Aeration strategy: a need for very high ethanol performance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> fed-batch process

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions—oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)—on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l^–1), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l^–1 in 45 h) and average productivity (3.3 vs 2.6 g l^–1 h^–1) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l^–1. We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l^–1 in 45 h) in order to achieve a highly competitive dynamic process.     

Production of single cell protein from ethanol by fed-batch process

The production of single cell protein (SCP) form ethanol is an interesting process to study from a biochemical engineering viewpoint. The cellular yield mainly depends upon the metabolic activity of the cells and the amount of substrate available. Fedbatch fermentations Were run in a 70 liter highly instrumented computer-coupled fermentor using Candida utilis. Respiratory quotient and culture fluorescence, measuring NADH, indicate by which pathway ethanol is utilized. By monitoring these parameters it is possible to control the ethanol concentration so that accumulation of acetate is minimized and the conversion of ethanol to cell mass is maximized.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of bakers yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol. For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, , is fixed at a level below the point of ethanol production, i.e., _crit. Optimally, growth should be maintained at _crit, but in practice, this is difficult because _crit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just above _crit by regulating ethanol concentration in the bioreactor. The models used for control design are shown, as are the experimental results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an overflow type metabolism.     

温度对超高浓度酒精生料发酵体系的影响

通过对超高底物浓度生料发酵中温度的影响研究发现,采用温度梯度的方法可大幅提高酵母的生产效率。以高粱为例,采用35%绝对干物浓度,在新型生料水解酶的配合下,通过合适的逐级降温培养方式,使用普通酒精干酵母,在90h内发酵醪液酒精浓度可达20%(V/V)以上。     

A high-throughput media design approach for high performance mammalian fed-batch cultures

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.     

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.     

超高浓度培养基条件下酵母细胞生长及酒精生成的准稳态动力学研究

在1．5L搅拌式发酵罐中,使用葡萄糖质量浓度分别为120、200、280g／L的培养基进行酿酒酵母Saccharomyces cerevisiae连续发酵生成酒精的动力学研究。研究发现,当培养基中葡萄糖浓度为200和280g／L时,发酵液中残糖浓度、酒精浓度以及菌体生物量从小幅度波动的准稳态发展到大幅度波动的振荡状态。提出了伴有周期性振荡现象准稳态过程的概念,并针对该过程,建立了兼有底物和产物抑制的酵母细胞生长和产物酒精生成动力学模型。     

Simultaneous saccharification and fermentation of sweet sorghum carbohydrates to ethanol in a fed-batch process

Summary The simultaneous saccharification and fermentation (SSF) of sweet sorghum carbohydrates to ethanol by Fusarium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae 2541 or Zymomonas mobilis CP4 in a fed-batch fermentation process was studied. While SSF was adequately carried out by the first microorganism the process achieved its maximum value by the mixed culture of the fungus and yeast. Under optimum conditions, ethanol yields and concentrations as high as 29.7 g of ethanol per 100 g of dry sorghum stalk and 7.5 % (w/v) respectively were obtained. These values together with the high yield of sorghum crop in Greece make this process promising and worthy of further investigation for the production of fuel bioethanol.     

分批补料对木糖发酵生产乙醇的影响

以树干毕赤酵母为发酵菌种,纯木糖为发酵底物,通过分批补料来提高糖利用率以及乙醇得率。结果表明,在24h内,最佳初始木糖浓度为80g/L,在28h的发酵周期中,可以将木糖浓度提高至90g/L,在32h发酵周期内可以将木糖浓度提高至100g/L。通过分批补料,乙醇浓度得到明显提高。当总糖浓度分别为80g/L、90g/L时,24h发酵周期内,分批补料次数以1次为宜,乙醇浓度分别达30.95g/L、32.60g/L,相比于不补料即一次性投料,乙醇浓度分别提高了9.36%、9.18%。总糖浓度100g/L,28h发酵周期内,补料2次效果最佳,乙醇浓度达37.49g/L,比一次性投料下提高了10.36%,较一次性投料达到相同发酵效果缩短了4h。     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

高效液相法测定蜂蜜中甘油的含量

建立了以混合溶剂直接提取测定蜂蜜中甘油含量的方法.采用ZORBAX Carbohydrate a-nalysis(4.6 mm×250 mm 5-Micro)色谱柱,以乙腈/水(80:20,v/v)为流动相,示差检测器,使用乙腈/甲醇/水混合作为溶剂快速检测甘油.结果表明,应用此法的甘油浓度在10.0 mg/L～250...     

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.     

Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae

To increase ethanol yield and decrease glycerol production in Saccharomyces cerevisiae, the strategies of direct cofactor-regulation were explored. During anaerobic batch fermentations, the yeast expressing Bacillus cereus gapN gene, encoding non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrognease, produced 73.8?g ethanol?l(-1), corresponding to 96% of theoretical maximum yield compared to 92% for the wild type. The yeast expressing Escherichia coli frdA gene encoding the NAD(+)-dependent fumarate reductase, exhibited a 22% (relative to the amount of substrate consumed) increase in glycerol yield in medium containing 2?g fumarate?l(-1). The yeast expressing mhpF gene, encoding acetylating NAD(+)-dependent acetaldehyde dehydrogenase, produced 74.5?g ethanol?l(-1), corresponding to 97.4% of theoretical maximum yield while glycerol decreased by 40% when acetic acid was added before inoculation. This strain represents a promising alternative for ethanol production with lignocellulosic hydrolysates where acetate is available at significant amounts.     

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

A two-stage enzymatic process for synthesis of extremely pure high oleic glycerol monooleate

This paper presents a research interest concentrating on aims to establish a feasible industrial process for enzymatic production of highly pure glycerol monooleate (GMO). The synthesis of high oleic glycerol monooleate by enzymatic glycerolysis of high oleic sunflower oil, using Novozyme 435 as the biocatalyst, in a binary solvent mixture of tert-butanol and tert-pentanol (80/20, v/v), at a lab scale has been studied. A yield of 75.31% monoacylglycerol has been achieved at the first stage. A yield of 93.3% GMO was finally reached after further purification at the second stage. To evaluate the possibility of the process for industrialization, production of GMO was performed at a pilot-plant scale under the correspondingly adjusted conditions. A yield of 68.17% and 93.4% of GMO was obtained, respectively, at the end of the three stages.     

Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites.     

Simulation and optimization of fed-batch culture for ethanol production from molasses

Two simulation methods for ethanol production from molasses by a flocculating yeast, Saccharomyces cerevisiae AM12, were investigated and molasses feeding was optimized. The first method was based on a deterministic model with fixed kinetic parameters and the second was based on regression analysis. The amount of ethanol produced in a fed-batch culture with multiple additions of molasses was simulated by both of these two methods. Simulated results of a fed-batch culture were compared with those of a simple batch culture by a model of regression analysis. The intermittent addition of molasses gave better production than a single addition at the beginning; more frequent addition may further improve production. The experimental results suggested the same. The effect of the amount of the added molasses on ethanol production was investigated by simulation. Repeated batch culture with and without intermittent addition of molasses in each batch was also done.List of Symbols C _e deviation of calculated results from experimental results - F m³ volume of feed medium added to the fermentor - P kg/m³ concentration of ethanol - P _M kg total amount of ethanol - S kg/m³ concentration of sugar - S ₀ kg/m³ concentration of sugar in the molasses feed medium - S _M kg total amount of sugar - V m³ culture volume - X kg/m³ concentration of cells - X _M kg total amount of cells - x _c calculated data - x _e experimental data - h^–1 specific rate of growth - kg-sugar/(kg-cell h) specific rate of sugar consumption - kg-ethanol/(kg-cell h) specific rate of ethanol production     

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.