Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26 g/g/h) higher than those presented by the ETS1 (0.22 g/g/h), ETS2 (0.17 g/g/h), and ETS3 (0.17 g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

Utilization of whey powder as substrate for low-cost preparation of ß-galactosidase as main product, and ethanol as by-product, by a litre-scale integrated process.

Whey powder, a by-product of dairy industry, is an attractive raw material for value-added products. In this study, utilization of whey powder as substrate for low-cost preparation of ß-galactosidase as main product and ethanol as by-product were investigated by a litre-scale integrated strategy, encompassing fermentation, isolation, permeabilization and spray drying. Firstly, through development of low-cost industrial culture and fed-batch strategies by Kluyveromyces lactis, 119.30U/mL ß-galactosidase activity and 16.96mg/mL by-product ethanol were achieved. Afterward, an up-dated mathematic model for the recycling permeabilization was established successfully and 30.4g cells sediment isolated from 5L fermentation broth were permeabilized completely by distilled ethanol from broth supernatant. Then ß-galactosidase product with 5.15U/mg from protection of gum acacia by spray drying was obtained. Furthermore, by-product ethanol with 31.08% (v/v) was achieved after permeabilization. Therefore, the integrated strategy using whey powder as substrate is a feasible candidate for industrial-scale implementation.

Assessment of volatile and non-volatile compounds in durian wines fermented with four commercial non-Saccharomyces yeasts.

BACKGROUND: Chemical compositions of durian wines fermented with Metschnikowia pulcherrima Flavia, Torulaspora delbrueckii Biodiva, Pichia kluyveri FrootZen and Kluyveromyces thermotolerans Concerto were investigated. RESULTS: Sucrose was not utilized by M. pulcherrima and P. kluyveri, resulting in little formation of ethanol (0.3-0.5%, v/v), while about 7% ethanol was produced by the other two yeasts. Volatiles such as esters and sulfur-containing compounds were synthesized or catabolized and distinctive differences existed among yeasts. Larger amounts of higher alcohols and ethyl esters were detected in wines fermented by T. delbrueckii and K. thermotolerans, whereas M. pulcherrima and P. kluyveri produced more acetate esters such as ethyl acetate (1034.43 and 131.05 mg L(-1) respectively) and isoamyl acetate (0.56 and 27.68 mg L(-1) respectively). Most endogenous sulfur volatiles such as disulfides declined to trace levels, but new ones such as thioesters were formed. Sulfur volatiles in wines fermented by T. delbrueckii accounted for 0.20% relative peak area (RPA), followed by K. thermotolerans (0.23% RPA), P. kluyveri (1.43% RPA) and M. pulcherrima (4.16% RPA). CONCLUSION: The findings showed that a more complex flavor could result from fermentation with different non-Saccharomyces yeasts and the typical durian odor would still remain.

Ethanol fermentation from molasses at high temperature by thermotolerant yeast Kluyveromyces sp. IIPE453 and energy assessment for recovery.

High temperature ethanol fermentation from sugarcane molasses B using thermophilic Crabtree-positive yeast Kluyveromyces sp. IIPE453 was carried out in batch bioreactor system. Strain was found to have a maximum specific ethanol productivity of 0.688 g/g/h with 92 % theoretical ethanol yield. Aeration and initial sugar concentration were tuning parameters to regulate metabolic pathways of the strain for either cell mass or higher ethanol production during growth with an optimum sugar to cell ratio 33:1 requisite for fermentation. An assessment of ethanol recovery from fermentation broth via simulation study illustrated that distillation-based conventional recovery was significantly better in terms of energy efficiency and overall mass recovery in comparison to coupled solvent extraction-azeotropic distillation technique for the same.

Application of comet assay for the assessment of DNA damage caused by chemical genotoxins in the dairy yeast Kluyveromyces lactis.

Kluyveromyces lactis, also known as dairy yeast, has numerous applications in scientific research and practice. It has been approved as a GRAS (Generally Recognized As Safe) organism, a probiotic, a biotechnological producer of important enzymes at industrial scale and a bioremediator of waste water from the dairy industry. Despite these important practical applications the sensitivity of this organism to genotoxic substances has not yet been assessed. In order to evaluate the response of K. lactis cells to genotoxic agents we have applied several compounds with well-known cyto- and genotoxic activity. The method of comet assay (CA) widely used for the assessment of DNA damages is presented here with new special modifications appropriate for K. lactis cells. The comparison of the response of K. lactis to genotoxins with that of Saccharomyces cerevisiae showed that both yeasts, although considered close relatives, exhibit species-specific sensitivity toward the genotoxins examined.

Stoichiometric identification with maximum likelihood principal component analysis.

This study presents an effective procedure for the determination of a biologically inspired, black-box model of cultures of microorganisms (including yeasts, bacteria, plant and animal cells) in bioreactors. This procedure is based on sets of experimental data measuring the time-evolution of a few extracellular species concentrations, and makes use of maximum likelihood principal component analysis to determine, independently of the kinetics, an appropriate number of macroscopic reactions and their stoichiometry. In addition, this paper provides a discussion of the geometric interpretation of a stoichiometric matrix and the potential equivalent reaction schemes. The procedure is carefully evaluated within the stoichiometric identification framework of the growth of the yeast Kluyveromyces marxianus on cheese whey. Using Monte Carlo studies, it is also compared with two other previously published approaches.

Cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025.

The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated in this work. This strain was preliminarily cultivated in a synthetic medium containing glucose and xylose and was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pretreatment and used as fermentation media. This hydrolysate is rich in glucose, xylose, and arabinose and contains traces of formic acid and acetic acid. In batch fermentations of CABH at pH 4.5, the strain produced only ethanol. The effects of temperature on the kinetic parameters of ethanol fermentation by K. marxianus CE025 using CABH were also evaluated. Maximum specific growth rate (µ(max)), overall yields of ethanol based on glucose consumption [Formula: see text] and based on glucose + xylose consumption (Y ( P/S )), overall yield of ethanol based on biomass (Y ( P/X )), and ethanol productivity (P (E)) were determined as a function of temperature. Best results of ethanol production were achieved at 30°C, which is also quite close to the optimum temperature for the formation of biomass. The process yielded 12.36 ± 0.06 g l(-1) of ethanol with a volumetric production rate of 0.257 ± 0.002 g l(-1) h(-1) and an ethanol yield of 0.417 ± 0.003 g g(-1) glucose.

The yeast Kluyveromyces marxianus and its biotechnological potential.

Strains belonging to the yeast species Kluyveromyces marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. As a consequence, several different biotechnological applications have been investigated with this yeast: production of enzymes (beta-galactosidase, beta-glucosidase, inulinase, and polygalacturonases, among others), of single-cell protein, of aroma compounds, and of ethanol (including high-temperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production. Compared to its congener and model organism, Kluyveromyces lactis, the accumulated knowledge on K. marxianus is much smaller and spread over a number of different strains. Although there is no publicly available genome sequence for this species, 20% of the CBS 712 strain genome was randomly sequenced (Llorente et al. in FEBS Lett 487:71-75, 2000). In spite of these facts, K. marxianus can envisage a great biotechnological future because of some of its qualities, such as a broad substrate spectrum, thermotolerance, high growth rates, and less tendency to ferment when exposed to sugar excess, when compared to K. lactis. To increase our knowledge on the biology of this species and to enable the potential applications to be converted into industrial practice, a more systematic approach, including the careful choice of (a) reference strain(s) by the scientific community, would certainly be of great value.

A reverse transcriptase PCR technique for the detection and viability assessment of Kluyveromyces marxianus in yoghurt.

A fast and sensitive reverse transcriptase PCR (RT-PCR) method was developed for the detection of viable Kluyveromyces marxianus in yoghurt. Yeast-specific primers were used with the RT-PCR to evaluate the suitability of 18S rRNA as a target for the detection of viable yeasts in pure culture and yoghurt. The RT-PCR assay was able to detect down to 10(2) CFU ml(-1) in yoghurt samples contaminated with viable yeast cells. Application of the RT-PCR method to commercial yoghurt samples demonstrated the utility of this technique for detection of low concentrations of viable yeast cells in naturally contaminated dairy products. The 18S rRNA molecule is an appropriate target for cell viability assessment because of its limited persistence after cell death and the resultant high level of sensitivity of the assay.

Yeast (different sources and levels) as protein source in diets of reared piglets: effects on protein digestibility and N-metabolism.

The aim of this study was to examine the feeding value of different yeasts as a substitute for soya bean meal, the main protein source in diets of weaned piglets. Tested two yeasts were already available on the market, Saccharomyces cerevisiae and Kluyveromyces lactis (beer and milk yeast), which replaced 40% of the soya bean meal in the diets. Furthermore, a yeast (Kluyveromyces fragilis) grown on whey, a side-product of cheese production, was used in increasing concentrations in the diets, so that increasing amounts of the soya bean meal (20%, 40% and 60%) could be replaced. As proved in these experiments, a replacement of 60% of the soya protein with whey yeast protein had positive effects on the performances (daily weight gain) and on the N-metabolism and did not have negative effects on the health or the faeces consistency. The whey yeast stands out because of its high protein quality (N-digestibility and N-retention). Furthermore, the replacement of soya bean meal with highly digestible yeasts is welcomed under the aspect of animal health, because of the reduction of anti-nutritive soya components (stachyose, glycinin) in diets of weaned piglets. The controlled production conditions of the yeasts result in a high feed safety; in addition, the yeast as an end-of-pipe-product is a resource conserving and valuable feed. A main stimulus for the use of yeasts, however, in a food production controlled by economic standpoints, is their price and the costs of other competing feeds.

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast.

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.

Decolorization of Remazol Black-B using a thermotolerant yeast, Kluyveromyces marxianus IMB3.

The ability of Kluyveromyces marxianus IMB3 to decolorize Remazol Black-B dye was investigated. The effect of environmental conditions, such as pH and temperature were examined. No noticeable effects on decolorization were observed when pH varied from 3.0-5.5. Maximum colour removal, 98%, was achieved at 37 degrees C. Little or no colour removal was detected when K. marxianus IMB3 was incubated under anaerobic conditions. Further investigation, in which decolorization was monitored under extreme temperatures and low pH (to inhibit growth) and using ten fold dense inoculum, revealed that decolorization was due to biosorption to the yeast cells and not due to a metabolic reaction.

Heterologous Kluyveromyces lactis beta-galactosidase production and release by Saccharomyces cerevisiae osmotic-remedial thermosensitive autolytic mutants.

The beta-galactosidase from Kluyveromyces lactis is a high molecular weight protein with commercial interest. A major drawback of its industrial production is the high cost associated with extraction and downstream processing due to its intracellular nature. In this work, the effectiveness of the utilization of Saccharomyces cerevisiae LD1 and LHDP1 strains, osmotic-remedial mutants which lyse at 37 degrees C, for the heterologous production and release into the extracellular medium of this protein has been proved. The highest absolute values of released beta-galactosidase have been obtained with the protease-deficient strain LHDP1 by osmotic shock.