Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae

Thermal damage, high osmolarity, and ethanol toxicity in the yeast Saccharomyces cerevisiae limit titer and productivity in fermentation to produce ethanol. We show that long-term adaptive laboratory evolution at 39.5°C generates thermotolerant yeast strains, which increased ethanol yield and productivity by 10% and 70%, in 2% glucose fermentations. From these strains, which also tolerate elevated-osmolarity, we selected a stable one, namely a strain lacking chromosomal duplications. This strain (TTY23) showed reduced mitochondrial metabolism and high proton efflux, and therefore lower ethanol tolerance. This maladaptation was bolstered by reestablishing proton homeostasis through increasing fermentation pH from 5 to 6 and/or adding potassium to the media. This change allowed the TTY23 strain to produce 1.3–1.6 times more ethanol than the parental strain in fermentations at 40°C with glucose concentrations ~300 g/L. Furthermore, ethanol titers and productivities up to 93.1 and 3.87 g·L ⁻¹·hr ⁻¹ were obtained from fermentations with 200 g/L glucose in potassium-containing media at 40°C. Albeit the complexity of cellular responses to heat, ethanol, and high osmolarity, in this study we overcome such limitations by an inverse metabolic engineering approach.     

自絮凝颗粒酵母乙醇连续发酵耦合废液全循环工艺的研究

在以CO2为驱动力的单级悬浮床生物反应器中,进行了自絮凝颗粒酵母乙醇连续发酵耦合废液全循环实验研究。以双酶法制备的玉米粉糖化液为底物,系统连续运行了28 d,每隔5 d将收集到的发酵液集中精馏处理,得到的废糟液直接用于玉米粉调浆制糖。实验数据表明,在稀释率为0.05 h-1条件下,发酵液中乙醇、残还原糖、残总糖质量浓度基本稳定,其平均值为82.97,30.02和40.87 g.L-1。对废液循环工艺过程中,发酵液中的8种高沸点有机酸进行了定量分析,发现发酵液中仅丙酮酸有明显积累,并最终在0.1~0.3 mol.L-1范围内波动。实验结果表明:系统中存在的高沸点副产物不对乙醇发酵产生明显抑制作用,乙醇连续发酵能够在废液全循环条件下良好运行。     

自絮凝酵母高浓度重复批次乙醇发酵

利用发酵性能优良的自絮凝酵母Saccharomyces cerevisiaeflo,研究开发了重复批次高浓度乙醇发酵系统,以节省下游加工过程的能耗。在终点乙醇浓度达到120g/L左右的条件下,发酵系统的乙醇生产强度达到8.2g/(L·h)。然而实验中发现,随着发酵批次的增多,自絮凝酵母沉降性能逐渐下降,从发酵液中沉降分离所需时间相应延长,导致发酵液中高浓度乙醇对酵母的毒害作用加剧,影响其发酵活性和发酵系统运行的稳定性,发酵装置运行11个批次后无法继续运行。实验结果表明,絮凝能力下降导致的酵母絮凝颗粒尺度减小是其沉降性能下降的主要原因。进一步研究发现,酵母的絮凝能力通过再培养可以恢复。在此基础上对发酵系统操作进行改进,每批发酵结束后可控采出一定比例菌体,调节系统的酵母细胞密度和乙醇生产强度以刺激酵母增殖,保持其絮凝能力。在达到相同发酵终点乙醇浓度条件下,虽然发酵系统的乙醇生产强度降低到4.0g/(L·h),但运行10d后絮凝颗粒酵母尺度趋于稳定,继续运行14d,未发现絮凝颗粒酵母尺度继续下降的现象,系统可以稳定运行。     

Role of phosphatidylinositol (PI) in ethanol production and ethanol tolerance by a high ethanol producing yeast

The effect of inositol addition on phospholipids, cell growth, ethanol production and ethanol tolerance in a high ethanol producing Saccharomyces sp were studied. Addition of inositol greatly influenced major phospholipid synthesis. With inositol in the fermentation medium, phosphatidylinositol (PI) content was increased, while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were decreased. However, without inositol in the fermentation medium, PI content dropped down within 24 h, then increased, but was lower than in the presence of inositol. When yeast cells had a higher content of PI, they produced ethanol much more rapidly and tolerated higher concentrations of ethanol. During ethanol shock treatment at 18% (v/v) ethanol, yeast cells with a higher concentration of PI lost their viability much more slowly than those with a lower concentration of PI, indicating that the PI content in these yeast cells can play an important role in ethanol production and ethanol tolerance. Fatty acids and ergosterol were not responsible for high ethanol tolerance and high ethanol production in this yeast strain. Received 22 September 1998/ Accepted in revised form 20 December 1998     

Continuous ethanol production by immobilized yeast reactor coupled with membrane pervaporation unit

A system comprised of an immobilized yeast reactor producing ethanol, with a membrane pervaporation module for continuously removing and concentrating the produced ethanol, was developed. The combined system consisted of two integrated circulation loops: In one the sugar-containing medium is circulated through the membrane pervaporation module. The two loops were interconnected in a way allowing for separate parameter optimization (e.g., flow rate, temperature, pH) for each loop.The fermentation unit was 2.0 L bioreactor with five equal segments, packed with 5-mm beads of immobilized yeasts. The bead matrix was a crosslinked polyacrylamide hydrazide gel coated with calcium alginate. The fast circulation loop of the bioreactor allowed for efficient liberation of CO(2) at the top of the immobilized yeast reactor. Continuous operation of the uncoupled reactor for over 50 days with inflowing defined medium or dilute molasses at a residence time of 1.25 h yielded ethanol at a rate of about 10 g/L h.The pervaporation unit was constructed from four 60-cm-long tubular membranes of silicone composite on a polysulfone support. The output from the fermentor was circulated through the inside of the tubes of a unit with a total surface area of 800 cm(2), having an average flux of 150 mL/h, and selectivities to ethanol vs. water up to 7. A vacuum of 30 mb was applied to the outside of the tubes, removing 20-30 g of ethanol per hour, which was collected in condensors. The continuous removal of ethanol, avoiding inhibition of the fermentation process, resulted in an improved productivity and allowed the use of high sugar concentrations (40% wt/vol) offering the potential of a compact system with reduced stillage.The combined system of ethanol production and removal enabled an operative steady state at which the liquid volume of the system, and the concentrations of ethanol within the reactor ( 4% wt/vol), as well as within the flux crossing the pervaporation membrane (17%-20% wt/vol) were kept constant. At the steady state, a 40% wt/vol sugar solution could be continuously added to the fermentor when 12%-20% wt/vol clear ethanol solution was continuously removed by the pervaporation unit. Membrane fouling was reversed by short washing steps, and continuous step operation was maintained by working with two different modules that were interchanged. In this manner, long term continuous operation (over 40 days) was achieved with a productivity of 20-30 g/L h, representing over a twofold increase relative to the continuously operated reactor uncoupled from the membrane and a fivefold increase in comparison with the value obtained fro a corresponding batch fermentation.     

Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40 degrees C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35 degrees C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38 degrees C.     

Performance comparison of ethanol and butanol production in a continuous and closed-circulating fermentation system with membrane bioreactor

Since both ethanol and butanol fermentations are urgently developed processes with the biofuel-demand increasing, performance comparison of aerobic ethanol fermentation and anerobic butanol fermentation in a continuous and closed-circulating fermentation (CCCF) system was necessary to achieve their fermentation characteristics and further optimize the fermentation process. Fermentation and pervaporation parameters including the average cell concentration, glucose consumption rate, cumulated production concentration, product flux, and separation factor of ethanol fermentation were 11.45?g/L, 3.70?g/L/h, 655.83?g/L, 378.5?g/m²/h, and 4.83, respectively, the corresponding parameters of butanol fermentation were 2.19?g/L, 0.61?g/L/h, 28.03?g/L, 58.56?g/m²/h, and 10.62, respectively. Profiles of fermentation and pervaporation parameters indicated that the intensity and efficiency of ethanol fermentation was higher than butanol fermentation, but the stability of butanol fermentation was superior to ethanol fermentation. Although the two fermentation processes had different features, the performance indicated the application prospect of both ethanol and butanol production by the CCCF system.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

初始条件对高浓度乙醇连续发酵过程的影响及振荡行为提高发酵效率的机理分析

前期实验在稀释速率为0.027h-1的高浓度乙醇连续发酵过程中,发现了一种长周期、宽振幅的参数振荡现象。本实验进一步考察了不同稀释速率下的连续发酵过程,发现在稀释速率为0.04h-1条件下,也能出现类似的振荡现象;在稀释速率为0.027h-1或0.04h-1的条件下,改变系统的初始状态可以得到振荡和稳态两种不同的发酵过程。比较振荡和稳态过程的实验数据后,发现在稀释速率为0.04h-1的条件下,与稳态过程相比,振荡过程的平均残糖浓度降低了14.8%,平均乙醇浓度提高了12.6%,平均设备生产强度提高了12.3%。进一步分析表明:与稳态过程相比,振荡过程动力学行为不仅存在滞后,而且在相同残糖和乙醇浓度条件下,所对应的平均比生长速率提高了53.8%。     

利用乙醇发酵副产CO2培养富含淀粉亚心型四爿藻作为其补充原料

建立了乙醇发酵耦联微藻培养系统,研究了利用酿酒酵母Saccharomyces cerevisiae乙醇发酵副产CO2为碳源,培养富含淀粉的亚心形四爿藻Tetraselmis subcordiformis,作为乙醇发酵补充原料的可行性。在连续光照培养条件下,间歇式培养7 d,反应器中藻细胞密度达到2.0 g/L左右,胞内淀粉含量约45％。微藻细胞收集后,经超声处理和酶法水解,葡萄糖释放量为胞内淀粉总量的71.1％。S. cerevisiae发酵微藻生物质水解液生产乙醇,其得率达到理论值的87.6％。表明乙醇发酵耦联微藻培养可行,既减少了CO2向环境的排放,又收获了富含淀粉的微藻生物质作为乙醇发酵的补充原料,节省粮食类淀粉质原料的消耗。     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Observed quasi-steady kinetics of yeast cell growth and ethanol formation under very high gravity fermentation condition

Using a generalSaccharomyces cerevisiae as a model strain, continuous ethanol fermentation was carried out in a stirred tank bioreactor with a working volume of 1,500 mL. Three different gravity media containing glucose of 120, 200 and 280 g/L, respectively, supplemented with 5 g/L yeast extract and 3 g/L peptone, were fed into the fermentor at different dilution rates. Although complete steady states developed for low gravity medium containing 120 g/L glucose, quasi-steady states and oscillations of the fermented parameters, including residual glucose, ethanol and biomass were observed when high gravity medium containing 200 g/L glucose and very high gravity medium containing 280 g/L glucose were fed at the designated dilution rate of 0.027 h⁻¹. The observed quasi-steady states that incorporated these steady states, quasi-steady states and oscillations were proposed as these oscillations were of relatively short periods of time and their averages fluctuated up and down almost symmetrically. The continuous kinetic models that combined both the substrate and product inhibitions were developed and correlated for these observed quasi-steady states.     

Genetic and physiological alterations occurring in a yeast population continuously propagated at increasing temperatures with cell recycling

This work investigated the effects of increasing temperature from 30°C to 47°C on the physiological and genetic characteristics of Saccharomyces cerevisiae strain 63M after continuous fermentation with cell recycling in a system of five reactors in series. Steady state was attained at 30°C, and then the temperature of the system was raised so it ranged from 35°C in the last reactor to 43°C in the first reactor or feeding reactor with a 2°C difference between reactors. After 15 days at steady state, the temperature was raised from 37°C to 45°C for 25 days at steady state, then from 39°C to 47°C for 20 days at steady state. Starter strain 63M was a hybrid strain constructed to have a MAT a/α, LYS/lys, URA/ura genotype. This hybrid yeast showed vigorous growth on plates at 40°C, weak growth at 41°C, positive assimilation of melibiose, positive fermentation of galactose, raffinose and sucrose. Of 156 isolates obtained from this system at the end of the fermentation process, only 17.3% showed the same characteristics as starter strain 63M. Alterations in mating type reaction and in utilization of raffinose, melibiose, and sucrose were identified. Only 1.9% of the isolates lost the ability to grow at 40°C. Isolates showing requirements for lysine and uracil were also obtained. In addition, cell survival was observed at 39–47°C, but no isolates showing growth above 41°C were obtained.     

Yeast cell-free enzyme system for bio-ethanol production at elevated temperatures

A yeast cell-free enzyme system containing an intact fermentation assembly and that is capable of bio-ethanol production at elevated temperatures in the absence of living cells was developed to address the limitations associated with conventional fermentation processes. The presence of both yeast glycolytic and fermentation enzymes in the system was verified by SDS-PAGE and LC–MS/MS Q-TOF analyses. Quantitative measurements verified sufficient quantities of the co-factors ATP (1.8 mM) and NAD⁺ (0.11 mM) to initiate the fermentation process. Bio-ethanol was produced at a broad temperature range of 30–60 °C but was highly specific to a pH range of 6.0–7.0. The final bio-ethanol production at 30, 40, 50, and 60 °C was 3.37, 3.83, 1.94, and 1.60 g/L, respectively, when a 1% glucose solution was used, and the yield increased significantly with increasing cell-free enzyme concentrations. A comparative study revealed better results for the conventional fermentation system (4.46 g/L) at 30 °C than the cell-free system (3.37 g/L); however, the efficacy of the cell-free system increased with temperature, reaching a maximum (3.83 g/L) at 40 °C, at which the conventional system could only produce 0.48 g/L bio-ethanol. Successful bio-ethanol production using a single yeast cell-based enzyme system at higher temperatures will lead to the development of novel strategies for efficient bio-ethanol production through SSF.     

Ethanol production by Saccharomyces cerevisiae in biofilm reactors

Biofilms are natural forms of cell immobilization in which microorganisms attach to solid supports. At ISU, we have developed plastic composite-supports (PCS) (agricultural material (soybean hulls or oat hulls), complex nutrients, and polypropylene) which stimulate biofilm formation and which supply nutrients to the attached microorganisms. Various PCS blends were initially evaluated in repeated-batch culture-tube fermentation with Saccharomyces cerevisiae (ATCC 24859) in low organic nitrogen medium. The selected PCS (40% soybean hull, 5% soybean flour, 5% yeast extract-salt and 50% polypropylene) was then used in continuous and repeated-batch fermentation in various media containing lowered nitrogen content with selected PCS. During continuous fermentation, S. cerevisiae demonstrated two to 10 times higher ethanol production in PCS bioreactors than polypropylene-alone support (PPS) control. S. cerevisiae produced 30 g L⁻¹ ethanol on PCS with ammonium sulfate medium in repeated batch fermentation, whereas PPS-control produced 5 g L⁻¹ ethanol. Overall, increased productivity in low cost medium can be achieved beyond conventional fermentations using this novel bioreactor design. Received 20 May 1997/ Accepted in revised form 29 August 1997     

Fermentation of lactose to ethanol with recombinant yeast in an immobilized yeast membrane bioreactor

A novel concept of membrane bioreactor in which living cells are sandwiched between ultrafiltration (UF) and reverse osmosis (RO) membranes was applied for lactose fermentation to ethanol by genetically engineered yeast cells. The productivity of the Lactophile 13B strains was higher than that of the Lactophile 13D strains. In both cases performance data similar to those for glucose fermentation to ethanol by Saccharomyces cerevisiae were obtained. However, the operational stability of recombinant yeast cells was improved in the new bioreactor in comparison to the stability of these cells in a shake flask.     

Continuous ethanol production by flocculating yeast in the fluidized bed bioreactor

Abstract: Continuous fermentation by a highly flocculant strain of the yeast Saccharomyces cerevisiae was carried out in a tower fluidized-bed bioreactor. The synthetic and molasses media with a total sugar concentration of 17% (w/v) were used for fermentation. Different dilution rates were tested. Stable cell densities of 50 kg m^-3(dry weight) were maintained for all dilution rates. The ethanol productivity was increasing linearly with dilution rates up to 15—20 kg m^-3 h^-1. Aeration of the culture stabilized flocculating activity and viability of yeast and also permitted long-term operation of the bioreactor.     

纤维素乙醇统合加工过程中的酵母多酶共展示体系研究进展

利用统合生物加工过程(Consolidated bioprocessing,CBP)生产纤维素乙醇是目前国内外的研究热点。CBP需要一种“集成化”微生物,既能生产水解木质纤维素的多种酶类又能利用水解木质纤维素产生的糖类发酵产乙醇。以酿酒酵母表面展示技术为依托,建立CBP菌株多酶共展示体系的研究主要分为以下两个方向：一是直接将纤维素酶展示在细胞表面,即非复合型纤维素酶体系;另一种是通过表面展示纤维小体(Cellulosome)将纤维素酶间接地锚定在细胞表面,即复合型纤维素酶体系,本文主要从以上两个方向阐述了近几年对于纤维素乙醇生物统合加工过程的研究进展。因纤维小体对纤维素的降解能力比非复合型纤维素酶体系更强,所以其在酿酒酵母细胞表面的组装研究受到越来越多的关注,为了更深入透彻地了解纤维小体的酵母展示技术,文中对纤维小体的结构与功能及其在纤维素乙醇发酵中的应用研究进行重点论述,并对该领域的发展方向进行展望。     

Variation of the ethanol yield during oscillatory concentrations changes in undisturbed continuous ethanol fermentation of sugar-cane blackstrap molasses

During the oscillatory phase of an undisturbed continuous ethanol fermentation of sugar-cane blackstrap molasses, the relative ethanol yield oscillated between 70 and 92% of the theoretical value (0.511), while its actual value was 85.6%. The ethanol yield based on catabolic activity oscillated between 0.290 and 1.174 g/kcal, while its actual value was 0.686 g/kcal. The specific production rate of ethanol increased when the specific growth rate of the yeast cells increased; a linear equation correlates the above specific rates.