Ethanol yields during the feeding phase in fed-batch fermentation of sugar-cane blackstrap molasses

The efficiency of ethanol yield increased from 61% to 88% of the theoretical value as the filling-up time was approached in fed-batch fermentation with Saccharomyces cerevisiae. Temporary accumulation of ethanol within the yeast cells may explain the above variation.The author is with the Instituto Mauá de Tecnologia, Estrada das Lágrimas 2035, 09580-900. Sao Caetano Do Sul, SP, Brazil     

Development of novel carrier using natural zeolite and continuous ethanol fermentation with immobilized Saccharomyces cerevisiae in a bioreactor

A natural zeolite, easily vitrified and blown at 1300 °C with a high porosity and diam. of 5–100 m, was used to immobilize Saccharomyces cerevisiae at 3.6 × 10⁸ cells ml^–1 carrier. When the abilities of natural zeolite carrier were compared with glass beads, the capacity for immobilization and alcohol fermentation activity were, respectively, 2-fold higher and 1.2-fold higher than that of glass beads. Continuous alcohol fermentation was stable for over 21 d without breakage of the carrier.     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

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.     

Effect of pH,aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O₂L^–1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L^–1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attainingS. cerevisiae cells suitable for invertase production were: temperature=30°C; pH=5.0; DO=3.3 mg O₂L^–1; (S)=0.5 g L^–1 and sucrose added into the fermenter according to the equations: (V–V_o)=t²/16 or (V–V_o)=(V_f–V_o)·(e^0.6t–1)/10.This work was supported by FAPESP     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

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%。     

Metabolic flux and cell cycle analysis indicating new mechanism underlying process oscillation in continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions

Process oscillation characterized by long oscillation period and large oscillation amplitude was observed in continuous ethanol fermentation with Saccharomyces cerevisiae under very high gravity conditions. Metabolic flux analysis was applied to the fermentation system, and the results indicated that carbon flux distributions at the metabolic notes oscillated, correspondingly, and the root reason for the process oscillation was the intracellular metabolism of yeast cells. Cell cycle analysis with the flow cytometry showed that no cell-cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation, and thus different mechanism existed compared with the oscillation observed in the continuous culture of Saccharomyces cerevisiae and triggered by the synchronization of the daughter and mother cells under specific conditions. Furthermore, the overall metabolic activity of the yeast cells was examined, which was found not exactly out of phase but lag behind ethanol concentration that accumulated within the fermentation system and its inhibition on the yeast cells as well, which supported the mechanistic speculation for the process oscillation: the lag response of yeast cells to ethanol inhibition.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Biosynthesis of invertase by Saccharomyces cerevisiae with sugarcane molasses as substrate

Biosynthesis of invertase by Saccharomyces cerevisiae 01K32 was inversely proportional to the concentration of sugarcane blackstrap molasses included in the medium. In a fermenter, an intracellular invertase activity of 440 U/g dry cells was obtained.     

Comparative metabolomic analysis on industrial continuous and batch ethanol fermentation processes by GC-TOF-MS

The intracellular metabolic profile characterization of Saccharomyces cerevisiae throughout industrial ethanol fermentation was investigated using gas chromatography coupled to time-of-flight mass spectrometry. A total of 143 and 128 intracellular metabolites in S. cerevisiae were detected and quantified in continuous and batch fermentations, respectively. The two fermentation processes were both clearly distinguished into three main phases by principal components analysis. Furthermore, the levels of some metabolites involved in central carbon metabolism varied significantly throughout both processes. Glycerol and phosphoric acid were principally responsible for discriminating seed, main and final phases of continuous fermentation, while lactic acid and glycerol contributed mostly to telling different phases of batch fermentation. In addition, the levels of some amino acids such as glycine varied significantly during both processes. These findings provide new insights into the metabolomic characteristics during industrial ethanol fermentation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

On the performances of noise filters in the restoration of oscillatory behavior in continuous yeast cultures

Continuous flow microbial fermentations under industrial conditions are subject to the influx of noise, mainly through the feed stream. Noise upsets the normal deterministic behavior. For continuous cultures of Saccharomyces cerevisiae exhibiting oscillatory responses, four kinds of commonly used noise filters, three algorithmic and one neural, have been compared for their ability to restore noise-free oscillations. An auto-associative neural filter was the best, similar to earlier observations for other organisms under non-oscillatory conditions. This enhances the general applicability of neural filters for industrial scale fermentations.     

Improvement of the ethanol productivity in a high gravity brewing at pilot plant scale

A 2³ full factorial design was used to study the influence of different experimental variables, namely wort gravity, fermentation temperature and nutrient supplementation, on ethanol productivity from high gravity wort fermentation by Saccharomyces cerevisiae (lager strain), under pilot plant conditions. The highest ethanol productivity (0.69 g l^–1 h^–1) was obtained at 20°P [°P is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C], 15°C, with the addition of 0.8% (w/v) yeast extract, 24 mg l^–1 ergosterol and 0.24% (v/v) Tween 80.     

Batch ethanol fermentation of molasses: a correlation between the time necessary to complete the fermentation and the initial concentrations of sugar and yeast cells

Batch fermentations of sugar-cane blackstrap molasses to ethanol, using pressed yeast as inoculum, demonstrated an exponential relationship between the time necessary to complete the fermentation and the initial concentrations of sugar and yeast cells. The parameters of the derived exponential equations depended on the experimental conditions.     

Production of recombinant hirudin in galactokinase-deficientSaccharomyces cerevisiae by fed-batch fermentation with continuous glucose feeding

The artificial gene coding for anticoagulant hirudin was placed under the control of theGAL10 promoter and expressed in the galactokinase-deficient strain (Δgal1) ofSaccharomyces cereivisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L.     

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.     

Growth kinetics ofSaccharomyces cerevisiae in batch and fedbatch cultivation using sugarcane molasses and glucose syrup from cassava starch

Growth kinetics ofSaccharomyces cerevisiae in glucose syrup from cassava starch and sugarcane molasses were studied using batch and fed-batch cultivation. The optimum temperature and pH required for growth were 30°C and pH 5.5, respectively. In batch culture the productivity and overall cell yield were 0.31 g L^–1 h^–1 and 0.23 g cells g^–1 sugar, respectively, on glucose syrup and 0.22 g L^–1 h^–1 and 0.18 g cells g^–1 sugar, respectively, on molasses. In fed-batch cultivation, a productivity of 3.12 g L^–1 h^–1 and an overall cell yield of 0.52 g cells g^–1 sugar in glucose syrup cultivation and a productivity of 2.33 g L^–1 h^–1 and an overall cell yield of 0.46 g cells g^–1 sugar were achieved in molasses cultivation by controlling the reducing sugar concentration at its optimum level obtained from the fermentation model. By using an on-line ethanol sensor combined with a porous Teflon^® tubing method in automating the feeding of substrate in the fed-batch culture, a productivity of 2.15 g L^–1 h^–1 with a yield of 0.47 g cells g^–1 sugar was achieved using glucose syrup as substrate when ethanol concentration was kept at a constant level by automatic control.     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

酿酒酵母L610利用菊糖生产乙醇发酵条件的研究

以1株能够直接利用菊糖产乙醇的酿酒酵母L610为出发菌株,对其利用菊糖生产乙醇的发酵条件进行了一系列研究。结果表明,L610最适乙醇发酵温度为37℃,且40℃高温发酵对其产乙醇能力无显著影响;L610对酸性发酵环境有良好的耐受性,当发酵液p H值降至3.5时,其糖醇转化率及乙醇产量仍保持较高水平;以0.025~0.10 vvm的通气量通气12 h有利于L610发酵菊糖产乙醇;L610对350 g/L的高浓度菊糖有良好的转化率,乙醇浓度和生产强度分别达到129 g/L和1.35 g/(L·h);当直接以300 g/L菊芋粗粉为唯一底物进行发酵时,L610发酵产乙醇浓度达到89.6 g/L,为理论产量的78.1%。本研究所取得的成果为酿酒酵母一步法发酵菊芋生产乙醇的工业化发展提供参考。