稀释速率对高浓度酒精连续发酵过程振荡行为的影响

在一搅拌罐和三段管式反应器组成的组合反应器系统中,使用葡萄糖浓度为280g/L,添加5g/L酵母膏和3g/L蛋白胨的底物,在总稀释速率分别为0.032h^-1,0.024h^-1,0.017h^-1,0.012h^-1和0.006h^-1的条件下,考察了稀释速率对高浓度酒精发酵系统振荡行为的影响。结果表明,振荡行为在特定的稀释速率条件下呈现,进而基于数学上的分岔理论,分析了振荡行为发生的可能性及对应的稀释速率范围,并与实验结果进行了比较,在此基础上,讨论了振荡行为对酒精发酵过程的影响。     

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.     

多级串联悬浮床反应器系统中自絮凝颗粒酵母乙醇连续发酵耦合废糟液直接全循环使用的研究

在一套四级串联悬浮床生物反应器系统中,以双酶法制备的玉米粉糖化液为底物,进行了废糟液全循环条件下自絮凝颗粒酵母乙醇连续发酵的实验研究。在实验中,每隔5 d将从末级反应器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。系统连续运行了120d,共进行了24批次实验,数据分析表明系统达到了平衡状态。在平均发酵时间为20h条件下,发酵终点乙醇浓度平均为11.7%（V/V）,残还原糖浓度平均为7.9g/L,装置运行平稳。这些工作为自絮凝颗粒酵母乙醇发酵耦合废糟液直接全循环使用、实现污染物源头减废、清洁生产奠定了理论和实验基础。     

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.     

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。     

串联填充管式反应系统中高浓度乙醇连续发酵

在一套由搅拌罐和管式反应器串联而成的组合式反应系统中,利用酿酒酵母进行连续发酵生产高浓度乙醇。后续管式反应器内通过装填聚氨酯颗粒和木块对酵母细胞进行吸附固定化,在乙醇抑制造成细胞活性大幅降低的情况下,通过大幅提高细胞浓度保证发酵效率,在稀释速率0.02h^-1和280g/L葡萄糖的条件下,系统的终点乙醇浓度为15.4 % (v/v)。研究表明在一定稀释速率之下,应该通过增加反应器的级数来降低稀释速率,以达到提高终点乙醇浓度,如简单地降低进料速率则可能增加整个系统所受的乙醇抑制,对提高终点乙醇浓度效果不显著。     

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.     

无载体固定化酵母细胞木薯淀粉质原料酒精连续发酵研究

以木薯粉糖化液为发酵底物,在总发酵体积(有效)为15L的悬浮床生物反应器中,对一株粟酒裂殖酵母变异株进行一级和二级连续发酵研究。结果表明,二级连续发酵系统可明显改善一级系统的不足,并取得了平均流加糖液浓度150g/L,发酵强度为97g/L.h,流出液酒精浓度727g/L,残糖浓度374g./L,总糖利用率达90%的较好结果;整个系统在连续一个月的运行中从未发现染菌现象,发酵操作稳定。     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

Analytical monitoring of alcoholic fermentation using NIR spectroscopy

Alcoholic fermentation under Saccharomyces cerevisiae yeasts is governed largely by glucose uptake, biomass formation, ethanol and glycerin production, and acidification. In this work, PLS calibration models were developed with a view to determining these analytical parameters from near infrared spectra and analytical data provided by the corresponding reference methods. The models were applied to a set of samples obtained from various fermentation processes. The glucose, ethanol, and biomass values predicted by the models exhibited a high correlation with those provided by the reference method.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Optimization of pretreatment and saccharification for the production of bioethanol from water hyacinth by Saccharomyces cerevisiae

Alkaline-oxidative (A/O) pretreatment and enzymatic saccharification were optimized for bioethanol fermentation from water hyacinth by Saccharomyces cerevisiae. Water hyacinth was subjected to A/O pretreatment at various NaOH and H(2)O(2) concentrations and reaction temperatures for the optimization of bioethanol fermentation by S. cerevisiae. The most effective condition for A/O pretreatment was 7% (w/v) NaOH at 100 °C and 2% (w/v) H(2)O(2). The carbohydrate content was analyzed after reaction at various enzyme concentrations and enzyme ratios using Celluclast 1.5 L and Viscozyme L to determine the effective conditions for enzymatic saccharification. After ethanol fermentation using S. cerevisiae KCTC 7928, the concentration of glucose, ethanol and glycerol was analyzed by HPLC using a RI detector. The yield of ethanol in batch fermentation was 0.35 g ethanol/g biomass. Continuous fermentation was carried out at a dilution rate of 0.11 (per h) and the ethanol productivity was 0.77 [g/(l h)].     

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.     

Influence of bioreactor hydraulic characteristics on a Saccharomyces cerevisiae fed-batch culture: hydrodynamic modelling and scale-down investigations

Yeast is a widely used microorganism at the industrial level because of its biomass and metabolite production capabilities. However, due to its sensitivity to the glucose effect, problems occur during scale-up to the industrial scale. Hydrodynamic conditions are not ideal in large-scale bioreactors, and glucose concentration gradients can arise when these bioreactors are operating in fed-batch mode. We have studied the effects of such gradients in a scale-down reactor, which consists of a mixed part linked to a non-mixed part by a recirculation pump, in order to mimic the hydrodynamic conditions encountered at the large scale. During the fermentation tests in the scale-down reactor, there was a drop in both biomass yield (ratio between the biomass produced and the glucose added) and trehalose production and an increase in both fermentation time (time between inoculation and beginning of stationary phase) and ethanol production. We have developed a stochastic model which explains these effects as the result of an induction process determined mainly by the hydrodynamic conditions. The concentration profiles experienced by the microorganisms during the scale-down tests were expressed and linked to the biomass yields of the scale-down tests.     

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。     

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).     

The effect of vitamins and amino acids on glucose uptake in aerobic chemostat cultures of three Saccharomyces cerevisiae strains

In the respiro-fermentative region of aerobic chemostat cultures at steady state, Saccharomyces cerevisiae CBS 8066 produced high concentrations of ethanol with concomitant low levels of residual glucose which followed Monod kinetics. By contrast, very high residual glucose concentrations were observed in cultures of S. cerevisiae strains ATCC 4126 and NRRL Y132 at dilution rates above 60% of the washout dilution rate, resulting in much lower ethanol concentrations, even though clearly glucose-limited at lower dilution rates in the respiratory region. The addition of a vitamin mixture resulted in decreased residual glucose concentrations in respiro-fermentative cultures of all three strains, but the effect was much more pronounced with strains ATCC 4126 and NRRL Y132. Meso-inositol was mainly responsible for this effect, although with strain ATCC 4126 other vitamins as well as an amino acid mixture were also required to minimise the steady-state residual glucose levels. The residual glucose concentration in continuous culture was, therefore, greatly dependent on the growth factor requirements of the particular yeast strain, which apparently increased on increasing the dilution rate into the respiro-fermentative region. The strain differences with respect to growth factor requirements at high dilution rates, which were not evident at low dilution rates, had a profound effect on the kinetics of glucose assimilation in aerobic chemostat culture.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Kinetics of lactose fermentation using a recombinant Saccharomyces cerevisiae strain

This work presents a multi-route, non-structural kinetic model for interpretation of ethanol fermentation of lactose using a recombinant flocculent Saccharomyces cerevisiae strain expressing both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces lactis. In this model, the values of different metabolic pathways are calculated applying a modified Monod equation rate in which the growth rate is proportional to the concentration of a key enzyme controlling the single metabolic pathway. In this study, three main metabolic routes for S. cerevisiae are considered: oxidation of lactose, reduction of lactose (producing ethanol), and oxidation of ethanol. The main bioprocess variables determined experimentally were lactose, ethanol, biomass, and dissolved oxygen concentrations. Parameters of the proposed kinetic model were established by fitting the experimental data obtained in a small lab-scale fermentor with the initial lactose concentrations ranging from 5 g/dm3 to 50 g/dm3. A very good agreement between experimental data and simulated profiles of the main variables (lactose, ethanol, biomass, and dissolved oxygen concentrations) was achieved.