% of its maximal activity, and the enzyme is still active at 90°C. The optimum pH of the enzyme has a broad pH range, between 6.0 and 7.5, and it is remarkably active for the alkaline region, exhibiting 89% of relative activity at pH 9.O. The enzyme was partially inactivated by different divalent metal ions. Because of its tolerance for high temperature and pH conditions, and the absence of contaminating cellulase activity, the xylanase produced byBacillus sp 3M appears to be attractive for use in the pulp and paper industry. Indeed, the efficiency of the enzyme application to the kraftEucalyptus pulp was studied for bleaching pretreatment, resulting in a moderate increase of pulp bleachability.

Membrane concentration of fungal cellulases

A 10 000 Da hollow-fiber membrane characterized by an average hydraulic resistance of 9·5 × 106 Pa s m−1 and an average transmembrane pressure of 1·5 × 106 Pa was used to concentrate a commercial fungal cellulase so solution. The level of enzymatic activity increased slowly for the first 3 h before it started rising steeply in an exponential concentration effect due to solute polarization at the inner layer of the membrane, producing a flow rate decrease of approximately 20%. Enzymatic activity yield and permeate flow were studied in a range between 940 and 1300 ml min−1 for cross-flow rate, 0·68 × 106 and 1·22 × 106 Pa for outlet pressure, and ultrafiltration time varying between 90 and 300 min. Pressure was revealed to be more important than cross-flow rate in the efficiency of the concentration. Also, a long lasting operation produced better results in terms of the studied responses than any combination of the other factors. The values of the specific activity of the cellulase components for an 8-h ultrafiltration period remained constant, indicating that no shear inactivation occurred.