Thermotolerant Yeast Kluyveromyces Marxianus Research Articles

Ethanol fermentation from xylose by metabolically engineered strains of Kluyveromyces marxianus

We constructed a xylose-fermenting recombinant strain of thermotolerant yeast Kluyveromyces marxianus, DMB3-7. Both xylose consumption and ethanol production were remarkably increased in DMB3-7 compared to the control strain at 30°C. Furthermore, DMB3-7 produced ethanol from xylose at both 42°C and 45°C, above which xylose metabolic activity decreased.

Improving ethanol and xylitol fermentation at elevated temperature through substitution of xylose reductase inKluyveromyces marxianus

Thermo-tolerant yeast Kluyveromyces marxianus is able to utilize a wide range of substrates, including xylose; however, the xylose fermentation ability is weak because of the redox imbalance under oxygen-limited conditions. Alleviating the intracellular redox imbalance through engineering the coenzyme specificity of NADPH-preferring xylose reductase (XR) and improving the expression of XR should promote xylose consumption and fermentation. In this study, the native xylose reductase gene (Kmxyl1) of the K. marxianus strain was substituted with XR or its mutant genes from Pichia stipitis (Scheffersomyces stipitis). The ability of the resultant recombinant strains to assimilate xylose to produce xylitol and ethanol at elevated temperature was greatly improved. The strain YZB014 expressing mutant PsXR N272D, which has a higher activity with both NADPH and NADH as the coenzyme, achieved the best results, and produced 3.55gl(-1) ethanol and 11.32gl(-1) xylitol-an increase of 12.24- and 2.70-fold in product at 42°C, respectively. A 3.94-fold increase of xylose consumption was observed compared with the K. marxianus YHJ010 harboring KmXyl1. However, the strain YZB015 expressing a mutant PsXR K21A/N272D, with which co-enzyme preference was completely reversed from NADPH to NADH, failed to ferment due to the low expression. So in order to improve xylose consumption and fermentation in K. marxianus, both higher activity and co-enzyme specificity change are necessary.

Production of Bioethanol from Carrot Pomace Using the Thermotolerant Yeast Kluyveromyces marxianus

Carrot pomace, a major agricultural waste from the juice industry, was used as a feedstock for bioethanol production by fermentation with the thermotolerant yeast Kluyveromyces marxianus. Treatment of the carrot pomace with AccelleraseTM 1000 and pectinase at 50 °C for 84 h, resulted in conversion of 42% of its mass to fermentable sugars, mainly glucose, fructose, and sucrose. Simultaneous saccharification and fermentation (SSF) at 42 °C was performed on 10% (w/v) carrot pomace; the concentration of ethanol reached 18 g/L and the yield of ethanol from carrot pomace was 0.18 g/g. The highest ethanol concentration of 37 g/L was observed with an additional charge of 10% supplemented to the original 10% of carrot pomace after 12 h; the corresponding yield was 0.185 g/g. Our results clearly demonstrated the potential of combining a SSF process with thermotolerant yeast for the production of bioethanol using carrot pomace as a feedstock.

Comparing cell viability and ethanol fermentation of the thermotolerant yeast Kluyveromyces marxianus and Saccharomyces cerevisiae on steam-exploded biomass treated with laccase

In this study, the thermotolerant yeast Kluyveromyces marxianus CECT 10875 was compared to the industrial strain Saccharomyces cerevisiae Ethanol Red for lignocellulosic ethanol production. For it, whole slurry from steam-exploded wheat straw was used as raw material, and two process configurations, simultaneous saccharification and fermentation (SSF) and presaccharification and simultaneous saccharification and fermentation (PSSF), were evaluated. Compared to S. cerevisiae, which was able to produce ethanol in both process configurations, K. marxianus was inhibited, and neither growth nor ethanol production occurred during the processes. However, laccase treatment of the whole slurry removed specifically lignin phenols from the overall inhibitory compounds present in the slurry and triggered the fermentation by K. marxianus, attaining final ethanol concentrations and yields comparable to those obtained by S. cerevisiae.

Different laccase detoxification strategies for ethanol production from lignocellulosic biomass by the thermotolerant yeast Kluyveromyces marxianus CECT 10875

In this work, laccase enzymes were evaluated to detoxify the whole slurry from steam-exploded wheat straw. For it, two different strategies, laccase treatment before or after enzymatic hydrolysis, were employed. The detoxification efficiency was analyzed on enzymatic hydrolysis and fermentation levels by the thermotolerant yeast Kluyveromyces marxianus. Laccases reduced phenolic compounds without affecting weak acids and furan derivates. A lower glucose recovery was observed when laccase treatments were carried out before enzymatic hydrolysis, phenomenon that was not showed after enzymatic hydrolysis. In contrast, both laccase treatment strategies enhanced ethanol concentrations, reducing significantly the lag phase of the yeast and allowing substrate loading increments of saccharification and fermentation broths.

Utilization capability of sucrose, raffinose and inulin and its less-sensitiveness to glucose repression in thermotolerant yeast Kluyveromyces marxianus DMKU 3-1042

Kluyveromyces marxianus possesses a useful potential to assimilate a wide variety of substrates at a high temperature, but the negative effect by coexisting glucose is critical for utilization of biomass containing various sugars. Such a negative effect on the activity of inulinase, which is the sole enzyme to hydrolyze sucrose, raffinose and inulin, has been demonstrated in K. marxianus without analysis at the gene level. To clarify the utilization capability of sucrose, raffinose and inulin and the glucose effect on inulinase in K. marxianus DMKU 3-1042, its growth and metabolite profiles on these sugars were examined with or without glucose under a static condition, in which glucose repression evidently occurs. Consumption of sucrose was not influenced by glucose or 2-deoxyglucose. On the other hand, raffinose and inulin consumption was hampered by glucose at 30°C but hardly hampered at 45°C. Unlike Saccharomyces cerevisiae, increase in glucose concentration had no effect on sucrose utilization. These sugar-specific glucose effects were consistent with the level of inulinase activity but not with that of the KmINU1 transcript, which was repressed in the presence of glucose via KmMig1p. This inconsistency may be due to sufficient activity of inulinase even when glucose is present. Our results encourage us to apply K. marxianus DMKU 3-1042 to high-temperature ethanol fermentation with biomass containing these sugars with glucose.

Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus

Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

The Crucial Role of Alcohol Dehydrogenase Adh3 inKluyveromyces marxianusMitochondrial Metabolism

The function of mitochondrial Adh3 in the thermotolerant yeast Kluyveromyces marxianus was investigated. An ADH3-disrupted mutant exhibited growth retardation on non-fermentable carbon sources, except for ethanol, and this was suppressed by supplementation with antioxidants. Detailed analysis of the phenotype revealed that the mutant showed an increase in the activity of NADH dehydrogenase, sensitivity to H(2)O(2), and accumulation of reactive oxygen species (ROS), and that these carbon sources increased the activity of succinate dehydrogenase. The increase in both activities may reflect enhanced expression of both dehydrogenases by elevation of their substrate levels. The ROS level became low when antioxidants were added. These findings suggest that the ADH3 mutation and such carbon sources cause an elevation of the substrate level of the respiratory chain and eventually of the ROS level via increased expression of primary dehydrogenases, which in turn causes cell growth retardation. Adh3 might thus play a crucial role in the control of the NADH/NAD(+) balance in mitochondria.

Bioethanol production from wheat straw by the thermotolerant yeast Kluyveromyces marxianus CECT 10875 in a simultaneous saccharification and fermentation fed-batch process

Solid content in the simultaneous saccharification and fermentation (SSF) broth should be as high as possible in order to reach higher ethanol concentration. In this work, several feeding strategies for ethanol production from steam-exploded wheat straw by Kluyveromyces marxianus CECT 10875 have been studied with the aim of obtaining higher ethanol concentrations. Previous fermentability tests as well as SSF processes showed the difficulty of using the slurry for ethanol production under the studied conditions. Notwithstanding, fed-batch SSF processes with water-insoluble solids (WIS) fraction resulted in better configuration, reaching the highest ethanol concentration (36.2 g/L) with an initial WIS content of 10% (w/v) and 4% (w/v) of substrate addition at 12 h, which meant 20% more ethanol when compared with batch SSF.

Effect of hydrothermolysis process conditions on pretreated switchgrass composition and ethanol yield by SSF with Kluyveromyces marxianus IMB4

Hot compressed liquid water was used to treat switchgrass in a method called hydrothermolysis to disrupt lignin, dissolve hemicellulose, and increase accessibility of cellulose to cellulase. Three temperatures (190, 200, and 210 °C) and hold times (10, 15, and 20 min) were tested. Switchgrass treated at 190 °C for 10 min had the greatest xylan recovery in the prehydrolyzate. Less than 0.65 g/L glucose were released into the prehydrolyzate for all pretreatment conditions, indicating most glucose was retained as cellulose in the solid substrate. 5-Hydroxymethylfurfural (HMF) and furfural formation in the prehydrolyzate were found to be less than 1 g/L for all treatments. The highest concentration of ethanol, 16.8 g/L (72% of theoretical), was produced from switchgrass pretreated at 210 °C and 15 min using simultaneous saccharification and fermentation (SSF) at 45 °C with the thermotolerant yeast Kluyveromyces marxianus IMB4 and 15 FPU cellulase/g glucan.

Simultaneous Saccharification and Fermentation of Orange Processing Waste to Ethanol Using Kluyveromyces marxianus

Thermotolerant yeast Kluyveromyces marxianus performance was evaluated at elevated temperatures (37C to 45C) in batch simultaneous saccharification and fermentations (SSFs) using deterpenized high-solids (23.5% dry solids) orange processing waste (CPW) and a mixture of commercial pectinolytic, cellulolytic, and hemicellulolytic enzymes. Effects of inoculum size, temperature, pH, and nutritional supplements on ethanol production were studied. SSFs done at 40C 1C inoculated with K. marxianus cultures containing 0.126 0.027 mg cells g-1 CPW did not perform as well as SSFs with a 10 higher inoculation (1.26 0.27 mg cells g-1 CPW). Using the low inoculum, the highest ethanol production (2.5 wt%) was achieved with the addition of calcium carbonate sufficient to maintain pH above 4.8 and supplemented with ammonium sulfate or yeast extract . SSFs with the higher K. marxianus inoculum produced significantly higher ethanol concentrations of 3.4 to 3.5 wt% (84% to 86% theoretical yield) after 48 h for all treatments at 37C and for the lower carbonate concentrations (pH 4.0 to 4.2) at 40C. These results were equivalent to the ethanol produced (3.5 wt%, 86% theoretical yield) in SSFs using Saccharomyces cerevisiae at the same inoculum level after 48 h at 37C. Ethanol was also produced in SSFs at 42C and 45C with K. marxianus, but in significantly lower (18% to 38%) amounts. S. cerevisiae does not perform well above 37C, and K. marxianus may therefore be suitable for SSF of CPW in tropical and subtropical climates where citrus is processed and costs to cool and maintain temperatures below 35C are higher than in temperate regions.

Fermentation of xylose by the thermotolerant yeast strains Kluyveromyces marxianus IMB2, IMB4, and IMB5 under anaerobic conditions

The effects of temperature and initial pH on anaerobic xylose utilization by the thermotolerant yeast Kluyveromyces marxianus IMB2, IMB4, and IMB5 were evaluated. Ethanol yield from xylose was greatest with IMB4, a temperature of 40 °C and an initial pH of 5.5. Xylitol yield was greatest with yeast strains IMB2 and IMB5, a temperature of 45 °C and an initial pH of 4.5 or 5.0. Additional fermentations of xylose at 40 and 45 °C, pH 5.5, and 20 times more cells were done using IMB4 since it exhibited the greatest ethanol yield. Ethanol and xylitol yields from xylose were greater at 40 °C than at 45 °C and volumetric production rates for ethanol and xylitol were 0.02 and 0.08 g/(l h), respectively. IMB4 xylitol yields were comparable to other xylitol producers, but with lower productivity. These strains are not good candidates for utilization of xylose under anaerobic conditions and other strategies should be explored.

Comparison of the Gene Expression Patterns of Alcohol Dehydrogenase Isozymes in the Thermotolerant YeastKluyveromyces marxianusand Their Physiological Functions

Four genes encoding alcohol dehydrogenase (Adh) isozymes in the thermotolerant yeast Kluyveromyces marxianus, a potent candidate for ethanol production at high temperatures, were investigated. Of these, KmADH3 and KmADH4 were cloned and sequenced, and their deduced amino acid sequences were compared with those of KmAdh1 and KmAdh2 and other Adhs of Kluyveromyces lactis and Saccharomyces cerevisiae. The four KmAdhs had high sequence similarity, though KmAdh3 and KmAdh4 possessed an amino-terminal extension as a mitochondrial targeting sequence, and appear to belong to the zinc-containing Adh family. These results and the results of Southern blot experiments suggest that there are at least four Adh isozymes in K. marxianus, two cytoplasmic enzymes and two mitochondrial enzymes. The expression profile revealed that KmADH genes are differently expressed depending on growth phase and carbon source, suggesting that these highly homologous Adhs play distinctive roles in cells.

Production of 2‐phenylethanol and 2‐phenylethylacetate from L‐phenylalanine by coupling whole‐cell biocatalysis with organophilic pervaporation

An integrated bioprocess for the production of the natural rose-like aroma compounds, 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc), from L-phenylalanine (L-phe) with yeasts was investigated. The hydrophobicity of the products leads to product inhibition, which can be compensated by in situ product removal (ISPR). An organophilic pervaporation unit, equipped with a polyoctylmethylsiloxane (POMS) membrane, was coupled via a bypass to a bioreactor and proved to be a suitable technique for the in situ removal of high-boiling products from culture broth. With batch cultures of the thermotolerant yeast Kluyveromyces marxianus CBS 600 in a standard medium at 35 degrees C, the use of pervaporation resulted in a double 2-PE concentration (2.2 g/L) and 1.3 g/L 2-PEAc, which only accumulated transiently in low concentrations during cultivation without ISPR. Using a previously optimized medium, the variation of the temperature from 30 degrees C to 40 degrees C caused an increase in the total conversion yield from 63% to 79%, corresponding to total product concentrations of 5.23 and 5.85 g/L, respectively. In the 40 degrees C batch experiment, the volumetric productivity (2-PE + 2-PEAc) during the exponential phase was 5.2 mmol/L h. While for 2-PE, there is still potential for further optimization, the more hydrophobic 2-PEAc was nearly completely removed from the aqueous culture broth (enrichment factor >400), resulting in highly aroma-enriched permeates. Due to the temperature-correlated performance of the pervaporation, the bioconversion was still efficient even at 45 degrees C (conversion yield: 69%). Surprisingly, at 45 degrees C, the molar ratio of the two products inverted and 2-PEAc turned out to be the main product (4.0 g/L), which opens easy control of the reaction's selectivity by external means. Retrofitting the process with interim heating and cooling equipment to use different temperature levels for cultivation and pervaporation resulted in a decreased yield and product concentration caused by multiple stress factors. The medium composition affected the pervaporation efficiency with molasses acting detrimental.

Proceso de sacarificación y fermentación simultáneas para la conversión de la fracción celulósica del residuo de la extracción del aceite de oliva en etanol

In this work, the residue generated in the new two-step centrifugation process for olive oil extraction is assessed for the production of bioethanol. Both olive pulp and fragmented stones fractions comprised in such residue are analyzed and tested at laboratory scale for bioconversion to ethanol by a simultaneous saccharification and fermentation (SSF) process. Firstly, optima conditions for the enzymatic hydrolysis step of steam-exploded pretreated sustrates were determined. Then, simultaneous saccharification and fermentation process was assayed using the thermotolerant yeast Kluyveromyces marxianus in different assay conditions. For the selected conditions, 9 kg of unpretreated pulp or 6 kg of pretreated fragmented stones (both based on dry matter) would be necessary to obtain 1 liter of ethanol.

Iminodiacetate and Nitrilotriacetate Degradation by Kluyveromyces marxianus IMB3

The thermotolerant yeast Kluyveromyces marxianus IMB3 was capable of utilising either iminodiacetate or nitrilotriacetate as a sole source of nitrogen for growth. Cell extracts contained iminodiacetate dehydrogenase and nitrilotriacetate monooxygenase activities, suggesting the presence in the yeast of orthologues of these bacterial enzymes. The activities were not detectable in complete medium-growth cells, nor in nitrogen-starved cells, suggesting an inducible biodedgradation pathway for biodegradation of these xenobiotics, which has not been previously reported in a eukaryotic cell system. This observation emphasises the hitherto unrealised importance of yeast strains in the biodegradation of xenobiotics in the environment.

Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis

In this work, a recycled paper-derived feedstock was used to produce ethanol by the simultaneous saccharification and fermentation (SSF) process using the thermotolerant yeast Kluyveromyces marxianus CECT 10875. At standard SSF conditions, the highest yield (about 80% of theoretical) was obtained at low substrate concentration and high enzyme loading. With increasing substrate concentration, mixing difficulties appeared which prevented an adequate SSF process performance and limited ethanol production. An SSF fed-batch procedure was then used which permitted an increase in substrate concentrations while maintaining SSF yields similar to that obtained at standard SSF, thus allowing an increased final ethanol production (about 18 g/l).

Ethanol adaptation in a thermotolerant yeast strain Kluyveromyces marxianus IMB3

The maximum ethanol concentration produced from glucose in defined media at 45 degrees C by the thermotolerant yeast Kluyveromyces marxianus IMB3 was 44 g L-1. Acclimatisation of the strain through continuous culture at ethanol concentrations up to 80 g L-1, shifted the maximum ethanol concentration at which growth was observed from 40 g L-1 to 70 g L-1. Four isolates were selected from the continuous culture, only one of which produced a significant increase in final ethanol concentration (50 +/- 0.4 g L-1), however in subsequent fermentations, following storage on nutrient agar plates, the maximum ethanol concentration was comparable with the original isolate, The maximum specific ethanol production rates (approximately 1.5 g (gh)(-1)) were also comparable with the original strain except for one isolate (0.7 g (gh)(-1)). The specific ethanol productivity decreased with ethanol concentration; this decrease correlated linearly (rval 0.92) with cell viability. Due to the transience of induced ethanol tolerance in the strain it was concluded that this was not a valid method for improving final ethanol concentrations or production rates.

Fermentation of molasses using a thermotolerant yeast, Kluyveromyces marxianus IMB3: simplex optimisation of media supplements.

The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45 degrees C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308-313, 1965) simplex optimisation method. The optimum concentration of the supplements were 0.576 g1(-1) magnesium sulphate, 0.288 g1(-1) potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate.

The effects of phosphoric acid pretreatment on conversion of cellulose to ethanol at 45�C using the thermotolerant yeast Kluyveromyces marxianus IMB3

Here we report on the effects of phosphoric acid pretreated cellulose as a substrate for ethanol production by K. marxianus IMB3 using simultaneous saccharification and fermentation systems at 45°C. With untreated, milled filter paper as substrate the maximum amount of ethanol produced was 25% of the maximum theoretical yield. After pre-treatment with 100% phosphoric acid, the yield increased to 42% of the maximum theoretical yield. When untreated microcrystalline cellulose was used as the fermentation substrate, yields of ethanol as 45°C amounted to 16% of the maximum theoretical yield whereas pretreatment of the substrate with phosphoric acid resulted in an increase in ethanol production to 69% of the maximum theoretical yield. This suggests that pretreatment of substrate with phosphoric acid would contribute to a reduction in the amount of exogenous enzyme needed.