Parameter Oscillation Attenuation and Mechanism Exploration for Continuous VHG Ethanol Fermentation

Transcription

1 Parameter Oscillation Attenuation and Mechanism Exploration for Continuous VHG Ethanol Fermentation F.W. Bai, 1,2 X.M. Ge, 1 W.A. Anderson, 2 M. Moo-Young 2 1 Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 11623, China; telephone: ; fax: ; fwbai@dlut.edu.cn 2 Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Received 26 December 27; revision received 1 May 28; accepted 24 June 28 Published online 15 July 28 in Wiley InterScience ( DOI 1.12/bit.2243 ABSTRACT: 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 28 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.27 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 2 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 without yeast cell immobilization effect did not affect the oscillatory behavior Correspondence to: F.W. Bai of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation. Biotechnol. Bioeng. 29;12: ß 28 Wiley Periodicals, Inc. KEYWORDS: continuous ethanol fermentation; very high gravity; packing; oscillation; yeast cell immobilization; attenuation Introduction Sustainable parameter oscillations were observed for continuous ethanol fermentations with Zymononos mobilis (Bruce et al., 1991; Daugulis et al., 1997; Ghommidh et al., 1989; Jöbses et al., 1986; Jarzebski, 1992; Lee et al., 1979; McLellan et al., 1999), and Saccharomyces cerevisiae (Bai et al., 24; Borzani, 21). These oscillations were characterized by long oscillation periods and large oscillation amplitudes. For example, the oscillation periods about 5 h for glucose, ethanol and biomass, and the oscillation amplitudes of 2 1 g L 1 for glucose and 5 9 g L 1 for ethanol were reported in the continuous ethanol fermentation with Z. mobilis (McLellan et al., 1999), while much longer oscillation periods of 16 h for sugar, ethanol, and biomass, and larger oscillation amplitudes of 1 7 g L 1 for sugar and 4 7 g L 1 for ethanol were observed in the continuous ethanol fermentation with S. cerevisiae (Borzani, 21). Our recent studies further indicated that these oscillations negatively affected ethanol fermentation performance with increase in residual sugar and decrease in the ethanol yield which is calculated without the deduction of the residual sugar in the ethanol fermentation industry (Bai et al., 28); therefore, these oscillations need be attenuated. ß 28 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 12, No. 1, January 1, 转载

2 In fact, parameter oscillations also exist in continuous ethanol fermentation in the industry where the tanks-inseries fermentation system is widely used to alleviate ethanol inhibition, particularly in the front and middle tanks, but have been ignored and believed to be the fluctuations caused by the variations of the operating parameters such as the flowrate and sugar concentration of the medium, fermentation temperature and ph value, although the current process control system can precisely control these parameters, making their variations too small to trigger any significant oscillations in the fermentation system. Extended fermentation time is adopted in order to attenuate these oscillations and achieve steady or quasi-steady state for the fermentation system. The extension of the fermentation time, without doubt, decreases the ethanol productivity. Mechanistic analysis indicated ethanol inhibition and the lag responses of yeast cells to the ethanol inhibition are the main reason triggering these oscillations (Bai et al., 24). Our recent studies further demonstrated that the lethal effect of high ethanol concentration on yeast cells, especially under high gravity (HG) and very high gravity (VHG) fermentation conditions, is the reason for the exaggerated oscillations observed in those rear bioreactors (submitted for publication). The mixing performance of bioreactors affects substrate as well as product inhibition. A CSTR can effectively alleviate substrate inhibition because high substrate concentration is diluted immediately when the medium is added into the bioreactor, while a PFR can mitigate product inhibition as the highest product concentration is achieved gradually along the axial direction of the bioreactor. For ethanol fermentation, CO 2, produced in an equal mole number with that of ethanol, makes the mixing performance of the fermentors, especially the main ones in a cascade fermentation system, more close to the CSTR model, which alleviates the substrate inhibition but worsens the product inhibition. Theoretically, a tanks-inseries system, with its mixing performance close to a PFR model if the number of the tanks is infinite, could alleviate product inhibition. In our previous studies, a bioreactor system composed of a tank and three tubular bioreactors in series was established for the continuous VHG ethanol fermentation, and oscillations occurred within the tubular bioreactors were attenuated after the tubular bioreactors were packed with 1/2 Intalox ceramic saddles, but the corresponding mechanism was simply speculated to be the backmixing alleviation of the tubular bioreactors caused by the packing which partitioned the tubular bioreactors into small chambers in series without experimental validation (Bai et al., 24). In this article, the mixing performance of the packed tubular bioreactors was studied by analyzing its residence time distribution (RTD) to verify the aforementioned mechanistic analysis for the oscillation attenuation. In the mean time, the impact of the packing s yeast cell immobilization on the oscillation attenuation was further studied. Materials and Methods Strain, Media, and Pre-Cultures Strain, media, and pre-culture were described in the reference (Bai et al., 24). Bioreactor System, Inoculation, and Ethanol Fermentation The diagram of the bioreactor system for the continuous VHG ethanol fermentation is illustrated in Figure 1. It was expected that both substrate and product inhibition could be alleviated through the combination of a tank fermentor and tubular bioreactors in such a series model. The inoculation and continuous VHG ethanol fermentation protocols for this bioreactor system were also introduced previously (Bai et al., 24). In addition to 1/2 Intalox ceramic saddles, 1 mm 1 mm 1 mm polyurethane particles, 5 mm 5mm 1 mm wood chips, and f1 mm 1 mm made of 8 mesh stainless steel wire mesh were selected to study the impact of yeast cell immobilization on oscillation attenuation. The working volumes of the tubular bioreactors were measured to be 42, 41, and 4 ml; 34, 33, and 32 ml; 6, 59, and 58 ml when packed with the polyurethane particles, wood chips, and, respectively. Analytical Methods The analytical methods for glucose, ethanol, biomass, and yeast cell viability were described in the reference (Bai et al., 24). RTDs were measured for the bioreactors in the system in order to evaluate their mixing performance. Xylose that cannot be metabolized by the yeast was used as a tracer and added into the medium at a concentration of 2 g L 1, and the step input response technique was applied to the system (Levenspiel, 1999). Xylose was analyzed by HPLC (Waters TM 6 Controller and Waters TM 6 Pump; Detector: Waters 41 Differential Refractometer; Column: Aminex 1 HPX-87H, 3 mm 7.8 mm; Eluant:.5 mol L 1 H 2 SO 4, flow rate:.4 ml min 1 ; Data treatment software: Waters Millennium 32). Results and Discussion Given the fact that packing occupied volumes of the tubular bioreactors, and correspondingly, decreased these tubular bioreactors working volumes, the average fermentation time was shortened when the same medium flow rate was applied to the bioreactor system. However, not only were lower glucose and higher ethanol were achieved after the tubular bioreactors were packed with 1/2 Intalox ceramic saddles, but the oscillations observed in the empty tubular 114 Biotechnology and Bioengineering, Vol. 12, No. 1, January 1, 29

3 Figure 1. Diagram of the bioreactor system for continuous VHG ethanol fermentation. (1) Substrate tank, (2) peristaltic pump, (3) stirred tank, (4 and 5) ph and temperature control units, (6) tubular bioreactors, (7 and 8) thermostat water inlets and outlets, (9) broth tank, (1) exhaust gas washing tank, (11) air flowmeters, () humidifiers, and (13) sampling ports. bioreactors were also effectively attenuated, as illustrated in Table I. The packing partitioned the tubular bioreactors into small chambers, each of them close to a CSTR. Although those chambers were randomly arranged, theoretically, the backmixing of these tubular bioreactors could be mitigated, and correspondingly, their ethanol inhibition could be alleviated, which might attenuate the oscillations as the mechanism triggering these oscillations was proposed to be the lag response of yeast cells to ethanol inhibition (Bai et al., 24). Mixing Performance of the Packed Tubular Bioreactors Figures 2 5 show the step input responses of each bioreactor, through which the variances of the RTDs were calculated to be.95 for the tank,.6 for the tank and first packed tubular bioreactor,.46 for the tank, first and second packed tubular bioreactor, and.3 for the bioreactor system. The variance of the RTD was experimentally measured to be.95 for the tank, while theoretically calculated to be 1. for an ideal CSTR. Thus, the tank was operated as a CSTR as predicted. If the packed tubular bioreactors were assumed as ideal CSTRs, the variances of the RTDs were calculated to be.7 for the tank and the first packed tubular bioreactor,.55 for the tank, the first and second packed tubular bioreactors, and.46 for the whole bioreactor system (Levenspiel, 1999). The experimentally measured variances were very close to these theoretical predictions, indicating the mixing performance of the packed tubular bioreactors was still close to the Table I. Comparison of the parameter oscillations for the empty columns and the columns packed with the Intalox ceramic saddles. Bioreactors First tubular bioreactor Second tubular bioreactor Third tubular bioreactor Parameters Empty Packed Empty Packed Empty Packed Glucose (g L 1 ) Oscillation rage Oscillation amplitude Oscillation average Ethanol (g L 1 ) Oscillation rage Oscillation amplitude Oscillation average Bai et al.: Continuous VHG Ethanol Fermentation 115 Biotechnology and Bioengineering

4 C/C Figure Time, h Step input response of the tank fermentor. Xylose was used as a tracer and added into the medium at a concentration of 2 g L 1. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h 1. C/C Figure Time, h Step input response of the tank, the first and second tubular bioreactors packed with the wood chips. Xylose was used as a tracer and added into the medium at a concentration of 2 g L 1. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h 1. CSTR model, and ethanol concentration gradients could not be established within these tubular bioreactors. The reason for this phenomenon was that the average fermentation time about 1 h was too long, and the corresponding superficial speed of the broth passed through the tubular bioreactors was too low to overcome the backmixing of ethanol and glucose. Therefore, the improvement of the ethanol fermentation performance of these tubular bioreactors as well as the attenuation of their oscillations was not from the impact of the packing on their backmixing performance. Impact of the Yeast Cell Immobilization Packing, as a supporting material, can immobilize cells. However, its role in attenuating oscillations was never reported. When the tubular bioreactors were packed with 1/2 Intalox ceramic saddles, its yeast cell immobilization effect was examined. It was found that only moderate yeast cell immobilization was achieved (Bai et al., 24), and the impact of the yeast cell immobilization on the oscillation attenuation could not be validated, without comparison with other packings. Very good yeast cell immobilization was achieved when the tubular bioreactors were packed with polyurethane particles, porous supporting materials (Baptista et al., 26; Kourkoutas et al., 24; Lorenz et al., 1987), while the, with smooth surfaces, could not immobilize yeast cells, and the wood chips, another widely used supporting material for cell immobilization (Moo-Young et al., 198), exhibited similar yeast cell immobilization effect with the Intalox ceramic saddles because of their similar surface properties. The impact of these packings on the oscillations are illustrated in Figures 6 8. Table II further summarizes and compares the difference of these packings in yeast cell immobilization. As can be seen, the best yeast cell immobilization was achieved for the porous polyurethane particles as predicted, with the yeast cell concentrations within the bioreactors of 6.1, 71.7, and 18.7 g (DCW) L 1 for the first, second, and third tubular bioreactors, comparing with the yeast cell concentrations of only 5., 1.7, and.8 g (DCW) L 1 detected in C/C Figure Time, h Step input response of the tank and the first tubular bioreactor packed with the wood chips. Xylose was used as a tracer and added into the medium at a concentration of 2 g L 1. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h 1. C/C Figure Time, h Step input response of the bioreactor system, including the tank and the three tubular bioreactors packed with the wood chips. Xylose was used as a tracer and added into the medium at a concentration of 2 g L 1. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h Biotechnology and Bioengineering, Vol. 12, No. 1, January 1, 29

5 a Glucose, g l c Biomass, g(dcw) l -1 b Ethanol, g l Intalox ceramic saddles Figure 6. Impact of the packings on the fermentation performance of the first tubular bioreactor. Oscillation profiles are shown for (a) glucose, (b) ethanol, and (c) biomass. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h 1. the effluent. A yeast cell mass balance was established for the first tubular bioreactor as the yeast cell concentration in the effluent out of it was almost equal to that in the effluent into it, but 1-fold higher yeast cell concentration was obtained within the bioreactor because of the yeast cell immobilization role of the porous polyurethane particles. For the second and third tubular bioreactors, although yeast cell mass balances were not approached within the duration of the experiment as the yeast cell concentrations in the effluent into them were higher than those in the effluent out of them, the impact of the imbalance of the yeast cell biomass on ethanol fermentation performance was negligible because the viability of the yeast cells within the second and third tubular bioreactors was lower, as illustrated in Table III. However, the significant oscillations of glucose and ethanol were observed for all the three tubular bioreactors, as illustrated in Figures 6 8, when the tubular bioreactors were packed with the porous polyurethane particles. The averages of glucose and ethanol of the third tubular bioreactor were 22.8 and 111. g L 1, but their oscillation ranges were and g L 1, making the absolute oscillation amplitudes 1 as high as 34. and 18.6 gl 1 for glucose and ethanol, almost the same levels as those observed for the empty column (Bai et al., 24). On the other hand, moderate yeast cell immobilization was achieved for the wood chips as well as for the Intalox ceramic saddles. The yeast cell concentrations within the first, second, and third bioreactors were 25.3, 26.4, and 35.1 g (DCW) L 1, and the yeast cell concentrations in the effluent out of the first, second, and third tubular bioreactors were 6., 4.2, and 2.2 g (DCW) L 1 when the bioreactors were packed with the wood chips. The yeast cell concentrations within the first, second, and third tubular bioreactors were 16., 27.6, and 39.3 g (DCW) L 1, and the yeast cell concentrations in the effluent out of the first, second, and third tubular bioreactors were 7.6, 2.9, and 1.9 g (DCW) L 1 when the bioreactors were packed with the Intalox ceramic saddles. An imbalance of the yeast cell biomass for the first tubular bioreactor in which the yeast 1 The absolute oscillation amplitudes were defined as the differences between the oscillation peaks and troughs. Bai et al.: Continuous VHG Ethanol Fermentation 117 Biotechnology and Bioengineering

6 cell concentrations in the effluent out of it were higher than those in the effluent into it, indicating these packings improved the inner environment of the bioreactor, making it more favorable for the yeast cell growth, which could be the reason for the improvement of the ethanol fermentation performance as well as the oscillation attenuation within the second and third tubular bioreactors. The oscillations were effectively attenuated when the broth passed through the third tubular bioreactor packed with the wood chips and the Intalox ceramic saddles, and quasi-steady states were observed, as the fluctuation ranges of its residual glucose were only g L 1 when packed with the wood chips and g L 1 when packed with the Intalox ceramic saddles. In order to further explore the mechanistic reason for the oscillation attenuation caused by the wood chips and the Intalox ceramic saddles, the viability of the yeast cells within the packed bioreactors was examined, and it was found that the percentage of the viable cells was extremely lower when the tubular bioreactors were packed with the porous polyurethane particles, particularly for the third tubular bioreactor within which almost no viable yeast cells were detected, although its yeast cell concentration was as high as 18.7 g (DCW) L 1. When the empty columns were employed, the second and third columns were aerated in order to prevent yeast cells from settling. Although the aeration rate of.5 vvm was very low, it was enough to improve the viability of the yeast cells suffered from serve ethanol inhibition (Alfenore et al., 24; Ryu et al., 1984), guaranteeing the percentage of the viable cells in the effluent out of the third tubular bioreactor to be around 4%. The aeration for the second and third tubular bioreactors was interrupted after they were packed, making their viable cell percentages decreased dramatically. However, the higher biomass concentrations resulted from the packings yeast cell immobilization role effectively compensated this impact, and much better fermentation performance as well as the oscillation attenuation was achieved after the bioreactors were packed with the wood chips and the Intalox ceramic saddles. Compared with the porous polyurethane particles, the wood chips and the Intalox ceramic saddles had a common characteristic: the yeast cells loosely deposited and immobilized onto their surfaces, making viability-lost yeast a 8 b 16 Glucose, g l Ethanol, g l wood chips polyurethane particles c Biomass, g(dcw) l wood chips Intalox ceramic saddles polyurethane particles Figure 7. Impact of the packings on the fermentation performance of the second tubular bioreactor. Oscillation profiles are shown for (a) glucose, (b) ethanol, and (c) biomass. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h Biotechnology and Bioengineering, Vol. 12, No. 1, January 1, 29

7 a 8 b 16 Glucose, g l Ethanol, g l c Biomass, g(dcw) l Figure 8. Impact of the packings on the fermentation performance of the third tubular bioreactor. Oscillation profiles are shown for (a) glucose, (b) ethanol, and (c) biomass. The VHG medium containing 28 g L 1 glucose was fed into the tank fermentor at the dilution rate of.27 h 1. cells caused by the lethal effect of the high ethanol concentrations inside these tubular bioreactors easily detached, renewed, and washed out. Although the polyurethane particles immobilized much more yeast cells, the renewal of the viability-lost yeast cells that were immobilized within their inner pores by adsorption was difficult, which was supported by the much lower viable yeast cell percentages of 22.7%, 4.7%, and.8% for the first, second, Table II. Comparison of the packings yeast cell immobilization effects. Packings Tubular bioreactors Wood chips Polyurethane particles Ceramic saddles First Yeast cells in the broth, X 1 (g(dcw) L 1 ) Yeast cells deposited onto the surface or adsorbed into the pores of the packings, X 2 (g(dcw) L 1 ) Total yeast cell concentration, X T (g(dcw) L 1 ) Second Yeast cells in the broth, X 1 (g(dcw) L 1 ) Yeast cells deposited onto the surface or adsorbed into the pores of the packings, X 2 (g(dcw) L 1 ) Total yeast cell concentration, X T (g(dcw) L 1 ) Third Yeast cells in the broth, X 1 (g(dcw) L 1 ) Yeast cells deposited onto the surface or adsorbed into the pores of the packings, X 2 (g(dcw) L 1 ) Total yeast cell concentration, X T (g(dcw) L 1 ) X 1 was measured at the end when the broth within the packed tubular bioreactors was drained out and yeast cells loosely immobilized onto the packing surfaces detached, and X 2 was measured after the packings were washed and the remained yeast cells were collected. X T ¼ X 1 þ X 2. Bai et al.: Continuous VHG Ethanol Fermentation 119 Biotechnology and Bioengineering

8 Table III. Comparison of the ethanol fermentation performance and yeast cell viabilities of the packed tubular bioreactors. Tubular bioreactors Wood chips Packings Polyurethane particles Ceramic saddles First Average residual glucose (g L 1 ) Average ethanol (g L 1 ) Cell viability (%) Inside bioreactor In effluent Second Average residual glucose (g L 1 ) Average ethanol (g L 1 ) Cell viability (%) Inside bioreactor In effluent Third Average residual glucose (g L 1 ) Average ethanol (g L 1 ) Cell viability (%) Inside bioreactor In effluent Viable cell percentages were detected at the end when the broth within the packed tubular bioreactors was drained out, and the yeast cells deposited onto the surfaces of the packings as well as occluded within the pores of the polyurethane particles were washed out and collected. time. For the second and third bioreactors, although higher viable yeast cell percentages were detected in the effluent out of the bioreactors packed with all the three packings, the biggest difference in the viable yeast cell percentages was observed in the effluent and within the bioreactors packed with the polyurethane particles, indicating less viable yeast cells were washed out when the bioreactors were packed with the wood chips and Intalox ceramic saddles than packed with the polyurethane particles. The significant difference in the viability of the immobilized yeast cells came from the different mechanism of yeast cell immobilization, which gives a reasonable explanation for the role of the wood chips and Intalox ceramic saddles in improving the ethanol fermentation performance as well as attenuating the oscillations. The Raschig rig packing with smooth surfaces that yeast cells could not deposited onto, was studied. As can be seen, no yeast cell immobilization was observed for the bioreactors packed with this packing, and the ethanol fermentation performance and oscillatory behavior of the tubular bioreactors was not affected, which further supported the role of the yeast cell immobilization in the improvement of the ethanol fermentation performance as well as the oscillation attenuations. and third bioreactors, comparing with the viable yeast cell percentages of 42.6%, 19.5%, and 9.8% within the bioreactors packed with the wood chips, and 32.5%, 7.9%, and 4.9% within the bioreactors packed with the Intalox ceramic saddles. In fact, almost no viable yeast cells were detected within the third bioreactor packed with the polyurethane particles, which was in a good accordance with the conversion of glucose within this bioreactor, 23.7 g L 1 in the effluent from the second tubular bioreactor into the third bioreactor and 22.8 g L 1 in the effluent out of the third bioreactor. The lower viable yeast cell percentages overwhelmingly affected the performance of the second and third bioreactors packed with the polyurethane particles, making them unable to attenuate their oscillations. For the first tubular bioreactor packed with the wood chips and Intalox ceramic saddles, the viable yeast cell percentages in the effluent were 4.1% and 29.5%, almost the same as the viable cell percentages of 42.6% and 32.5% inside the bioreactor, indicating that the viability-lost yeast cells could be washed out in time. However, the viable yeast cell percentage within the first tubular bioreactor was measured to be 22.7% when it was packed with the polyurethane particles, not only significantly lower than the viable yeast cell percentages when the bioreactor was packed with the wood chips and the Intalox ceramic saddles, but also lower than the viable yeast cell percentage of 26.8% detected in the effluent out of the bioreactor, which meant more viability-lost yeast cells were retained within the pores of the polyurethane particles and could not be washed out in Conclusions When the tubular bioreactors were packed with the Intalox ceramic saddles, their parameter oscillations were effectively attenuated. Through the RTD analysis, the impact of the backmixing alleviation on the oscillation attenuation was ruled out, because the ethanol concentration gradients could not be established within the packed tubular bioreactors. As the packing immobilized yeast cells, the impact of the yeast cell immobilization on the oscillation attenuation was studied by applying packings with different yeast cell immobilization effects. The experimental results indicated that only the Intalox ceramic saddles and wood chips that provided surfaces for yeast cells to deposit or absorb loosely onto, making the immobilized yeast cells easily renewed, could improve the ethanol fermentation performance as well as attenuate the parameter oscillations. Although the porous polyurethane particles immobilized more yeast cells, they could not attenuate the oscillations because of the extremely low yeast cell viability within the bioreactors packed with this packing. That no improvement on ethanol fermentation performance as well as on oscillation attenuation was observed for the tubular bioreactors packed with the Raschig rigs without yeast cell immobilization effect further supported this mechanistic analysis. References Alfenore S, Cameleyre X, Benbadis L, Bideaux C, Uribelarrea JL, Goma G, Molina-Jouve C, Guillouet SE. 24. Aeration strategy: A need for very Biotechnology and Bioengineering, Vol. 12, No. 1, January 1, 29

9 high ethanol performance in Saccharomyces cerevisiae fed-batch process. Appl Microbiol Biotechnol 63: Bai FW, Chen LJ, Anderson WA, Moo-Young M. 24. Parameter oscillations in very high gravity medium continuous ethanol fermentation and their attenuation on multi-stage packed column bioreactor system. Biotechnol Bioeng 88: Bai FW, Anderson WA, Moo-Young M. 28. Ethanol fermentation technologies from sugar and starch materials. Biotechnol Adv 26: Baptista CMSG, Coias JMA, Oliveira ACM, Oliveira NMC, Rocha JMS, Dempsey MJ, Lannigan KC, Benson PS. 26. Natural immobilization of microorganisms for continuous ethanol production. Enzym Microbal Technol 4: Borzani W. 21. Variation of the ethanol yield during oscillatory concentrations changes in undisturbed continuous ethanol fermentation of sugarcane blackstrap molasses. World J Microbiol Biotechnol 17: Bruce LJ, Axford DB, Ciszek B, Daugulis AJ Extractive fermentation by Zymomonas mobilis and the control of oscillatory behavior. Biotechnol Lett 13: Daugulis AJ, McLellan PJ, Li J Experimental investigation and modeling of oscillatory behavior in the continuous culture of Zymomonas mobilis. Biotechnol Bioeng 56: Ghommidh C, Vaija J, Bolarinwa S, Navarro JM Oscillatory behavior of Z. mobilis in continuous cultures: A simple stochastic model. Biotechnol Lett 11: Jarzebski AB Modeling of oscillatory behavior in continuous ethanol fermentation. Biotechnol Lett 14: Jöbses IML, Egberts GTC, Luyben KCAM, Roels JA Fermentation kinetics of Zymomonas mobilis at high ethanol concentrations: Oscillations in continuous cultures. Biotechnol Bioeng 18: Kourkoutas Y, Bekatorou A, Banat IM, Marchant R, Koutinas AA. 24. Immobilization technologies and supporting materials suitable in alcohol production: A review. Food Microbiol 21: Lee KJ, Tribe DE, Rogers PL Ethanol production by Zymomonas mobilis in continuous culture at high glucose concentrations. Biotechnol Lett 1: Levenspiel O The tanks-in-series model. In: Anderson W, editor. Chemical reaction engineering. 3rd edn. New York: John Wiley & Sons. Lorenz O, Haulena F, Rose G Immobilization of yeast cells in polyurethane ionomers. Biotechnol Bioeng 29: McLellan PJ, Daugulis AJ, Li J The incidence of oscillatory behavior in the continuous fermentation of Zymomonas mobilis. Biotechnol Prog 15: Moo-Young M, Lamptey J, Robinson CW Immobilization of yeast cells on various supports for ethanol fermentation. Biotechnol Lett 2: Ryu DDY, Kim YJ, Kim JH Effect of air supplement on the performance of continuous ethanol fermentation system. Biotechnol Bioeng 26: 16. Bai et al.: Continuous VHG Ethanol Fermentation 1 Biotechnology and Bioengineering