Kluyveromyces Marxianus Cells Research Articles

Detection of Kluyveromyces marxianus and other spoilage yeasts in yoghurt using a PCR-culture technique

A combined PCR-culture technique was developed for the detection of viable yeasts in yoghurt samples. Yoghurt samples were inoculated with either viable or heat-inactivated Kluyveromyces marxianus cells, and analyzed before and after incubation for 24 h at 25 °C under agitation. DNA was extracted from the samples and amplified using yeast-specific primers targeted at the gene coding for the 18S rRNA. A 251-bp fragment was amplified by the Polymerase Chain Reaction from the yoghurt samples containing initial yeasts counts of 10 cfu g − 1 or higher, whereas no PCR product was generated from control uninoculated yoghurt samples. Comparison of PCR results obtained before and after the incubation step was used to assess yeast viability. Viability was also confirmed by plating on Sabouraud–Dextrose–Chloramphenicol Agar. Moreover, comparison of the results obtained using PCR-culture and plate count methods for the analysis of commercial yoghurt samples, demonstrated that the PCR-culture technique developed in this work can be very useful for the rapid detection of viable spoilage yeasts in dairy industries.

The NADP+-dependent glutamate dehydrogenase of the yeast Kluyveromyces marxianus responds to nitrogen repression similarly to Saccharomyces cerevisiae

NADP+-dependent glutamate dehydrogenase (NADP+-Gdh) is the first step in ammonia assimilation pathway in Saccharomyces cerevisiae and the knowledge of its regulation is the key for many biotechnological purposes such as single cell protein production. The regulation of NADP+-Gdh activity in Kluyveromyces marxianus cells was evaluated under different ammonia supply in batch cultivations. The results showed that K. marxianus NADP+-Gdh activity is induced over a narrow range of extracellular ammonia supply, being repressed by both high ammonia concentration and the glutamate formed. This activity is not growth-associated and may function mainly to trace low amounts of ammonia after growth cessation. The results demonstrated that NADP+-Gdh may not be the main enzyme for ammonia assimilation in K. marxianus, as it has been postulated for K. lactis, instead is subjected to the same regulatory mechanism described for S. cerevisiae.

NMR confirmation of an alkali-insoluble glucan from Kluyveromyces marxianus cultivated on a lactose-based medium

Yeast cells of Kluyveromyces marxianus grown in a sterile lactose-based medium, were extracted with sodium hydroxide to give a fraction of alkali-insoluble yeast (AIY) glucan. From 1H- and 13C-NMR characterization it was concluded that the glucan consisted mostly of β(1 → 3) linkages and was similar to that obtained from Saccharomyces cerevisiae used in the preparations for skin revitalization and repair. The molecular weight of the glucan was determined to be approximately 3 × 105 Da. Therefore, K. marxianus grown on a lactose-based medium similar to whey, a waste stream of the dairy industry, could be a commercial source of β(1 → 3) glucans for use in the food and pharmaceutical industries.

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO2 of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.

Immobilization system of Kluyveromyces marxianus cells in barium alginate for inulin hydrolysis

A suspension of Kluyveromyces marxianus cells (256 mg ml −1) with inulinase activity were immobilized in barium alginate treated with glutaraldehyde. After five runs of inulin hydrolysis, the immobilized cells system had up to 85% residual activity. When beads were heat treated at 65 °C for 5 min, the hydrolysis rate of inulin was improved without affecting the stability of the system in five hydrolysis batches. Immobilized enzyme was thermostable at temperatures between 45 and 65 °C. The system efficiency ( η) was highly dependent on bead diameter, reaching values up to 0.79 with a bead diameter of 1.43 mm. Inulin diffused into alginate matrix with a diffusion coefficient ( D E) of 2.5×10 −6 cm 2 s −1, resulting in apparent kinetic values of the immobilized enzyme of K′ m=0.522 mM and V′ max=113.7 μmol min −1; while values for free enzyme were 0.15 mM and 250 μmol min −1. Thiele module values ( Φ) were close to 1, indicating that the limitations of the system were due to diffusional resistance of substrate.

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977).

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was demonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.

A simple and rapid method for lithium acetate-mediated transformation of Kluyveromyces marxianus cells

Efficient plasmid transformation of Kluyveromyces marxianus cells of 1.9 × 103 transformant μg−1 DNA with an episomal plasmid was achieved by the use of a simple lithium acetate method with the addition of 10 mM DTT and an increased heat shock temperature of 47 °C. This method is shown to be also efficient for replicative plasmids. Therefore, we suggest its use as a routine method to transform K. marxianus cells.

The effect of copper(II) ions on the growth and bioaccumulation properties of some yeasts

The influence of copper(II) ions on the growth and bioaccumulation properties of adapted and growing cells of Saccharomyces cerevisiae, Kluyveromyces marxianus, Schizosaccharomyces pombe and Candida sp. was studied. The level of copper(II) accumulation and microbial growth were dependent on the pH and initial copper(II) concentration. Optimum pH values for maximum copper(II) accumulation were determined as 4.0, 5.0, 4.0 and 4.0 for S. cerevisiae, K. marxianus, S. pombe and Candida sp., respectively. The results indicated that Candida sp. and K. marxianus are able to accumulate large amounts of copper(II) from feed medium at higher copper(II) concentrations without loosing their biological activities.

Cell wall surface properties and flocculence of a Kluyveromyces marxianus strain

Yeast flocculation is under genetic control and is described as a cell wall interaction. This characteristic of yeast cells has been traditionally used in industrial fermentation processes. The surface characteristics of the cell walls are expected to be a determinant factor in the aggregation mechanism. Results confirming this have been reported for Saccharomyces strains. It is important to extend these studies to other genera. Among them, due to its potential industrial interest, Kluyveromyces strains must be considered. In this work are reported results relating cell wall surface properties (hydrophobicity and electrophoretic mobility) with the flocculation ability of a strain of Kluyveromyces marxianus. The effect of proteolytic enzymes, pH, salts and sugars on flocculation was also studied. The results obtained clearly demonstrate that cell wall hydrophobicity is a major determinant in the flocculation ability of the Kluyveromyces marxianus cells.

Lactose hydrolysis in an aqueous two-phase system by whole-cell β-galactosidase of Kluyveromyces marxianus: Partition and separation characteristics

The selection of suitable aqueous two-phase system for lactose hydrolysis by whole-cell β-galactosidase is described. Partitioning of substrate, products, biocatalyst and viable, permeabilized and glutaraldehyde-treated cells of Kluyveromyces marxianus in various systems containing polyethylene glycol and dextran was studied. Some characteristics of the selected systems were also established.

Optimization of β-galactosidase extraction from Kluyveromyces marxianus

The influence of several parameters, such as temperature, pH, and concentration of buffer and solvent, on the release of β-galactosidase from Kluyveromyces marxianus cells was studied. In optimal conditions (37°C, pH 9.5–10.5) greater than 90% of the intracellular β-galactosidase activity was released into 0.1-0.5 m phosphate buffer after 1.5-2.0 h treatment with 1% chloroform. The described method is simple, effective, relatively fast, and selective.

Inhibition of transport systems in yeast by photodynamic treatment with Toluidine blue

Photodynamic treatment of yeast with the sensitizer Toluidine blue results in loss of cell viability. In previous investigations it was suggested that plasma membrane damage might be responsible for the loss of colony forming capacity. In this context the influence of photodynamic treatment on transmembrane transport systems was studied. It appeared that the uptake of the sugars glucose, lactose and galactose, the amino acids arginine, phenylalanine, glycine and aspartic acid and of the inorganic compound phosphate was inhibited by photodynamic treatment. The different elements of the energy providing system necessary for active transport, viz the plasma membrane ATPase and the protonmotive force, were not significantly affected by Toluidine blue and light, indicating that inhibition of transport is not caused by a reduction of the membrane potential or the transmembrane pH gradient. These observations suggest that the transport carriers themselves were damaged by treatment with Toluidine blue and light. This could be confirmed in experiments, in which the lactose and galactose transport proteins of treated and untreated cells were reconstituted in plasma membrane vesicles. It appeared that the carriers, obtained from photodynamically treated Kluyveromyces marxianus cells, had lost their transport capacity.

Flocculation of Kluyveromyces marxianus is induced by a temperature upshift.

An upshift of the growth temperature from 26 to 40 degrees C in the presence of calcium leads to the aggregation of Kluyveromyces marxianus cells and to the formation of flocs. Analysis of cell wall proteins, either by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of extractable mannoproteins or by immunolocalization, revealed an accumulation of a protein with Mr 37 kDa(p37), upon flocculation. Immunological studies confirmed the homology of this protein with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When mRNA isolated from cells growing at 40 degrees C was translated in vitro, a 35 kDa newly labelled protein was synthesized and immunoprecipitation assays showed that this protein is recognized by p37-antiserum, suggesting that the 35 kDa polypeptide might be an unglycosylated precursor for of p37. The results indicated that the presence of this cell wall mannoprotein closely related to GAPDH is dependent on the growth temperature, suggesting its role as adhesin.

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.

The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperature range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be ca. pH 5. At this pH, the specific growth rate was 0.35 h(-1) and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6.

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.

Technical note: Application of immobilized cells of Kluyveromyces marxianus for continuous hydrolysis to fructose of fructans in Jerusalem artichoke extracts

Dead cells of Kluyveromyces marxianus having inulase (β‐D‐fructo fructanohydrolase E.C.3.2.1.7) activity were immobilized in alginate beads and used as a biocatalyst in a packed bed reactor and a stirred batch reactor. Fructosans of Jerusalem artichoke tubers, after extraction, were utilized for continuous or semi‐continuous production of fructose. In a bed reactor packed with 100 ml of beads, a volumetric productivity of 36 g/l/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a half life of 28 days was observed for the biocatalyst. Using artichoke extract containing 20% fructan solution, 98% conversion could be achieved in a batch reactor in 20 hr. Repeated cycling of beads resulted in considerable loss of catalyst from the reactor and subsequent loss in catalytic activity, thus giving a half life of only 14 days.

Continuous Hydrolysis of Fructans in Jerusalem Artichoke Extracts using Immobilized Nonviable Cells of Kluyveromyces marxianus

ABSTRACTNon‐viable immobilized cells of Kluyveromyces marxianus in alginate beads having inulase (ß‐2,1‐fructan fructano‐hydrolyase E.C.3.2.1.7) acitivity were used as biocatalyst in a packed bed reactor. Extracts of Jerusalem artichoke tubers were contacted with the biocatalyst for continuous conversion of the fructan component to fructose. In a bed reactor packed with 100 mL of beads, a volumetric productivity of 136 g/L/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a 55% loss in the original activity was observed, giving a half life for the biocatalyst of 28 days.

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β- d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol −1, respectively. K m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.

Improvement of inulinase stability of calcium alginate immobilized Kluyveromyces marxianus cells by treatment with hardening agents

To improve the inulinase (2,1-β- d-fructan fructanohydrolase, EC 3.2.1.7) stability of calcium alginate-immobilized Kluyveromyces marxianus cells, treatment with hardening agents has been investigated. Treatment of immobilized cells with some polycationic polymers resulted in little decrease in volumetric reactor productivity, but was most effective in increasing the inulinase stability of the immobilized cells. Inulinase stability of glutaraldehyde-hardened immobilized cells increased two-fold, and for hexamethylenediamine + glutaraldehyde and polyethyleneimine + glutaraldehyde-hardened cells increased six-fold compared with that of the unhardened cells.