Parameter oscillations in a very high gravity medium continuous ethanol fermentation and their attenuation on a multistage packed column bioreactor system

The quasi-steady-states, marked by small fluctuations of residual glucose, ethanol, and biomass concentrations, and sustainable oscillations marked by big fluctuations of these monitored fermentation parameters were observed during the continuous ethanol fermentation of Saccharomyces cerevisiae when very high gravity media were fed and correspondingly high ethanol concentrations reached. A high ethanol concentration was shown to be one of the main factors that incited these oscillations, although the residual glucose level affected the patterns of these oscillations to some extent. The lag response of S. cerevisiae to high ethanol stress that causes the shifts of morphology, viability loss, and death of yeast cells is assumed to be one of the probable mechanisms behind these oscillations. It was predicted that the longer the delay of this response was, the longer the oscillation periods would be, which was validated by the experimental data and the comparison with the oscillatory behaviors reported for the ethanologen bacterium, Zymomonas mobilis. Furthermore, three tubular bioreactors in series were arranged to follow a stirred tank bioreactor to attenuate these oscillations. However, exaggerated oscillations were observed for the residual glucose, ethanol, and biomass concentrations measured in the broth from these tubular bioreactors. After the tubular reactors were packed with Intalox ceramic saddle packing, these oscillations were effectively attenuated and quasi-steady-states were observed during which there were very small fluctuations of residual glucose, ethanol, and biomass within the entire experimental run.     

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。     

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。     

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。     

自絮凝酵母颗粒连续发酵生产酒精的新工艺

用既有优良酒精发酵性能,又具有强自絮凝能力的融合酵母株ＳＰＳＣ,在单釜有效容积为１０ｍ３的四釜并联气升环流悬浮床生物反应器系统中,进行了连续发酵生产酒精的研究。以玉米为原料,双酶法制糖,过滤得到清糖液作为底物,在稀释速率为０１／ｈ的条件下,终点发酵液中酒精浓度为７０～８０ｇ／Ｌ,残余还原糖和残余总糖分别为２～３ｇ／Ｌ和３～５ｇ／Ｌ,悬浮床反应器的设备生产强度达到７～８ｇ（ＥｔＯＨ）／（Ｌ·ｈ）。     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

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。     

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。     

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

ABSTRACT: BACKGROUND: Very high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15 % (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production. RESULTS: Batch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 [PLUS-MINUS SIGN] 3.1, 252 [PLUS-MINUS SIGN] 2.9 and 298 [PLUS-MINUS SIGN] 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at [MINUS SIGN]100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 [PLUS-MINUS SIGN] 3.1 and 252 [PLUS-MINUS SIGN] 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 [PLUS-MINUS SIGN] 1.3 and 120.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 [PLUS-MINUS SIGN] 0.4 and 17.0 [PLUS-MINUS SIGN] 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol was produced at 72 h when ORP was controlled at [MINUS SIGN]150 mV for the VHG fermentation with medium containing 298 [PLUS-MINUS SIGN] 3.8 g/L glucose, since yeast cell viability was improved more significantly. CONCLUSIONS: No lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at [MINUS SIGN]150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at [MINUS SIGN]100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.     

Ethanol fermentation in a yeast immobilized tubular fermentor

In this article, a mathematical model describing the kinetics of ethanol fermentation in a whole cell immobilized tubular fermentor is proposed. Experimental results show reasonable agreement with the proposed model. A procedure for treating the fermentation data for determining the ethanol inhibition constants k(1) and k(2) is described. The ethanol productivity of the immobilized cell fermentor is compared with those of traditional fermentors. Experimental studies indicate that with Saccharomyces cerevisiae (NRRL Y132) culture, ethanol productivity in the range 21.2-83.7 g ethanol L(-1) h(-1) at ethanol concentration of 76-60 g/L can be achieved. This is comparable to or higher than those reported in the literature for yeast. The product yield factor of 0.5 g ethanol/g glucose was obtained. The immobilized cell fermentor does not show washout at dilution rates of 7 h(-1) and shows good stability over a 650-h operating period.     

Increased phenotypic stability and ethanol tolerance of recombinant Escherichia coli KO11 when immobilized in continuous fluidized bed culture

The recombinant Escherichia coli B strain KO11, containing chromosomally-integrated genes for ethanol production, was developed for use in lignocellulose-to-ethanol bioconversion processes but suffers from instability in continuous culture and a low ethanol tolerance compared to yeast. Here we report the ability cell immobilization to improve its phenotypic stability and ethanol tolerance during continuous culture on a 50 g/L xylose feed. Experiments conducted in a vertical tubular fermentor operated as a liquid-fluidized bed with the cells immobilized on porous glass microspheres were compared to control experiments in the same reactor operated as a chemostat without the support particles. Without cell immobilization the ethanol yield fell sharply following start-up, declining to 60% of theoretical after only 8-9 days of continuous fermentation. While immobilizing the cells did not prevent this decline, it delayed its onset and slowed its rate. With immobilization, a stable high ethanol yield (>85%) was maintained for at least 10 days, thereafter declining slowly, but remaining above 70% even after up to 40 days of fermentation. The ethanol tolerance of E. coli KO11 cells was substantially increased by immobilization on the glass microspheres. In ethanol tolerance tests, immobilized cells released from the microspheres had survival rates 2.3- to 15-fold higher than those of free cells isolated from the same broth. Immobilization is concluded to be an effective means of increasing ethanol tolerance in E. coli KO11. While immobilization was only partially effective in combating its phenotypic instability, further improvements can be expected following optimization of the immobilization conditions.     

自絮凝颗粒酵母酒精高浓度连续发酵的研究

在四釜串联气升环流悬浮床生物反应器系统中 ,进行了絮凝颗粒酵母酒精连续发酵的研究。以CO2 为驱动动力 ,发酵液液蒸馏废液全循环 ,稀释率为 0 2 h。发酵成熟醑酒精平均浓度为 96 6g L ,残余还原糖和总糖分别为 1 2g L和 4 1g L。     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

A novel process of ethanol production accompanied by extraction of sugar in cane chips

By connecting a rhomboid unit for fermentation and an exchangeable tubular unit for extraction, a novel bioreactor was designed to produce high concentration of ethanol solution from non-peeled sugar cane chips by means of a cyclic system for exchanging old chips with new ones. The rhomboid and tubular units were packed with 1.0–1.5 mm biocatalyst entrapped yeast-cells with Al alginateand cane chips of 2.0–3.0 mm in width, respectively. The volume ratio of the two units was 1.0. At the start of the first cycle, 2 g/l of Al₂(SO₄)₃ 14–18 H₂O solution was added to the bioreactor, where the ratio of the solution to the working volume was 0.85. Both sugar extraction and fermentation were anaerobically performed at pH 2.5 and a temperature of 30°C by circulating the solution through the two units. After each cycle, the tubular unit was exchanged for a new unit packed with new chips. The combined solution of free ethanol and the ethanol obtained by pressing the old chips was re-used as the circulating solution.When the volume ratio of the biocatalyst to the total working volume was 0.1 and the amount of dried cane chips in the tubular unit was 200 g/l, 16% (w/v) ethanol was produced after 7 cycles. Each cycle was established at about 20 h. The number of free cells in the circulating solution was only 2×10⁷/ml after 7 cycles.     

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.     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.     

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。     

Differences in response of Zymomonas mobilis and Saccharomyces cerevisiae to change in extracellular ethanol concentration

In high cell density batch fermentations, Zymomonas mobilis produced 91 g L(-1) ethanol in 90 min but culture viability fell significantly. Similar viability losses in rapid fermentations by yeast have recently been shown to be attributable in part to the high rate of change of the extracellular ethanol concentration. However, in simulated rapid fermentations in which ethanol was pumped continuously to low cell density Z. mobilis suspensions, increases in the rate of change of ethanol concentration in the range 21-83 g L(-1) h(-1) did not lead to accelerated viability losses. The lag phase of Zymomonas cultures exposed to a 30-g L(-1) step change in ethanol concentration was much shorter than that of Saccharomyces cerevisiae, providing evidence that the comparative insensitivity of Zymomonas to high rates of change of ethanol concentration is due to its ability to adapt to changes in ethanol concentration more rapidly than yeast. (c) 1994 John Wiley & Sons, Inc.     

The long-term effects of ethanol on immobilized cell reactor performance using K. fragilis

The effects of ethanol on reactor performance were studied in a small, 5-cm packed height, "differential" type immobilized cell reactor. Lactose utilizing yeast cells, Kluyveromyces fragilis, were absorbed to a porous adsorbant sponge matrix in a gas continuous reactor. Step changes in the feed ethanol concentration to the column (10-130 g/L) were used to test the reactor response over extended periods of time (about 30-50 h per dosage level) followed by a return to basal zero inlet ethanol feed. Effluent cell density and effluent cell viability were measured at intervals. An inhibitory response in ethanol productivity to feed dosage ethanol levels above 20 g/L was detected almost immediately, with a near steady state response noted within 2.5 h of initiating the dosage. Feed ethanol levels above 50 g/L resulted in a subsequent gradual decrease in reactor productivity over time, which was associated with a decrease in the fraction of viable shed cells in the reactor effluent. The reactor response to a step removal of the ethanol inhibition was also monitored. Quick and complete rebounding of the fermentation rate to the original basal rate was noted following dosage concentrations of under 50 g/L ethanol. Recovery rates slowed following ethanol dosage levels above 50 g/L. Viable shed cell density improved overtime during the slow recovery periods. Growth rates (as determined by shed cell density) were more strongly inhibited than productivity. Growth responded more slowly to changes in ethanol environment as growth rates at 30 h fell to about 40% of the rates measured 7.5 h after initiation of a dosage level. It is concluded that ethanol contributions to cell injury and death (and consequent ICR performance degradation) may be more important than ethanol inhibition of productivity rates in the long-term operation of immobilized cell reactors at ethanol concentrations over 50 g/L.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.