Xylose and yeasts: A story beyond xylitol production.

Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose, where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment (pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport, except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the studied yeast and the conditions in which these are studied.

The yeast Scheffersomyces amazonensis is an efficient xylitol producer.

This study assessed the efficiency of Scheffersomyces amazonensis UFMG-CM-Y493T, cultured in xylose-supplemented medium (YPX) and rice hull hydrolysate (RHH), to convert xylose to xylitol under moderate and severe oxygen limitation. The highest xylitol yields of 0.75 and 1.04 g g-1 in YPX and RHH, respectively, were obtained under severe oxygen limitation. However, volumetric productivity in RHH was ninefold decrease than that in YPX medium. The xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in the YPX cultures were strictly dependent on NADPH and NAD+ respectively, and were approximately 10% higher under severe oxygen limitation than under moderate oxygen limitation. This higher xylitol production observed under severe oxygen limitation can be attributed to the higher XR activity and shortage of the NAD+ needed by XDH. These results suggest that Sc. amazonensis UFMG-CM-Y493T is one of the greatest xylitol producers described to date and reveal its potential use in the biotechnological production of xylitol.

Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads.

Cell immobilization has shown to be especially adequate for xylitol production. This work studies the suitability of the air lift bioreactor for xylitol production by Debaryomyces hansenii immobilized in Ca-alginate operating in fed-batch cultures to avoid substrate inhibition. The results showed that the air lift bioreactor is an adequate system since the minimum air flow required for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation by this yeast, also maintaining the integrity of the alginate beads and the viability of the immobilized cells until 3 months of reuses. Maximum productivities and yields of 0.43 g/l/h and 0.71 g/g were achieved with a xylose concentration of 60 g/l after each feeding. The xylose feeding rate, the air flow, and the biomass concentration at the beginning of the fed-batch operation have shown to be critical parameters for achieving high productivities and yields. Although a maximum xylitol production of 139 g/l was obtained, product inhibition was evidenced in batch experiments, which allowed estimating at 200 and 275 g/l the IC50 for xylitol productivity and yield, respectively. The remarkable production of glycerol in the absence of glucose was noticeable, which could not only be attributed to the osmoregulatory function of this polyol in conditions of high osmotic pressure caused by high xylitol concentrations but also to the role of the glycerol synthesis pathway in the regeneration of NAD(+) in conditions of suboptimal microaeration caused by insufficient aeration or high oxygen demand when high biomass concentrations were achieved.

Studies on xylitol production by metabolic pathway engineered Debaryomyces hansenii.

Debaryomyces hansenii is one of the most promising natural xylitol producers. As the conversion of xylitol to xylulose mediated by NAD(+) cofactor dependent xylitol dehydrogenase (XDH) reduces its xylitol yield, xylitol dehydrogenase gene (DhXDH)-disrupted mutant of D. hansenii having potential for xylose assimilating pathway stopping at xylitol, was used to study the effects of co-substrates, xylose and oxygen availability on xylitol production. Compared to low cell growth and xylitol production in cultivation medium containing xylose as the only substrate, XDH disrupted mutants grown on glycerol as co-substrate accumulated 2.5-fold increased xylitol concentration over those cells grown on glucose as co-substrate. The oxygen availability, in terms of volumetric oxygen transfer coefficient, kLa (23.86-87.96 h(-1)), affected both xylitol productivity and yield, though the effect is more pronounced on the former. The addition of extra xylose at different phases of xylitol fermentation did not enhance xylitol productivity under experimental conditions.

Improving downstream processes to recover tartaric acid, tartrate and nutrients from vinasses and formulation of inexpensive fermentative broths for xylitol production.

BACKGROUND: Vinasses, the main liquid wastes from the distillation process of grape marc and wine lees, are acidic effluents with high organic content, including acids, carbohydrates, phenols, and unsaturated compounds with high chemical oxygen demand, biological oxygen demand and solid concentrations. These wastes can be revalued to provide additional benefits when they are employed as feedstock of some compounds including tartaric acid, calcium tartrate and economic nutrients for the elaboration of fermentable broths. RESULT: This study attempts to recover tartaric acid and calcium tartrate from vinasses. All the tartaric acid initially solubilised was recovered in both processes. The residual streams can be successfully employed as economic nutrients for the xylose to xylitol bioconversion, achieving higher global volumetric productivities (Q(P, xylitol) = 0.232 g L(-1) h(-1)) and products yields (Y(xylitol/S) = 0.57 g g(-1)) than fermentations carried out using commercial nutrients (Q(P, xylitol) = 0.193 g L(-1) h(-1) and Y(xylitol/S) = 0.55 g g(-1) respectively). CONCLUSION: Tartaric acid can be recovered from vinasses in the form of tartaric acid crystals and calcium tartrate. The residual streams generated in the process can be used as economic nutrients for the production of xylitol by D. hansenii.

Development of cost-effective media to increase the economic potential for larger-scale bioproduction of natural food additives by Lactobacillus rhamnosus , Debaryomyces hansenii , and Aspergillus niger.

Yeast extract (YE) is the most common nitrogen source in a variety of bioprocesses in spite of the high cost. Therefore, the use of YE in culture media is one of the major technical hurdles to be overcome for the development of low-cost fermentation routes, making the search for alternative-cheaper nitrogen sources particularly desired. The aim of the current study is to develop cost-effective media based on corn steep liquor (CSL) and locally available vinasses in order to increase the economic potential for larger-scale bioproduction. Three microorganisms were evaluated: Lactobacillus rhamnosus , Debaryomyces hansenii , and Aspergillus niger . The amino acid profile and protein concentration was relevant for the xylitol and citric acid production by D. hansenii and A. niger , respectively. Metals also played an important role for citric acid production, meanwhile, D. hansenii showed a strong dependence with the initial amount of Mg(2+). Under the best conditions, 28.8 g lactic acid/L (Q(LA) = 0.800 g/L.h, Y(LA/S) = 0.95 g/g), 35.3 g xylitol/L (Q(xylitol) = 0.380 g/L.h, Y(xylitol/S) = 0.69 g/g), and 13.9 g citric acid/L (Q(CA) = 0.146 g/L.h, Y(CA/S) = 0.63 g/g) were obtained. The economic efficiency (E(p/euro)) parameter identify vinasses as a lower cost and more effective nutrient source in comparison to CSL.

Maintenance and growth requirements in the metabolism of Debaryomyces hansenii performing xylose-to-xylitol bioconversion in corncob hemicellulose hydrolyzate.

In order to improve the biotechnological production of xylitol, the metabolism of Debaryomyces hansenii NRRL Y-7426 in corncob hemicellulose hydrolyzate has been investigated under different conditions, where either maintenance or growth requirements predominated. For this purpose, the experimental results of two sets of batch bioconversions carried out alternatively varying the starting xylose concentration in the hydrolyzate (65.6 < or = S(0) < or = 154.7 g L(-1)) or the initial biomass level (3.0 < or = X(0) < or = 54.6 g(DM) L(-1)) were used to fit a metabolic model consisting of carbon material and ATP balances based on five main activities, namely fermentative assimilation of pentoses, semi-aerobic pentose-to-pentitol bioconversion, biomass growth on pentoses, catabolic oxidation of pentoses, and acetic acid and NADH regeneration by the electron transport system. Such an approach allowed separately evaluating the main bioenergetic constants of this microbial system, that is, the specific rates of ATP and xylose consumption due to maintenance (m(ATP) = 21.0 mmol(ATP) C-mol(DM) (-1)h(-1); m(Xyl) = 6.5 C-mmol(Xyl) C-mol(DM) (-1)h(-1)) and the true yields of biomass on ATP (Y(ATP) (max) = 0.83 C-mol(DM) mol(ATP) (-1)) and on xylose (Y(Xyl) (max) = 0.93 C-mol(DM) C-mol(Xyl) (-1)). The results of this study highlighted that the system, at very high S(0) and X(0) values, dramatically increased its energy requirements for cell maintenance, owing to the occurrence of stressing conditions. In particular, for S(0) > 130 g L(-1), these activities required an ATP consumption of about 2.1 mol(ATP) L(-1), that is, a value about seven- to eightfold that observed at low substrate concentration. Such a condition led to an increase in the fraction of ATP addressed to cell maintenance from 47% to 81%. On the other hand, the very high percentage of ATP addressed to maintenance (> 96%) at very high cell concentration (X(0) > or = 25 g(DM) L(-1)) was likely due to the insufficient substrate to sustain the growth.

Purification of xylitol obtained by fermentation of corncob hydrolysates.

Hydrolysates obtained by autohydrolysis-posthydrolysis of corncobs were detoxified with charcoal, concentrated, supplemented with nutrients, and fermented with Debaryomyces hansenii. After biomass removal, the fermented media contained 0.1137 kg of nonvolatile components (NVC)/kg of liquor, which corresponded mainly to xylitol (0.6249 kg/kg of NVC) but also to minor amounts of inorganic components (measured as ashes), proteins, nonfermented sugars (xylose and arabinose), uronic acids, arabitol, and other nonvolatile components (ONVC). The media were subjected to further processing (sequential stages of adsorption, concentration, ethanol precipitation, concentration, and crystallization) to obtain food-grade xylitol. Adsorption experiments were carried out at various solid-to-liquor ratios. Under selected conditions (1 kg of charcoal/15 kg of liquors), the xylitol content increased to 0.6873 kg/kg of NVC, and almost total decoloration was achieved. The resulting liquor was concentrated by evaporation to increase its NVC content to 0.4032 kg/kg of liquor (corresponding to a xylitol concentration of 0.280 kg/kg of liquor), and ethanol was added to precipitate a part of the NVC (mainly proteins, but also uronic acids, ashes, and other nonvolatile compounds). Refined liquors (containing 0.7303 kg of xylitol/kg of NVC) were concentrated again, and ethanol was added (to reach 40-60% volume of the stream) to allow crystallization at -10 or -5 degrees C. Under selected conditions, 43.7% of xylitol contained in the initial fermentation broth was recovered in well-formed, homogeneous crystals, in which xylitol accounted for 98.9% of the total oven-dry weight. Material balances are presented for the whole processing scheme considered in this work.