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 280g 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 1.5g l?1 were achieved under the oscillatory state, with an average ethanol productivity of 2.14g h?1l?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 2.00g h?1l?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?1l?1, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h?1.

Taking continuous ethanol fermentation with the self-flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300mm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300mm grown in presence or absence of 0.05 g L 1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 mm exhibited highest ethanol production (110.0 g L 1) and glucose uptake rate (286.69 C mmol L 1 h 1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system.

Yeast strains of Saccharomyces cerevisiae have been extensively studied in recent years for fuel ethanol production, in which yeast cells are exposed to various stresses such as high temperature, ethanol inhibition, and osmotic pressure fromproduct and substrate sugars aswell as the inhibitory substances released from the pretreatment of lignocellulosic biomass. An in-depth understanding of the mechanism of yeast stress tolerance contributes to breeding more robust strains for ethanol production, especially under very high gravity conditions. Taking advantage of the "omics" technology, the stress response and defense mechanism of yeast cells during ethanol fermentationwere further explored, and the newly emerged tools such as genome shuffling and global transcription machinery engineering have been applied to breed stress resistant yeast strains for ethanol production. In this review, the latest development of stress tolerance mechanisms was focused, and improvement of yeast stress tolerance by both random and rational tools was presented.

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.

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.

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.

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. Biotechnol. Bioeng. 2009; 102: 113-121.

The effects of zinc supplementation were investigated in the continuous ethanol fermentation using selfflocculating yeast. Zinc sulfate was added at the concentrations of 0.01, 0.05 and 0.1 g l?1, respectively. Reduced average floc sizes were observed in all the zinc-supplemented cultures. Both the ethanol tolerance and thermal tolerancewere significantly improved by zinc supplements, which correlatedwell with the increased ergosterol and trehalose contents in the yeast flocs. The highest ethanol concentration by 0.05 g l?1 zinc sulfate supplementation attained 114.5 g l?1, in contrast to 104.1 g l?1 in the control culture. Glycerol production was decreased by zinc supplementations, with the lowest level 3.21 g l?1, about 58% of the control. Zinc content in yeast cellswas about 1.4 Mol g?1 dry cellweight, about sixfold higher than that of control in all the zinc-supplemented cultures, and close correlation of zinc content in yeast cells with the cell viability against ethanol and heat shock treatment was observed. These studies suggest that exogenous zinc addition led to a reprogramming of cellular metabolic network, resulting in enhanced ethanol tolerance and ethanol production.

High ethanol tolerance is a desired property of industrial yeast strains for efficient ethanol fermentation. In this study, the impact of medium composition on ethanol tolerance of the self-flocculating yeast SPSC01 was investigated using a chemically defined medium. Singlefactor experiments revealed that besides magnesium and calcium, zinc also exhibited significant protective effect against ethanol toxicity; addition of 0.02 g/l zinc sulfate significantly increased cell viability in the ethanol shock treatment. Metal ions of manganese, cobalt, and ferrous failed to promote ethanol tolerance, although addition of 0.02 g/l cobalt increased ethanol production without apparent influence on ethanol tolerance. Furthermore, Uniform Design method was employed to obtain the medium with high cell viability, and the key nutrient factors in the medium composition were revealed to be (NH4)2SO4, K2HPO4, vitamin mixtures, and the metal ions of magnesium, calcium and zinc. The optimized combination of metal ions addition was (g/l): MgSO4 0.4, CaCl2 0.2, ZnSO4 0.01. The highest cell viability (90.2%) of SPSC01 against ethanol shock treatment was observed in the optimized medium, which demonstrated significant improvement of ethanol tolerance of the self-flocculating yeast.

This article critically reviews some ethanol fermentation technologies from sugar and starch feedstocks, particularly those key aspects that have been neglected or misunderstood. Compared with Saccharomyces cerevisiae, the ethanol yield and productivity of Zymomonas mobilis are higher, because less biomass is produced and a higher metabolic rate of glucose is maintained through its special Entner–Doudoroff pathway. However, due to its specific substrate spectrum as well as the undesirability of its biomass to be used as animal feed, this species cannot readily replace S. cerevisiae in ethanol production. The steady state kinetic models developed for continuous ethanol fermentations show some discrepancies, making them unsuitable for predicting and optimizing the industrial processes. The dynamic behavior of the continuous ethanol fermentation under high gravity or very high gravity conditions has been neglected, which needs to be addressed in order to further increase the final ethanol concentration and save the energy consumption. Ethanol is a typical primary metabolite whose production is tightly coupled with the growth of yeast cells, indicating yeast must be produced as a co-product. Technically, the immobilization of yeast cells by supporting materials, particularly by gel entrapments, is not desirable for ethanol production, because not only is the growth of the yeast cells restrained, but also the slowly growing yeast cells are difficult to be removed from the systems. Moreover, the additional cost from the consumption of the supporting materials, the potential contamination of some supporting materials to the quality of the co-product animal feed, and the difficulty in the microbial contamination control all make the immobilized yeast cells economically unacceptable. In contrast, the self-immobilization of yeast cells through their flocculation can effectively overcome these drawbacks.