Studies on immobilized Saccharomyces cerevisiae. II. Effect of temperature distribution on continuous rapid ethanol formation in molasses fermentation

Recently, considerable interest has been shown in the study and analysis of immobilized cell reactors. One of the major uses of such a reactor system is expected to be in ethanol production from carbohydrates. One distinct disadvantage of this system is carbon dioxide gas holdup associated with unsteady-state temperature distribution across the reactor. Taking into account the earlier published data and assuming steady-state-substrate balance, and unsteady-state energy balance, and an average gas holdup of 20% with the heat retained by the gas neglected, the average reaction rate in the differential element was computed. Finally, a mathematical model to predict steady-state temperature profile along the reactor was developed. It was verified with experimental data obtained from an immobilized yeast reactor column (1 m x 14.5 cm). The experimental data fit well those computed from the model within an accuracy of 5%.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

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.     

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.     

Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

Ethanol fermentation in a magnetically fluidized bed reactor with immobilized Saccharomyces cerevisiae in magnetic particles

Ethanol fermentation by immobilized Saccharomyces cerevisiae cells in magnetic particles was successfully carried out in a magnetically stabilized fluidized bed reactor (MSFBR). These immobilized magnetic particles solidified in a 2 % CaCl(2) solution were stable and had high ethanol fermentation activity. The performance of ethanol fermentation of glucose in the MSFBR was affected by initial particle loading rate, feed sugar concentration and dilution rate. The ethanol theoretical yield, productivity and concentration reached 95.3%, 26.7 g/L h and 66 g/L, respectively, at a particle loading rate of 41% and a feed dilution rate of 0.4 h(-1) with a glucose concentration of 150 g/L when the magnetic field intensity was kept in the range of 85-120 Oe. In order to use this developed MSFBR system for ethanol production from cheap raw materials, cane molasses was used as the main fermentation substrate for continuous ethanol fermentation with the immobilized S. cerevisiae cells in the reactor system. Molasses gave comparative ethanol productivity in comparison with glucose in the MSFBR, and the higher ethanol production was observed in the MSFBR than in a fluidized bed reactor (FBR) without a magnetic field.     

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.     

Continuous and static fermentation of glucose to ethanol by immobilized Saccharomyces cerevisiae cells of different ages.

Glucose was converted to ethanol by calcium-alginate-entrapped Saccharomyces cerevisiae NRRL Y-2034 cells that were 24, 48, 72, and 96 h old in continuous-flow and static repeated-batch fermentors. In general, older yeast cells were more efficient than younger ones. In most cases, the continuous fermentations were better than the static ones in producing maximum ethanol yields (5.11 g/10 g of glucose) over extended time periods. The best static fermentation (with 24-h-old cells) converted 100% of the glucose to ethanol for about 12 days, whereas the best continuous fermentation (with 96-h-old cells) converted 100% of the glucose for a remarkable period of about 3 months.     

Metabolic flux variation of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment.

The technique of metabolic flux analysis was implemented to elucidate the flux balancing of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment. The results showed that the majority of the substrate (97.70 +/- 0.49%) was funneled into the glycolytic pathway, while the remainder was subdivided between the pentose phosphate pathway and pathways for polysaccharide synthesis. At the pyruvate node, 87.30 +/- 1.38% of the flux was channeled through the reaction governed by pyruvate decarboxylase. Fluxes through the pyruvate dehydrogenase bypass were maintained at a constant level (82.65 +/- 1.47%) irrespective of the configuration of the fermentation setup. Activity through the TCA "cycle" was replenished by the reaction catalyzed by pyruvate carboxylase and by the transport of cytosolic oxaloacetate across the mitochondrial membrane. The CO(2) evolution rate varied as fermentation progressed; however, the yield coefficient of CO(2) remained at a constant value. Although a constant yield of ethanol (0.42 g of ethanol/g of glucose) was obtained, operations of the TCA cycle were gradually switched from partially reductive to partially oxidative pathways from the first fermenter to the fourth fermenter.     

Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae

A diffusion-based microreactor system operated with a reaction volume of 8 μL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (μPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 μm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.     

Increasing ethanol productivity during xylose fermentation by cell recycling of recombinant Saccharomyces cerevisiae

The influence of cell recycling of xylose-fermenting Saccharomyces cerevisiae TMB3001 was investigated during continuous cultivation on a xylose-glucose mixture. By using cell recycling at the dilution rate ( D) of 0.05 h(-1), the cell-mass concentration could be increased from 2.2 g l(-1) to 22 g l(-1). Consequently, the volumetric ethanol productivity increased ten-fold, from 0.5 g l(-1) h(-1) to 5.35 g l(-1) h(-1). By increasing the biomass concentration, the xylose consumption rate increased from 0.75 g xylose l(-1) h(-1) without recycling to 1.9 g l(-1) h(-1) with recycling. The specific ethanol productivity was in the range of 0.23-0.26 g g(-1) h(-1) with or without cell recycling, showing that an increased cell-mass concentration did not influence the efficiency of the yeast.     

酿酒酵母 L-阿拉伯糖转化乙醇代谢工程的研究进展简

L-阿拉伯糖是木质纤维素原料中一种重要的五碳糖组分,但传统的乙醇生产菌株酿酒酵母（ Saccharomyces cerevisiae）不能利用L 阿拉伯糖。通过代谢途径工程手段,在酿酒酵母中引入L 阿拉伯糖初始代谢途径可以获得能利用L 阿拉伯糖乙醇发酵的重组菌株。并且,通过代谢途径的疏通以及吸收系统的优化可以强化重组菌株代谢L 阿拉伯糖的能力。笔者从以上角度综述了近年来酿酒酵母转化L 阿拉伯糖生产乙醇的研究进展。     

Determination of the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation.

Considerable controversy exists concerning the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation. This controversy results from problems in the measurement of the intracellular concentration of compounds like ethanol, which are being produced rapidly by metabolism and potentially diffuse rapidly from the cell. We used a new method for the determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate the aqueous cell volume. This method avoided many of the technical problems in previous reports. Our results indicate that the extracellular concentrations of ethanol in fermenting suspensions of S. cerevisiae are less than or equal to those in the intracellular environment and do not increase to the high levels previously reported even during the most active stages of batch fermentation.     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

Candida shehatae and Saccharomyces cerevisiae work synergistically to improve ethanol fermentation from sugarcane bagasse and rice straw hydrolysate in immobilized cell bioreactor

Sugarcane bagasse (SCB) and rice straw (RS), abundant lignocellulosic agro‐industrial residues in South‐East Asia, are potent feedstocks for bioethanol production as they contain significant amount of glucose and xylose monomers after fractionation and subsequent enzymatic hydrolysis. To simultaneously convert glucose and xylose to ethanol, it requires co‐cultivation of Saccharomyces cerevisiae and Candida shehatae which are hexose and pentose‐fermenting yeasts, respectively. Xylose‐fermenting strain grows slower than glucose‐fermenting one, therefore low efficiency of xylose‐to‐ethanol conversion was found. To enhance the efficiency of ethanol fermentation, the present work proposed to improve xylose assimilation by using co‐immobilization of two strains in a packed bed bioreactor and to increase oxygenation of the medium by applying a recycled batch system when the recycle stream was intervened by a mixing system in a naturally aerated vessel. Initially, conversion of glucose and xylose to ethanol using pure culture was investigated. Subsequently, influence of different immobilization techniques was investigated. Cells entrapment in Ca‐alginate beads provided considerably high ethanol yield over cells immobilized on delignified cellulose, and thus it was selected to use as inoculum in an immobilized cell bioreactor (ICB). The results showed that continuous ethanol production yielded 0.38 and 0.40 g/g corresponding to 74.5% and 78.4% theoretical yields from SCB and RS hydrolysate, respectively. However, recycled batch system produced significantly improved ethanol yield to 0.49 g/g and 0.50 g/g corresponding to 96.1% and 98.0% theoretical yields for SCB and RS hydrolysate, respectively. In this study, higher ethanol concentration and less unfermented sugar concentration was successfully achieved in the ICB with recycled batch system when using SCB and RS hydrolysate as the substrate.     

Growth and growth estimation of Saccharomyces cerevisiae in solid-state ethanol fermentation

The anaerobic growth of a respiration-deficient mutant of Saccharomyces cerevisiae on solid medium was estimated by the CO₂ evolution rate (CER). The cell growth and ethanol production were calculated by a growth-model associated with CER. The estimated cell growth agreed with the observed data. The calculation and the observed CER suggested that the maximum ethanol production and maximum cell groth are restricted by the initial moisture content of the solid medium.