High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40 degrees C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

液泡蛋白酶B对酿酒酵母高温乙醇发酵效率的影响

【目的】提高酿酒酵母的高耐温性,从而提高菌株在高温下的乙醇发酵性能。【方法】利用染色体整合过表达酿酒酵母液泡蛋白酶B编码基因PRB1。【结果】在41 °C高温条件下进行乙醇发酵,过表达PRB1基因的重组酿酒酵母菌株可在31 h内消耗全部的葡萄糖,而对照菌株在相同时间内仅消耗不到一半的葡萄糖。【结论】利用蛋白酶B基因过表达可构建耐高温酿酒酵母菌株,提高在高温条件下乙醇的发酵效率。     

Physiological diversity and trehalose accumulation in Schizosaccharomyces pombe strains isolated from spontaneous fermentations during the production of the artisanal Brazilian cachaça

Twenty-seven Schizosaccharomyces pombe isolates from seven cacha?a distilleries were tested for maximum temperature of growth and fermentation, osmotolerance, ethanol resistance, invertase production, and trehalose accumulation. Two isolates were selected for studies of trehalose accumulation under heat shock and ethanol stress. The S. pombe isolates were also characterized by RAPD-PCR. The isolates were able to grow and ferment at 41 degrees C, resisted concentrations of 10% ethanol, and grew on 50% glucose medium. Four isolates yielded invertase activity of more than 100 micromol of reducing sugar x mg(-1) x min(-1). The S. pombe isolates were able to accumulate trehalose during stationary phase. Two isolates, strains UFMG-A533 and UFMG-A1000, submitted to a 15 min heat shock, were able to accumulate high trehalose levels. Strain UFMG-A533 had a marked reduction in viability during heat shock, but strain UFMG-A1000 preserved a viability rate of almost 20% after 15 min at 48 degrees C. No clear correlation was observed between trehalose accumulation and cell survival during ethanol stress. Strain UFMG-A1000 had higher trehalose accumulation levels than strain UFMG-A533 under conditions of combined heat treatment and ethanol stress. Molecular analysis showed that some strains are maintained during the whole cacha?a production period; using the RAPD-PCR profiles, it was possible to group the isolates according to their isolation sites.     

Isolation and Identification of Acetic Acid Bacteria for Submerged Acetic Acid Fermentation at High Temperature

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.     

Thermotolerance behavior in sugar cane syrup fermentations of wild type yeast strains selected under pressures of temperature,high sugar and added ethanol

Summary The selected yeast strains were examined for their ability to grow, to retain cell viability and to ferment diluted sugar cane juice (15 % total sugar, w/v) to ethanol at 40°C. The degree of agitation (aeration) affects the thermotolerance while the method used for isolation of the strains appears to have no significant effect. The yeast isolated are aerobically fermentative with increased levels of fermentation and growth resulting from agitation (aeration), the exact level of these increases being dependent on the strain used.     

Evaluation of thermotolerant yeasts in controlled simultaneous saccharifications and fermentations of cellulose to ethanol

Simultaneous saccharification and fermentation (SSF) experiments were performed at selected temperatures (37, 41, and 43 degrees C) to obtain comprehensive material balance and performance data for several promising strains of thermotolerant yeast. Parameters measured were ethanol concentration, yeast cell density, and residual sugar and cellulose concentrations. The three yeasts Saccharomyces uvarum, Candida brassicae, and C. lusitaniae and two mixed cultures of Brettanomyces clausenii with S. cerevisiae (mixed culture I) and C. Iusitaniae with S. uvarum (mixed culture II) exhibited rapid rates of fermentation, high ethanol yields, strong viability, or high cellobiase activity. Overall, mixed culture II at 41 degrees C performed better than either component yeast by themselves because it combined a cellobiose fermenting capability with the high ethanol tolerance and rapid glucose fermentation of conventional industrial yeasts. Thus, the mixed cultures provide good initial rates by preventing buildup of cellobiose (a strong inhibitor of enzyme activity) while attaining high ultimate yields of ethanol for high cellulase concentrations. However, C. brassicae and S. uvarum gave similar results to mixed culture II at 37 degrees C.     

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40°C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47°C. At 43°C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43°C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50°C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.     

Temperature-dependent kinetic model for nitrogen-limited wine fermentations

A physical and mathematical model for wine fermentation kinetics was adapted to include the influence of temperature, perhaps the most critical factor influencing fermentation kinetics. The model was based on flask-scale white wine fermentations at different temperatures (11 to 35 degrees C) and different initial concentrations of sugar (265 to 300 g/liter) and nitrogen (70 to 350 mg N/liter). The results show that fermentation temperature and inadequate levels of nitrogen will cause stuck or sluggish fermentations. Model parameters representing cell growth rate, sugar utilization rate, and the inactivation rate of cells in the presence of ethanol are highly temperature dependent. All other variables (yield coefficient of cell mass to utilized nitrogen, yield coefficient of ethanol to utilized sugar, Monod constant for nitrogen-limited growth, and Michaelis-Menten-type constant for sugar transport) were determined to vary insignificantly with temperature. The resulting mathematical model accurately predicts the observed wine fermentation kinetics with respect to different temperatures and different initial conditions, including data from fermentations not used for model development. This is the first wine fermentation model that accurately predicts a transition from sluggish to normal to stuck fermentations as temperature increases from 11 to 35 degrees C. Furthermore, this comprehensive model provides insight into combined effects of time, temperature, and ethanol concentration on yeast (Saccharomyces cerevisiae) activity and physiology.     

Selection and study of potent lactose-fermenting yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen most potent strains isolated from milk products fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11-12, and 10-12%, respectively (4 degrees C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30 degrees C for 2-3 days, ethanol concentration was 4-5%.     

Combined effects of ethanol, high homogenization pressure, and temperature on cell fatty acid composition in Saccharomyces cerevisiae

The specific aims of this research were to evaluate the combined effects of ethanol and high-pressure homogenization at different temperatures on cell viability in Saccharomyces cerevisiae and to study the induced modification of fatty acid composition. The decrease in viability was weak at 10 degrees C while a homogenization pressure over 1000 bar (1 bar = 100 kPa) induced a significant reduction in viability when the cells were incubated at 20 and 30 degrees C. The cell tolerance to pressure decreased with an increase in ethanol concentration and temperature. Ethanol, particularly intracellular ethanol accumulated by S. cerevisiae, played an important role in the response to homogenization pressure and in modification of the cell fatty acid composition. In fact, an unusually elevated accumulation of ethyl esters in lipid extracts of yeast cells subjected to high homogenization pressure, especially in the presence of exogenous ethanol and at 30 degrees C, was observed. Moreover, only unsaturated and traces of short chain fatty acids were esterified with ethanol.     

Fermentation efficiency of thermally dried kefir

Three thermal drying methods (conventional, vacuum and convective) were used for drying of kefir biomass and their effect on cell viability, fermentation rate and other kinetic parameters of lactose and whey fermentation were studied. Convective drying rate was higher than conventional and even higher than vacuum at each studied temperature (28, 33 and 38 degrees C). After that, fermentations were performed by kefir biomass dried by the three drying methods. Ethanol concentration, ethanol productivity and ethanol yield are higher in whey fermentations performed by kefir biomass dried with convective drying method. Regarding lactic acid production, fermentation performed by kefir biomass dried with conventional drying method gave higher concentrations, compared to other drying methods. Storage of kefir biomass convectively dried at 33 degrees C for 4months, without any precaution decreases its fermentability and thus reduces ethanol (31%) and lactic acid productivity (20%), but remains a promising technology, since a significant part of its initial fermentative activity is retained.     

Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l^− 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (q_s) and gluconate production (q_p) reduced progressively. Interestingly, gradual q_s and q_p reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.

     

Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices

The objective of this study was to compare high pressure resistance of Listeria monocytogenes strains at 25 degrees C and 50 degrees C at 350 MPa and to use high pressure (250 MPa and 350 MPa) at 30 degrees C and 40 degrees C for the inactivation of the relatively most pressure resistant strain inoculated in pasteurized apple, apricot, cherry and orange juices. L. monocytogenes CA was found to be the relatively most pressure resistant strain and increasing pressurization from 250 MPa to 350 MPa at 30 degrees C had an additional three to four log cycle reduction in viability, still leaving viable cells after 5 min. When 350 MPa at 40 degrees C for 5 min was applied more than eight log cycle reduction in cell population of all fruit juices was achieved. This study demonstrated that low temperature (40 degrees C) high pressure (350 MPa) treatment has the potential to inactivate relatively pressure resistant L. monocytogenes strains inoculated in different fruit juices within 5 min.     

Highly efficient bioethanol production by a Saccharomyces cerevisiae strain with multiple stress tolerance to high temperature, acid and ethanol

Use of super strains exhibiting tolerance to high temperature, acidity and ethanol is a promising way to make ethanol production economically feasible. We describe here the breeding and performance of such a multiple-tolerant strain of Saccharomyces cerevisiae generated by a spore-to-cell hybridization technique without recombinant DNA technology. A heterothallic strain showing a high-temperature (41°C) tolerant (Htg(+)) phenotype, a derivative from a strain isolated from nature, was crossed with a homothallic strain displaying high-ethanol productivity (Hep(+)), a stock culture at the Thailand Institute of Scientific and Technological Research. The resultant hybrid TJ14 displayed ability to rapidly utilize glucose, and produced ethanol (46.6g/l) from 10% glucose fermentation medium at high temperature (41°C). Not only ethanol productivity at 41°C but also acid tolerance (Acd(+)) was improved in TJ14 as compared with its parental strains, enabling TJ14 to grow in liquid medium even at pH 3. TJ14 maintained high ethanol productivity (46.0g/l) from 10% glucose when fermentation was done under multiple-stress conditions (41°C and pH 3.5). Furthermore, when TJ14 was subjected to a repeated-batch fermentation scheme, the growth and ethanol production of TJ14 were maintained at excellent levels over ten cycles of fermentation. Thus, the multiple-stress (Htg(+) Hep(+) Acd(+)) resistant strain TJ14 should be useful for cost-effective bioethanol production under high-temperature and acidic conditions.     

玫烟色拟青霉适温性及抗多菌灵特性的种内变异

在15 ℃～35 ℃的温度范围内比较测定了玫烟色拟青霉(Paecilomyces fumosoroseus) 6株野生菌的菌落生长、产孢量及孢子活力.结果显示,3个指标均以25 ℃左右最好,但同一温度下菌株间或同一菌株在不同温度下的差异显著, 菌株Pfr116和Pfr6206具有相对稳定优良的生长、产孢及活孢率性状.对不同浓度多菌灵对6株野生菌的菌落生长和单孢菌落形成的影响测定表明,菌株间存在较大差异.不同多菌灵浓度对单孢菌落形成的抑制率观察值可用逻辑斯蒂模型较好地拟合(r²≥0.90).用所获参数估计的最低禁菌浓度MIC值显示,Pfr4205和Pfr116对多菌灵表现为低抗性（MIC≤20.0 μg·ml^-1）;Pfr153、Pfr612和Pfr2175属于低水平中抗,MIC值仅略大于20 μg·ml^-1;而Pfr6206的MIC值高达93.5 μg·ml^-1,接近MIC>100 μg·ml^-1的高抗标准.因此,Pfr6206为适温性和抗多菌灵特性俱佳的菌株,可作为抗多菌灵和适应不同季节或地区的害虫生防菌剂的候选菌株.     

Solid-state fermentation of sweet sorghum to ethanol

Solid-state fermentation of chopped sweet sorghum particles to ethanol was studied in static flasks using an ethanol tolerant yeast strain. The influence of various process parameters, such as temperature, yeast cell concentration, and moisture content, on the rate and extent of ethanol fermentation was investigated. Optimal values of these parameters were found to be 35 degrees C, 7 x 10(8) cells/g raw sorghum, and 70% moisture level, respectively.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

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.     

Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects

The effect of temperatures ranging from 15 to 35 degrees C on a culture of Brettanomyces bruxellensis was investigated in regards to thermodynamics, metabolism, and kinetics. In this temperature range, we observed an increase in growth and production rates. The growth behavior was well represented using the Arrhenius model, and an apparent activation energy of 16.61 kcal/mol was estimated. A stuck fermentation was observed at 35 degrees C as represented by high cell death. The carbon balance established that temperature had no effect on repartition of the glucose consumption between biomass and products. Hence, the same biomass concentration was obtained for all temperatures, except at 35 degrees C. Moreover, using logistic and Luedeking-Piret models, we demonstrated that production rates of ethanol and acetic acid were partially growth associated. Parameters associated with growth (alpha eth and alpha aa) remained constant with changing temperature, whereas, parameters associated with the population (beta eth and beta aa) varied. Optimal values were obtained at 32 degrees C for ethanol and at 25 degrees C for acetic acid.