表面活性剂对D-阿拉伯糖醇发酵的影响

为提高D-阿拉伯糖醇的产量,研究不同类型表面活性剂对德巴利汉逊酵母(Debaryomyces hansenii)发酵生产D-阿拉伯糖醇的影响。结果表明:阳离子和阴离子表面活性剂对D-阿拉伯糖醇的生成几乎没有影响,部分非离子表面活性剂对D-阿拉伯糖醇的生产有促进作用,其中Trition X-100的影响最为显著。在不同发酵时间加入不同浓度的Trition X-100均对D-阿拉伯糖醇的生产有促进作用,当发酵24 h添加30 g/LTrition X-100时,D-阿拉伯糖醇的产量达到最高(92.9 g/L),相比于对照增加了27.2%。     

Casein Digestion by Debaryomyces hansenii Isolated from Cheese

To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on β-casein. The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both α_s- and β-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.     

Phosphate limitation stress induces xylitol overproduction by Debaryomyces hansenii

The physiological responses of xylose-grown Debaryomyces hansenii were studied under different nutritive stress conditions using continuous cultivation at a constant dilution rate of 0.055 h⁻¹. Metabolic steady-state data were obtained for xylose, ammonium, potassium, phosphate and oxygen limitation. For xylose and potassium limitation, fully oxidative metabolism occurred leading to the production of biomass and CO₂ as the only metabolic products. However, potassium-limiting cultivation was the most severe nutritional stress of all tested, exhibiting the highest xylose and O₂ specific consumption rates along with the lowest biomass yield, 0.22 g g⁻¹ xylose. It is suggested that carbon was mainly channelled to meet the cellular energy requirements for potassium uptake. For the other limiting nutritional conditions increasing amounts of extracellular xylitol were found for ammonium, phosphate and oxygen limitation. Although xylitol excretion is not significant for ammonium limitation, the same is not true for phosphate limitation where the xylitol productivity reached 0.10 g l⁻¹ h⁻¹, about half of that found under oxygen-limiting conditions, 0.21 g l⁻¹ h⁻¹. This work is the first evidence that xylitol production by D. hansenii might not only be a consequence of a redox imbalance usually attained under semi-aerobic conditions, but additional physiological mechanisms must be involved, especially under phosphate limitation. Cell yields changed drastically as a function of the limiting nutrient, being 0.22, 0.29, and 0.39 g g⁻¹ xylose for potassium, oxygen and phosphate limitation, respectively, and are a good indicator of the severity of nutritive stress.     

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 °C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.     

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.     

Relationship between growth and pH gradients of individual cells of Debaryomyces hansenii as influenced by NaCl and solid substrate

AIMS: To examine the relationship between the growth and pH gradients of Debaryomyces hansenii at a single-cell level. METHODS AND RESULTS: Using bioimaging techniques, the cell areas and early pH gradients (Delta pH(10)), i.e. the pH gradients determined 10 min after initiation of experiments, were determined for single cells of two D. hansenii strains in fluid and on solid (agar) substrate with and without 8% (w/v) NaCl. The combination of NaCl and solid substrate prolonged the growth initiation of both D. hansenii strains additively. In all our experiments, primarily two groups of cells existed; a vital group consisting of growing single cells with intact early pH gradients, and a group of dead cells without early pH gradients. CONCLUSIONS: Our results show that growth initiation of the D. hansenii cells is severely affected by NaCl and to a lesser extent by the type of substrate in an additive and strain dependent way. Moreover, the early pH gradient of a vital D. hansenii cell cannot be correlated with the rate of its subsequent growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the growth and pH gradients of D. hansenii on solid surfaces in the presence of NaCl.     

Xylitol production from barley bran hydrolysates by continuous fermentation with Debaryomyces hansenii

The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l^–1 h^–1 were obtained for dilution rates of 0.008–0.088 h^–1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l^–1 h^–1) was reached at a dilution rate of 0.284 h^–1.     

Activation of fermentation by salts in Debaryomyces hansenii

The presence of 1.0 M KCl or NaCl during growth of Debaryomyces hansenii results in increased ethanol production. An additional increase of fermentation was observed when the salts were also present during incubation under nongrowing conditions. Extracts of cells grown in the presence of salt showed increased alcohol dehydrogenase and phosphofructokinase activities, indicating that these enzymes are responsible for the increased fermentation capacity. This is confirmed by measurements of the glycolytic intermediates. The increased fermentation capacity of the cells grown with salts seems to enable them to cope with the additional energy required for uptake and/or efflux of cations.     

Differential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.     

A procedure for enrichment and isolation of mutants of the salt-tolerant yeast Debaryomyces hansenii having altered glycerol metabolism

Abstract The salt-tolerant yeast Debaryomyces hansenii produces and accumulates glycerol when subjected to salt stress, whereby the buoyant density of the cells is changed. This property allows for enrichment of mutants with altered glycerol metabolism by density gradient centrifugation. Colonies derived from cells with rapidly changing density following an osmotic shock were screened for increased glycerol production by observing their ability to support growth of a glycerol-requiring strain of Escherichia coli . The glycerol overproducing phenotype of two isolates was confirmed by chemical analysis.     

Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. The molecular mechanisms underlying its extreme osmotolerance and halotolerance have drawn considerable attention in the recent past. However, progress in this regard has been limited due to lack of availability of a transformation system and molecular tools to study the functions of the genes in D. hansenii . Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain and the DhHIS4 gene as the selectable marker. By screening the D. hansenii genomic library, we have isolated several autonomous replication sequences that can be used for constructing a replicating vector. Moreover, our study is the first to demonstrate gene disruption in D. hansenii by homologous recombination.     

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⁻¹h⁻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.     

Isolation of a psychrotolerant Debaryomyces hansenii strain from fermented tea plant (Camellia sinensis) leaves

Abstract

A psychrotolerant, halotolerant and alkalophilic yeast was isolated from fermented leaves of Camellia sinensis Kuntze, the tea plant. The yeast strain, named Tea-Y1, was both phenotypically and genotypically identified as belonging to the species Debaryomyces hansenii. This assignment was confirmed by scanning and transmission electron microscopy. The analysis of growth curves demonstrated the ability this yeast strain to grow in a temperature range between 4°C and 28°C, with an optimum of 23°C. The ecology of this yeast in the C. sinensis phyllosphere, as well as its possible role in tea fermentation and storage, with particular reference to iced tea, are discussed.     

汉逊德巴利酵母发酵葡萄糖生产D-阿拉伯糖醇

从378株耐高渗酵母中,筛选到1株由葡萄糖发酵高产D-阿拉伯糖醇的酵母。通过生理生化和分子生物学的鉴定,证实该菌株为Debaryomyces hansenii,保藏编号CICIM Y 0504。研究该酵母摇瓶发酵的主要影响因素,确定其摇瓶发酵条件为:葡萄糖200 g/L,酵母膏10 g/L,初始pH值3,装液量20 mL/250 mL,温度30℃。在此条件下发酵120 h,D-阿拉伯糖醇浓度达90.37 g/L,转化率45.18%。在15 L发酵罐对该酵母进行扩大培养,结果表明,初始葡萄糖浓度200 g/L的分批发酵产D-阿拉伯糖醇64.07 g/L,转化率33.94%;葡萄糖浓度控制在30～50 g/L的分批补料发酵产D-阿拉伯糖醇125 g/L,转化率37.5%。研究结果对葡萄糖发酵生产D-阿拉伯糖醇工业化的实现具有重要启示。     

Carbon material and bioenergetic balances of xylitol production from corncobs by Debaryomyces hansenii

The effect of oxygenation on xylitol production by the yeast Debaryomyces hansenii has been investigated in this work using the liquors from corncob hydrolysis as the fermentation medium. The concentrations of consumed substrates (glucose, xylose, arabinose, acetate and oxygen) and formed products (xylitol, arabitol, ethanol, biomass and carbon dioxide) have been used, together with those previously obtained varying the hydrolysis technique, the level of adaptation of the microorganism, the sterilization procedure and the initial substrate and biomass concentrations, in carbon material balances to evaluate the percentages of xylose consumed by the yeast for the reduction to xylitol, alcohol fermentation, respiration and cell growth. The highest xylitol concentration (71 g/L) and volumetric productivity (1.5 g/L.h) were obtained semiaerobically using detoxified hydrolyzate produced by autohydrolysis-posthydrolysis, at starting levels of xylose (S(0)) and biomass (X(0)) of about 100 g/L and 12 g(DM)/L, respectively. No less than 80% xylose was addressed to xylitol production under these conditions. The experimental data collected in this work at variable oxygen levels allowed estimating a P/O ratio of 1.16 mol(ATP)/mol(O). The overall ATP requirements for biomass production and maintenance demonstrated to remarkably increase with X(0) and for S(0) >or= 130 g/L and to reach minimum values (1.9-2.1 mol(ATP)/C-mol(DM)) just under semiaerobic conditions favoring xylitol accumulation.     

Dimorphic behaviour of Debaryomyces hansenii grown on barley bran acid hydrolyzates

Debaryomyces hansenii exhibited yeast-to-mycelium dimorphism in the continuous fermentation of xylose-containing media made from acid hydrolyzates of barley bran. The lower the dilution rate, the earlier the yeast-to-mycelia transition occurred. Within a selected range of dilution rates, the yeast morphology was reversibly affected by the dissolved O₂: low aeration caused the transition from oval cells to hyphae, and further increases in dissolved O₂ concentration resulted in recuperation of the oval shape. Under the operational conditions assayed, xylitol was the major fermentation product when the yeast was in both morphological forms, whereas the production of ethanol was increased when the yeast grew under hyphal morphology and oxygen limitation. The lower xylose consumption corresponded to the yeast-to-mycelia transition. In media made with commercial sugars (xylose or glucose), the yeast-to-mycelia transition was induced by adding selected amounts of acid-soluble lignin.