Continuous ethanol production from pineapple cannery waste using immobilized yeast cells

The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).     

Selection and adaptation of Saccharomyces cerevisae to increased ethanol tolerance and production

A total of 24 yeast strains were tested for their capacity to produce ethanol, and of these, 8 were characterized by the best ethanol yields (73.11-8 1.78%). The most active mutant Saccharomyce s cerevisiae ER-A, resistant to ethanol stress, was characterized by high resistance to acidic (pH 1.0 and 2.0), oxidative (1 and 2% of H2O2), and high temperature (45 and 52 degrees C) stresses. During cultivation under all stress conditions, the mutants showed a considerably increased viability ranging widely from about 1.04 to 3.94-fold in comparison with the parent strain S. cerevisiae ER. At an initial sucrose concentration of 150 g/l in basal medium A containing yeast extract and mineral salts, at 300C and within 72 h, the most active strain, S. cerevisiae ER-A, reached an ethanol concentration of 80 g/1, ethanol productivity of 1.1 g/Il/h, and an ethanol yield (% of theoretical) of 99.13. Those values were significantly higher in comparison with parent strain (ethanol concentration 71 g/1 and productivity of 0,99 g/l/h). The present study seems to confirm the high effectiveness of selection of ethanol-resistant yeast strains by adaptation to high ethanol concentrations, for increased ethanol production.     

Studies on immobilized Saccharomyces cerevisiae. I. Analysis of continuous rapid ethanol fermentation in immobilized cell reactor

Rapid fermentation of cane molasses into ethanol has been studied in batch, continuous (free-cell and cell-immobilized systems) by a strain of Saccharomyces cerevisiae at temperature 30 degrees C and pH 5.0. The maximum productivity of ethanol obtained in immobilized system was 28.6 g L(-1) h(-1). The cells were immobilized by natural mode on a carrier of natural origin and retention of 0.132 g cells/g carrier was achieved. The immobilized-cell column was operated continuously at steady state over a period of 35 days. Based on the parameter data monitored from the system, mathematical analysis has been made and rate equations proposed, and the values of specific productivity of ethanol and specific growth rate for immobilized cells computed. It has been established that immobilized cells exhibit higher specific rate of ethanol formation compared to free cells but the specific growth rate appears to be comparatively low. The yield of ethanol in the immobilized-cell system is also higher than in the free-cell system.     

Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis

Pichia stipitis NRRL Y-7124 is a xylose-fermenting yeast able to accumulate ca. 57 g/L ethanol. Because optimum process conditions are important, data were collected to determine the effects of temperature and pH on growth and fermentation rates and product accumulations. Temperatures (26-35 degrees C) providing optimum biomass and ethanol productivities did not necessarily provide maximum ethanol accumulation. Xylitol and residual xylose concentrations increased with temperature. Maximum ethanol selectivity was achieved at 25-26 degrees C with minimal sacrifice to production rates. The temperature optimum for xylose could not be generalized to glucose fermentations, in which ethanol productivity and accumulation were optimum at 34 degrees C. The optimum pH range for growth and fermentation on xylose was 4-7 at 25 degrees C.     

一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: II. Analysis of cell synchronization and metabolism

Sustained oscillations of biomass, ethanol, and ammonium concentrations, specific growth rate, and specific uptake rates of ethanol, ammonium, and oxygen were found in continuous cultures of Saccharomyces cerevisiae under controlled dissolved oxygen (DO), pH, and temperature conditions. The period of oscillations was approximately 2.5-3 h at a pH of 5.5 and 2-2.5 h at a pH of 6.5. Oscillations were observed only under conditions of low carbon (glucose below the minimum detectable level), nitrogen nutrient (ammonium concentration varied between 0.00001 and 0.0015M), and ethanol concentration (0.002-0.085 g/L) in the bioreactor.The oscillatory behavior at pH 5.5 was also characterized by partially synchronized cell growth and reproduction. Not only did the total percentage of budding cells oscillate with the same period as observed for the global biomass and nutrient concentrations, but the peaks in the individual subpopulations of initial budding, middle budding, and late budding cells appeared sequentially during the oscillation period. This provides strong evidence of the hypothesis that variations in metabolism during different periods in the cell cycle of a partially synchronized cell population are responsible for the observed oscillatory bioreactor behavior.The specific nutrient uptake rates for ammonium and oxygen as well as the net specific ethanol uptake rate oscillated with the same period as the biomass oscillations. These results show a dramatic increase in the ammonium and oxygen consumption rates prior to the initial budding of the synchronized subpopulation and a decrease in these rates during the late budding phase. At a pH of 5.5, the late budding phase is characterized by high specific ethanol productivity; however, the ethanol productivity lags the late budding phase at a pH pf 6.5. The observed time-varying metabolism in the oscillatory operating regime appears to be the result of the metabolic changes which occur during the cell cycle. Models which can predict the oscillatory biomass concentration and nutrient levels in this regime must be capable of predicting the concentrations and metabolic rates of the subpopulations as well.     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

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.     

Continuous ethanol production from Jerusalem artichoke tubers. I. Use of free cells of Kluyveromyces marxianus

The Continuous fermentation of Jerusalem artichoke juice to ethanol by free cells of Kluyveromyces marxianus UCD (FST) 55-82 has been studied in a continuous-stirred-tank bioreactor at 35 degrees C and pH 4.6. A maximum yield of 90% of the theoretical was obtained at a dilution rate of 0.05 h(-1). About 95% of the sugars were utilized at dilution rates lower than 0.15 h(-1). Volumetric ethanol productivity and volumetric biomass productivity reached maximum values of 7 g ETOH/L/h and 0.6 g dry wt/L/h, respectively, at a dilution rate of 0.2 h(-1). The maintenance energy coefficient for K. marxianus culture was found to be 0.46 g sugar/g biomass/h/ Oscillatory behavior was following a change in dilution rate from a previous steady state and from batch to continuous culture. Values of specific ethanol production rate and specific sugar uptake were found to increase almost linearly with the increase of the dilution rate. The maximum specific ethanol production rate and maximum specific sugar uptake rate were found to be 2.6 g ethanol/g/ cell/h and 7.9 sugars/g cell/h, respectively. Washout occurred at a dilution rate of 0.41 h(-1).     

选育耐受复合抑制剂酿酒酵母提高乙醇产量

能够耐受纤维素预处理中抑制剂的酿酒酵母对高效、经济生产纤维素乙醇至关重要。利用诱变结合驯化工程选育了一株可耐受复合抑制剂(1.3g/L糠醛、5.3g/L乙酸及1.0g/L苯酚)的工业酿酒酵母YYJ003。在pH 4.0的含有抑制剂的培养基中,耐受菌株乙醇产率是原始菌株的7.8倍,糠醛转化速率提高了5倍。在pH 5.5的复合抑制剂条件下,YYJ003发酵时间(16h)比野生菌株发酵时间(22h)缩短6h。在pH 4.0的未脱毒的玉米秸秆水热法预处理水解液中YYJ003的乙醇产率达到0.50g/g(乙醇/葡萄糖),乙醇产速达到4.16g/(L·h),而对照菌株无乙醇产出。     

Response of Saccharomyces cerevisiae to ethanol stress involves actions of protein Asr1p

During the fermentation process of Saccharomyces cerevisiae, yeast cells must rapidly respond to a wide variety of external stresses in order to survive the constantly changing environment, including ethanol stress. The accumulation of ethanol can severely inhibit cell growth activity and productivity. Thus, the response to changing ethanol concentrations is one of the most important stress reactions in S. cerevisiae and worthy of thorough investigation. Therefore, this study examined the relationship between ethanol tolerance in S. cerevisiae and a unique protein called alcohol sensitive RING/PHD finger 1 protein (Asr1p). A real-time PCR showed that upon exposure to 8% ethanol, the expression of Asr1 was continuously enhanced, reaching a peak 2 h after stimulation. This result was confirmed by monitoring the fluorescence levels using a strain with a green fluorescent protein tagged to the C-terminal of Asr1p. The fluorescent microscopy also revealed a change in the subcellular localization before and after stimulation. Furthermore, the disruption of the Asr1 gene resulted in hypersensitivity on the medium containing ethanol, when compared with the wild-type strain. Thus, when taken together, the present results suggest that Asr1 is involved in the response to ethanol stress in the yeast S. cerevisiae.     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus

Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/ L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary-stirred-tank (CST) bioreactor using cells of K. marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66 h(-1) for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g/biomass/h and 1.21 g sugars/g biomass/h, respectively.     

Filament formation and ethanol production by Zymomonas mobilis in adsorbed cell bioreactors

Zymomonas mobilis immobilized on microporous ion exchange resins has previously been shown to allow the attainment of high ethanol productivities in packed-bed bioreactors. The formation of bacterial filaments after several days of continuous operation, however, had resulted in excessive pressure increases across the reactor bed. The present work examines techniques for controlling filament formation by Z. mobilis in two reactor sizes (161 mL and 7.85 L) and a feed glucose concentration of 100 g/L. By controlling the fermentation temperature at 20-25 degrees C it has been possible to eliminate filament formation by Z. mobilis and to operate the larger bioreactor for 232 h with an ethanol productivity of 50 g/L h (based on total reactor volume). The rate of ethanol production has been shown to be very sensitive to temperature in the range 20-30 degrees C, and it is likely that slightly higher temperatures than those used in this study will improve ethanol productivity while still permitting long-term operation.     

Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast

Water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate has been utilized as a substrate for ethanol production using Pichia stipitis NRRL Y-7124. Hydrolysate fermentability was considerable improved by boiling, and overliming up to pH 10.0 with solid Ca(OH)(2) in combination with sodium sulfite. The percent total sugar utilized and ethanol yield (Y(p/s)) for the untreated hydrolysate were 20.15+/-0.17% and 0.19+/-0.003 g(p) g(s)(-1), respectively, compared with 76.0+/-0.32% and 0.35 g(p) g(s)(-1), respectively for the treated material. The fermentation was very effective at an aeration rate of 0.02 v/v/m, temperature 30+/-0.2 degrees C and pH 6.0+/-0.2. However, the volumetric productivity (Q(p)) was still considerably less than observed in a simulated synthetic hydrolysate medium with a sugar composition similar to the hemicellulose acid hydrolysate. L-Arabinose was not fermented but assimilated. The presence of acetic acid in the hydrolysate decreased the ethanol yield and productivity considerably.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.     

Growth characteristics of Candida utilis on volatile substrate in a multistage tower fermentor.

The influence of increasing ethanol concentration in the feed on growth and physiological activity of the yeast Candida utlis was studied. The measurements were made at steady states of continuous culture under constant values of dilution rate, temperature, and pH in all stages of the fermentor; Synthetic ethanol was used as the sole source of carbon and energy in the concentration range 10-100 g/liter. The maximum biomass concentration in the effluent and maximum productivity was achieved at 75 g ethanol/liter in the feed. In respect to ethanol losses in the outlet and biomass yield, the optimum ethanol concentration in the input of the growth medium was found to be about 50 g/liter using a four-stage system.     

Microwave-assisted enzymatic synthesis of beef tallow biodiesel

Optimal conditions for the microwave-assisted enzymatic synthesis of biodiesel have been developed by a full 2² factorial design leading to a set of seven runs with different combinations of molar ratio and temperature. The main goal was to reduce the reaction time preliminarily established by a process of conventional heating. Reactions yielding biodiesel, in which beef tallow and ethanol used as raw materials were catalyzed by lipase from Burkholderia cepacia immobilized on silica-PVA and microwave irradiations within the range of 8–15 W were performed to reach the reaction temperature. Under optimized conditions (1:6 molar ratio of beef tallow to ethanol molar ratio at 50°C) almost total conversion of the fatty acid presented in the original beef tallow was converted into ethyl esters in a reaction that required 8 h, i.e., a productivity of about 92 mg ethyl esters g⁻¹ h⁻¹. This represents an increase of sixfold for the process carried out under conventional heating. In general, the process promises low energy demand and higher biodiesel productivity. The microwave assistance speeds up the enzyme catalyzed reactions, decreases the destructive effects on the enzyme of the operational conditions such as, higher temperature, stability, and specificity to its substrate, and allows the entire reaction medium to be heated uniformly.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

Effects of temperature and pH on growth and antioxidant content of the microalga Scenedesmus obliquus

Reactive forms of oxygen can damage DNA (among other molecules), thus triggering, e.g., atherogenesis and carcinogenesis. However, such dietary antioxidants as lutein and β-carotene can effectively inactivate them; these compounds were found to high levels in a novel strain (M2-1) of the microalga Scenedesmus obliquus. The independent and combined effects of pH and temperature on its rates of growth and production of antioxidants were experimentally assessed, via a full factorial experimental design; the effects of each parameter independently, and of their interactions were accordingly quantified by ANOVA. Our results indicated that temperature plays a more important role on the maximum specific growth rate than pH; in terms of antioxidant content, pH and, to a lesser extent, temperature also have relevant effects. Consequently, the highest rate of biomass specific growth (0.294 ± 0.013 day(-1)) and biomass productivity (0.837 ± 0.054 mg L(-1) day(-1)) were associated with relatively low pH (6) and relatively high temperature (30°C). Conversely, the antioxidant production rate increased with pH; hence, the highest productivity (0.638 mg L(-1) day(-1)) was attained at pH 8 and 30°C. At the best operating conditions for antioxidant content, the levels of lutein and β-carotene were 203.57 ± 1.41 and 18.20 ± 0.33 mg mL(-1), respectively; the maximum production of either one occurred at the early exponential phase.