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Redox potential-driven repeated batch ethanol fermentation under very-high-gravity conditions
Process Biochemistry 47 (2012) 523–527
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Redox potential-driven repeated batch ethanol fermentation under very-high-gravity conditions Sijing Feng 1 , Shyam Srinivasan 1 , Yen-Han Lin ∗ Department of Chemical Engineering, University of Saskatchewan, Saskatoon, SK, Canada
a r t i c l e
i n f o
Article history: Received 13 September 2011 Received in revised form 22 December 2011 Accepted 23 December 2011 Available online 3 January 2012 Keywords: Redox potential Very-high-gravity fermentation Ethanol Self-cycling Repeated batch
a b s t r a c t When Saccharomyces cerevisiae was cultivated under ∼200 g glucose/l condition, the time point at which glucose was completely utilized coincided with the moment at which the slope of a redox potential profile changed from negative or zero to positive. Based on this feature, a redox potential-driven glucose-feeding fermentation operation was developed, and resulted in a self-cycling period of 14.25 ± 0.4 h. The corresponding ethanol concentration was maintained at 88.4 ± 1.0 g/l with complete glucose conversion, and the cell viabilities increased from 80% in the transition period to 97.2 ± 1.1%, implying the occurrence of yeast acclimatization. In contrast, a pre-determined 36-h manually adjusted period was chosen to oscillate yeast cells under ∼250 g glucose/l conditions, which resulted in 106.76 ± 0.7 g ethanol/l and 15.19 ± 1.3 g glucose/l remaining at the end of each cycle. Compared to the equivalent batch and continuous ethanol fermentation processes, the annual ethanol productivity of the reported fermentation operation is 2.4% and 13.2% greater, respectively in ∼200 g feeding glucose/l conditions. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fermentation processes traditionally follow either a batch or a continuous system. While batch systems require medium replenishment and re-inoculation at the end of each cycle, continuous systems have resulted in lower conversion and productivity [1,2]. A new process that can avoid the limitations of batch and continuous processes as well as take into consideration the inner workings of a cell is required. Self-cycling fermentation (SCF) is one such process. SCF is a cyclic process operating on a feedback control strategy that is in essence an oscillating process. In theory, cycles in a SCF operation are based on the metabolic rates of the fermenting cell population thus taking into account the biochemical aspect of any bioprocess. There is no external parameter controlling the cyclic process hence giving the name self-cycling. The details of the SCF process have been discussed by several authors [3–5]. For a fermentation process to be self-cycling, a parameter that is representative of the state of the cells in the fermenter should be measured, on the basis of this measurement fresh media feeding and broth removal would be accomplished. Previous studies used dissolved oxygen (DO) concentration or off-gas carbon dioxide evolution rate as the measured variable to incorporate the feedback control strategy for SCF
∗ Corresponding author. Fax: +1 306 966 4777. E-mail address: [email protected] (Y.-H. Lin). 1 These authors contributed equally. 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.12.018
processes under aerobic conditions. The reason for the inoperability of a DO-driven SCF process is mainly on the basis of sensor design and have been explained in detail elsewhere [3]. Prior research in the area of SCF processes has been discussed for various bacteria and yeasts other than Saccharomyces cerevisiae [4]. Most of these studies, where the DO concentration has been used as a control variable to activate self-cycling operation, were concerned with carbohydrate fermentation lasting for a maximum of 40–50 h with cycle times in the range of 2.5–4 h [3–5]. Sauvageau et al. [4] also proposed SCF as a physical fractionation process, in addition to methods like chemical induction and starvation, to bring about synchronization in cell populations. In contrast to chemical induction and starvation, physical fractionation processes can work with high cell population densities and maintain the synchronization beyond many generations [4,6]. According to Sheppard and Dawson [7], in a synchronized cell population individual cells are said to undergo division simultaneously and follow a particular rhythm or a periodic oscillation in contrast to normal cell populations where individual cells follow through different stages in their life cycle in their natural rhythm. Though previous research [4,7] proposed SCF as a means to synchronize cell populations, no concrete proof of the fact was provided through a standard like flow cytometric analysis. Instead, the most common observations in SCF processes were system measured parameters, including biomass, glucose and ethanol concentration, being forced to oscillate. Similar results were also reported by Her et al. and Bai et al. [8,9]. Ethanol fermentation by S. cerevisiae is facultative to nearly anaerobic fermentation process wherein the DO level is so low that
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a DO electrode becomes insensitive to changes in DO concentration in the fermenter. In contrast, redox potential reflects the momentary metabolic status of oxidization or reduction of yeast cells in a fermentation process. Both NADH, which serves as the electron donor, and DO, which serves as electron acceptor resulting from agitation or sparging, are the major contributors to the change of redox potential [10]. Due to the design of redox potential sensor, it offers a longer and more reliable measurement than a DO sensor. Very-high-gravity (VHG) ethanol fermentation is characterized by osmotic stresses and ethanol toxicity that are induced by high initial glucose concentration and high final ethanol concentration respectively. This eventually results in lower cell activity. Previous studies have credited a longer lag phase and incomplete glucose utilization to osmotic stress and ethanol toxicity respectively. The need for a synchronized cell population in ethanolic fermentation processes has been reinforced by different authors [7,11]. Once the toxic ethanol concentration threshold is exceeded, an abrupt reduction of cell viability occurs [10]. High ethanol toxicity and low cell viability would reduce the cells’ activities, resulting in sluggish fermentation. We hypothesized that the repeated batch operation of a fermentation system through SCF can result in increased ethanol tolerance in S. cerevisiae, thereby increasing glucose utilization and ethanol production under VHG conditions. To test the hypothesis, VHG fermentation was carried out utilizing two different feed glucose concentrations, ∼200 and ∼250 g/l, and the redox potential was used as a control variable to activate the system. 2. Materials and methods 2.1. Strain and growth media The active dry yeast (Ethanol RedTM ) was obtained from Lesaffre Yeast Corp (Milwaukee, MI, USA). The dry yeast was rehydrated with 50 ml sterilized water, and cultured in YPD agar (10 g/l yeast extract, 10 g/l peptone, 20 g/l dextrose, and 20 g/l agar). Two sub-culture steps were performed to purify yeast strains for later use. Yeast extract was obtained from HiMedia Laboratories (Mumbai, India). All other chemicals were of reagent grade or higher purity. The growth medium consisted of yeast extract (1%, w/v), sodium glutamate (0.1%, w/v), MgSO4 ·7H2 O (2 mM), KH2 PO4 (3.67 mM), urea (16 mM), (NH4 )2 SO4 (1 mM), and mineral salts, including 24 M H3 BO3 , 1.5 M Na2 MoO4 , 20 M MnSO4 ·H2 O, 10 M CuSO4 , 1.8 M KI, 100 M FeCl3 ·6H2 O, 82 M CaCl2 ·2H2 O, and 1000 M ZnSO4 ·7H2 O. The growth medium was steam sterilized at 121 ◦ C for 15 min. 2.2. Fermentation and redox potential measurement Fermentation was carried out in jar fermenters (Model: Omni Culture, New York, NY, USA) with 1-l working volumes, and the agitation rate was kept at 150 rpm for all runs. Each fermenter was equipped with an autoclavable redox potential electrode that was custom-made and ordered through Cole-Palmer Inc. (12 mm × 250 mm, Vernon Hills, IL, USA). Redox potentials were acquired by using LabView (Version 8.5, National Instrument, Austin, TX, USA), and a PID control algorithm was implemented to control the redox potential at a desired level by adjusting the redox potential-controlled scheme (to be described in Section 3). The yeast culture was cultivated in a shake flask until its mid-exponential phase, and then inoculated into a fermenter. The inoculum level was 5% of the working volume, and the pitching rate was adjusted to ca. 107 viable yeast cells per ml for all experiments. Each experiment was performed in duplicate. 2.3. Determination of self-cycling period Under 200 ± 3.3 g glucose/l condition, as the slope of the redox potential changed from negative or zero to positive, one half of the fermentation broth was withdrawn and an equal volume of fresh media was replenished. The self-cycling period was estimated by measuring time elapsed between peaks of two contiguous cycles. In the 250 ± 5.3 g glucose/l case, a manually adjusted period of 36 h was pre-determined according to Liu et al. [12] and the cycle of withdrawal and replenishment was repeated as described above. 2.4. Sample analysis A 5-ml fermentation broth aliquot was withdrawn every 6–8 h. In some cycles, samples were collected every 2 h. One portion of the aliquot was used to enumerate yeast cell population under a microscope. Viable and dead cells were differentiated
by using the methylene-violet staining procedure [13]. The viability was then calculated by taking the number of viable cells divided by the total number of both viable and dead cells and multiplied it by 100. The remaining portion of the aliquot was centrifuged (9000 × g, 4 ◦ C) for 5 min. Glucose and ethanol in the supernatant were quantified by high performance liquid chromatography (HPLC) (Model: HP 1100 series, Agilent Technologies, Mississauga, ON, Canada) employing a refractive index (RI) detector (Model: 1200 series from Agilent Technologies). An ion exclusion column (Model: ORH-801, Transgenomic, Inc., Omaha, NE, USA) was used to separate the metabolites. The mobile phase consisted of 40 mM H2 SO4 (HPLC grade) at a flow rate of 0.25 ml/min. Column and RI detector temperatures were maintained at 65 and 35 ◦ C, respectively.
3. Results and discussion Based on the slope change of the redox potential profile, yeast cells were subjected to SCF operation, resulting in a self-regulated repeated batch fermentation for the ∼200 g glucose/l case. In contrast, under the ∼250 g glucose/l condition, the effects of ethanol toxicity forced the self-cycling period to be manually selected as 36 h in order to lower residual glucose concentration between oscillating cycles. 3.1. Reproducibility of redox potential-driven repeated batch fermentation The experiments were initiated in batch mode followed by SCF operation. The SCF operation was designed to activate when the redox potential profile began changing its slope from negative or zero to positive at each cycle. Once activated, one half of the working volume in the fermenter was withdrawn, followed by the addition of an equal volume of fresh media. The SCF operation was then repeated until the media in the feed container was exhausted. If redox potential became lower than the set point value, i.e. −100 mV, an appropriate amount of filter-sterilized air, determined by the PID control algorithm, was sparged into the fermenter to maintain redox potential at the desired level. As illustrated in Fig. 1, under 200 ± 3.3 g glucose/l condition, the first cycle took 27.5 h to lower redox potential to a level at which no residual glucose was detected and SCF operation was activated. The self-cycling period, the time period between two consecutive cycles, dropped from ∼18 h and finally stabilized at 14.21 ± 0.4 h. After 265 h of fermentation, constant concentrations of biomass and ethanol were obtained as 4.02 ± 0.4 and 88.39 ± 1.0 g/l, respectively. The feature of the current operation is that there was no residual glucose and the slope of redox potential profile changed from negative or zero to positive at the end of each cycle (Fig. 2). It also can be seen that higher biomass concentration also indicates more viable cells, which results in a shorter self-cycling period. It was postulated that after four cycles of SCF operation, yeast cells adapted to the fermentation environment and the inhibitory effect of ethanol on yeast propagation is reduced. In contrast, under 250 ± 5.3 g glucose/l condition, ∼55 g/l residual glucose remained at the point at which the slope of the redox potential profile began changing from negative to positive (Fig. 1). In order to lower the amount of residual glucose, we decided to manually activate SCF operation every 36 h, which was equivalent to a dilution rate of 0.028 h−1 . The selection of this dilution rate was based on one of our previous VHG fermentation works [12] where the initial glucose concentration was 250 ± 2.56 g/l. After 370 h of fermentation, constant harvest concentrations of biomass, ethanol, and glucose at the each end of cycle were recorded as 3.1 ± 0.1, 15.19 ± 1.3, and 106.76 ± 0.7 g/l, respectively. Due to the operating characteristics of SCF, one half of the fermenter broth was harvested at the beginning of each cycle and the same volume of fresh media was refilled. The lag time was eliminated and the osmotic stresses were alleviated since a significant portion of the acclimatized cells was left in the fermenter at the
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Fig. 1. Profiles of glucose and ethanol concentrations, redox potential, and biomass concentration under 200 ± 3.3 and 250 ± 5.3 g glucose/l condition. The maximum ethanol concentrations are 88.4 ± 1.0 and 106.76 ± 0.7 for the ∼200 and ∼250 g glucose/l cases respectively.
end of each cycle. Cell viabilities are also indicative of accumulating ethanol concentration; however, once ethanol concentrations were above 85 g/l, a rapid decline in the cell viabilities was observed. It should not exclude that the effect of depletion of dissolved oxygen in the fermentation broth may also contribute to the abrupt reduction of yeast population, thereby lowering fermentation rate. 3.2. Mode of redox potential-driven repeated batch fermentation Section 3.1 describes that depending on the initial glucose concentration, two fermentation modes were obtained: self-cycling and manually adjusted. The ethanol fermentation by means of S. cerevisiae is regarded as a primary fermentation (i.e. ethanol is one of the primary metabolites directly derived from glucose). In other words, a higher initial glucose concentration would result in a higher final ethanol concentration. However, when ethanol concentration in a fermenter exceeds the ethanol toxic concentration, 85–90 g glucose/l in this study, a drastic reduction in yeast
viability becomes apparent [10]. As a result, incomplete fermentation (i.e. unspent glucose after the termination of fermentation) or sluggish fermentation (i.e. no residual glucose after a lengthy period of fermentation) is commonly observed. It is estimated that up to 10% of total glucose is being utilized for the formations of biomass and metabolic by-products, and the attainable ethanol yield is between 90 and 93% during ethanol fermentation [14]. Stoichiometrically, one mole of glucose is converted to two moles of ethanol. For the case of 200 and 250 g/l initial glucose concentrations, the theoretical ethanol concentrations are 91.98 (=200 × 0.9 × 0.511) and 108.08 g/l (=235 × 0.9 × 0.511) respectively. Note that for ∼250 g glucose/l condition, there were ∼15 g glucose/l remained in the fermenter. With reference to Fig. 1 (case of ∼200 g glucose/l), although the final ethanol concentration of 88.39 ± 1.0 g/l measured at the end of each cycle was within the ethanol toxic concentration range (i.e., 85–90 g/l), the corresponding biomass showed a continually increasing trend for the first four cycles, and the yeast viability
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Fig. 2. Illustration of redox potential and glucose concentration profiles occurring at the 10th cycle for 200 ± 3.3 g glucose/l case and the 5th cycle for 250 ± 5.3 g glucose/l case. This depicts the distinct differences existing among 200 and 250 g glucose/l in an SCF operation and the need to implement manual broth removal and refilling for the 250 g glucose/l case.
increased from 80% and finally stabilized at 97.2 ± 1.1% (Fig. 3). This indicated that the ethanol toxicity imposed minor to unnoticeable influence on yeast propagation under current conditions. It is postulated that yeast cells adapt to the toxic ethanol environment in the fermenter. As a result, SCF operation mode was engaged, resulting in repeated batch fermentation. In contrast, under ∼250 g/l initial glucose concentration, 50–65 g/l unspent glucose remained in the fermenter when the redox potential reached its lowest level (Figs. 1 and 2). This particular level occurred at the 19th hour of each cycle, at which point the biomass and ethanol concentrations were ∼3.1, and ∼85 g/l, respectively, and the yeast viability was 85 ± 5.1% (Fig. 3). Since there were 20–26% of initial glucose remained in the fermenter, a manually adjusted period of 36 h (reciprocal of 0.028 h−1 as derived from Liu et al. [12], in which a similar initial glucose concentration was used) was selected in an attempt to lower the residual glucose. As fermentation proceeded, a constant biomass concentration
Fig. 3. Cell viability profiles for the case of 200 ± 3.3 and 250 ± 5.3 g glucose/l during redox potential-driven repeated batch fermentation. While the initial viabilities drop in a similar pattern for both 200 and 250 cases, for the 200 g glucose/l case the viabilities eventually rise to 97.2 ± 1.1% indicating an acclimatization effect while for 250 g glucose/l case, the viabilities remained at 85 ± 5.1% indicating that cells do not get acclimatized to the high ethanol concentration environment.
(3.1 ± 0.1 g/l) between the 19th and the 36th hour was recorded (Fig. 1); however, the corresponding yeast viability decreased significantly to 75 ± 1.4%. The ethanol concentration at the end of each cycle (measured at the 36th hour) was 106.76 ± 0.7 g/l, and the residual glucose concentration was 15 ± 1.3 g/l. This indicated that yeast was at its stationary to death phase. Under ∼200 g glucose/l condition, it took ∼14 h for the redox potential in the fermenter to start rising from the lowest level; whereas about 19 h was required for the case of ∼250 g glucose/l. Such a time delay was related to the osmotic stress attributable to high initial glucose concentration [10]. The activation of SCF operation under ∼200 g glucose/l condition could be correlated to complete glucose utilization because the oxidizing power outperforms the reducing power in the fermenter. Consequently, an increase of redox potential from its lowest level triggers the SCF operation. In contrast, for the case of ∼250 g glucose/l, the residual glucose (50–65 g/l) in conjunction with the ethanol concentration (85 ± 1.4 g/l measured at the 19th hour, falls in the ethanol toxicity range, 85–90 g/l) retarded yeast growth in the fermenter. As a result, an increasing redox potential profile reflects insufficient provision for reducing power (contributed by yeast cells) in order to counteract oxidizing power (contributed by DO supplied through agitation or sparging). Because over 20% of unused glucose imposes difficulty for the subsequent downstream processing, it is necessary to increase glucose consumption. The simplest approach would be extending the duration of fermentation, as demonstrated in this article. Hence, the SCF operation was discarded; instead, a forced repeated batch operation was implemented for 250 g/l feeding glucose condition. 3.3. Measurement of yeast synchronization A quantifier to measure whether the culture in a fermenter is under synchronization operation is necessary. Blumenthal and Zahler [15] formulated a synchrony index (F) to represent the degree of synchronization during cell division: F=
Nt − 2t/g N0
(1)
In the formula, Nt , N0 , t, and g stand for cell concentration at time t, cell concentration at time 0, culture time, and doubling time of the cultivated cell respectively. When F equals to one, it represents a perfect synchronization (i.e., t approaches to zero). When F equals to zero, an exponential growth is established (i.e., t approaches to g). Given that the maximum specific growth rate of S. cerevisiae was assumed to be 0.454 h−1 , the equivalent doubling time was then calculated to be 1.53 h (=0.693/0.454). During ethanol fermentation, t is greater than g, making Eq. (1) inapplicable. Sauvageau et al. [4] modified Eq. (1) by reversing the definition of t and g, and letting the ratio of Nt to N0 be two under SCF operation. They concluded that synchronization was achieved if F value was greater than or equal to 0.6. However, the details of selecting 0.6 as a threshold F value were not provided. Though, the definition of Eq. (1) by Sauvageau et al. ensured that the F value was always positive, it did not explain the rationale behind using a constant value of two for the ratio of Nt to N0 . As a matter of fact, such a ratio was less than two based on measured biomass concentration (Fig. 1). Moreover, since the term synchronization corresponds to intracellular activity, cell synchronization as it is defined through Eq. (1) by Sauvageau et al. [4] alone cannot be used to quantify cell synchronization. So, we chose to use the term repeated batch operation for both ∼200 and ∼250 g glucose/l cases as opposed to synchronization operation. In order to distinguish between the two cases, we further differentiate the terms self-regulated and forced repeated batch operation. For ∼200 g/l feed glucose case, there was no manual interference with regards to the self-cycling operation, so the phenomenon was
S. Feng et al. / Process Biochemistry 47 (2012) 523–527
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Table 1 Comparison of annual ethanol productivity for batch, redox potential-driven repeated batch (also called self-cycling shown below), and continuous fermentation under ∼200 g glucose/l feed concentration. Vw = 100 m3 , Tw = 7920 h, redox potential = −100 mV. Down time, Td (h)
Fermentation mode
Batch or cycle time, Tf (h)
Batcha Self-cycling Continuousb
24 6 14.25 0.25 6 h in ageing vessel with 0.028 h−1 dilution rate (D)
a b
Ethanol concentration, C (g/l in single batch or cycle)
Annual productivity, Py (tons)
89.3 ± 0.2 88.4 ± 1.0 96.2 ± 2.6 (ageing vessel)
2358 2414 2133
Adapted from Lin et al. [10]. Adapted from Liu et al. [12].
termed as self-regulated repeated batch operation. In the ∼250 g/l feed glucose case, the cycle times were chosen manually in order to reduce residual glucose, so the observed phenomenon was termed as forced repeated batch operation. 3.4. Applicability of redox potential-driven repeated batch fermentation In order to determine the effect of redox potential-driven repeated batch fermentation on ethanol production under VHG conditions, a comparison of ethanol productivities among batch [10], redox potential-driven repeated batch (also called selfcycling), and continuous fermentation [12] operations was performed. The formulae used to calculate ethanol productivities are shown in Eqs. (2)–(4), and the calculated results are compiled in Table 1. Batch :
Py =
Tw × Vw × C Tf + Td
(2)
Self-cycling :
Tw Vw Py = ×C × Tf + Td 2
(3)
Continuous :
Py = D × C × Vw × Tw
(4)
Table 1 shows that redox potential-driven repeated batch fermentation (current work) increases ethanol productivity by 2.4% and 13.2% with respect to batch and continuous fermentation operation. One also observes that the increased productivity is partly related to shorter filling time (about 0.25 h) between cycles during repeated batch operation; whereas, it typically takes 6 h of down time between batch operation. 4. Conclusions Redox potential-driven SCF operation as a means to maintain repeated batch operation was studied for VHG ethanol fermentation processes with feed glucose concentrations of ∼200 and ∼250 g/l. Apart from the differences in residual glucose levels, the cell viability differences between initial glucose concentrations were idiosyncratic and indicative of the toxic effects of ethanol on yeast. It was concluded that (1) the redox potential-driven repeated batch fermentation operated under ∼200 g glucose/l condition improved ethanol tolerance of yeast cells and maintained high cell activities towards the end of each cycle, while such an operation failed to improve the cells’ maximum ethanol tolerance under ∼250 g glucose/l condition; (2) redox potential was a reliable measure of progress of ethanolic fermentation and it could be used as a control measure to trigger a self-cycling process. Elimination of lag phase through the reported fermentation operation has also shown to improve the annual ethanol productivity from its batch and continuous counterparts.
Appendix A. Nomenclature
C D F g Nt N0 Py t Td Tf Tw Vw
final ethanol concentration, g/l dilution rate, h−1 synchrony index cycle time, h biomass concentration at the end of cycle, g/l biomass concentration in the beginning of cycle, g/l annual ethanol productivity, tons/year doubling time, h down time, h fermentation time, h annual operating time, h working volume, m3
References [1] Bailey JE, Ollis DF. Biochemical engineering fundamentals. 2nd ed. New York: McGraw-Hill, Inc.; 1986. p. 383, 409. [2] Dawson PS. Continuously synchronized growth. J Appl Chem Biotechnol 1972;22:79–103. [3] Brown W, Cooper DG. Adapting the self-cycling fermentor to anoxic conditions. Environ Sci Technol 1999;33:1458–63. [4] Sauvageau D, Storms Z, Cooper D. Synchronized populations of Escherichia coli using simplified self-cycling fermentation. J Biotechnol 2010;149: 67–73. [5] Sheppard J, Cooper DG. Development of computerized feedback control for the continuous phasing of Bacillus subtilis. Biotechnol Bioeng 1990;36: 539–45. [6] Kepes F, Kepes A. Long-lasting synchrony of the division of enteric bacteria. Biochem Biophys Res Commun 1981;99:761–7. [7] Sheppard J, Dawson P. Cell synchrony and periodic behaviour in yeast populations. Can J Chem Eng 1999;77:893–902. [8] Her S-L, Duan K-J, Sheu D-C, Lin C-T. A repeated batch process for cultivation of Bifidobacterium longum. J Ind Microbiol Biotechnol 2004;31: 427–32. [9] Bai F, Chen L, Anderson W, Moo-Young M. Parameter oscillations in a very high gravity medium continuous ethanol fermentation and their attenuation on a multistage packed column bioreactor system. Biotechnol Bioeng 2004;88:558–66. [10] Lin Y-H, Chien W, Duan K. Correlation between reduction–oxidation potential profiles and growth patterns of Saccharomyces cerevisiase during very-high gravith fermentation. Process Biochem 2010;45:765–70. [11] Belli G, Gari E, Aldea M, Herrero E. Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae. Mol Microbiol 2001;39:1022–35. [12] Liu C, Lin Y-H, Bai F. Developement of redox potential-controlled schemes for very-high-gravity fermentation. J Biotechnol 2011;153:42–7. [13] Smart K, Chambers K, Lambert I, Jenkins C. Use of methylene violet staining procedure to determine yeast viability and vitality. J Am Soc Brew Chem 1999:57. [14] Ingledew W, Lin Y-H. Ethanol from starch-based feedstocks. In: Moo-Young M, editor. Comprehensive biotechnology, vol. 3, 2nd ed. Elsevier; 2011. p. 37. [15] Blumenthal L, Zahler S. Index for measurement of synchronization of cell populations. Science 1962;135:724.
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Redox potential-driven repeated batch ethanol fermentation under very-high-gravity conditions Sijing Feng 1 , Shyam Srinivasan 1 , Yen-Han Lin ∗ Department of Chemical Engineering, University of Saskatchewan, Saskatoon, SK, Canada
a r t i c l e
i n f o
Article history: Received 13 September 2011 Received in revised form 22 December 2011 Accepted 23 December 2011 Available online 3 January 2012 Keywords: Redox potential Very-high-gravity fermentation Ethanol Self-cycling Repeated batch
a b s t r a c t When Saccharomyces cerevisiae was cultivated under ∼200 g glucose/l condition, the time point at which glucose was completely utilized coincided with the moment at which the slope of a redox potential profile changed from negative or zero to positive. Based on this feature, a redox potential-driven glucose-feeding fermentation operation was developed, and resulted in a self-cycling period of 14.25 ± 0.4 h. The corresponding ethanol concentration was maintained at 88.4 ± 1.0 g/l with complete glucose conversion, and the cell viabilities increased from 80% in the transition period to 97.2 ± 1.1%, implying the occurrence of yeast acclimatization. In contrast, a pre-determined 36-h manually adjusted period was chosen to oscillate yeast cells under ∼250 g glucose/l conditions, which resulted in 106.76 ± 0.7 g ethanol/l and 15.19 ± 1.3 g glucose/l remaining at the end of each cycle. Compared to the equivalent batch and continuous ethanol fermentation processes, the annual ethanol productivity of the reported fermentation operation is 2.4% and 13.2% greater, respectively in ∼200 g feeding glucose/l conditions. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fermentation processes traditionally follow either a batch or a continuous system. While batch systems require medium replenishment and re-inoculation at the end of each cycle, continuous systems have resulted in lower conversion and productivity [1,2]. A new process that can avoid the limitations of batch and continuous processes as well as take into consideration the inner workings of a cell is required. Self-cycling fermentation (SCF) is one such process. SCF is a cyclic process operating on a feedback control strategy that is in essence an oscillating process. In theory, cycles in a SCF operation are based on the metabolic rates of the fermenting cell population thus taking into account the biochemical aspect of any bioprocess. There is no external parameter controlling the cyclic process hence giving the name self-cycling. The details of the SCF process have been discussed by several authors [3–5]. For a fermentation process to be self-cycling, a parameter that is representative of the state of the cells in the fermenter should be measured, on the basis of this measurement fresh media feeding and broth removal would be accomplished. Previous studies used dissolved oxygen (DO) concentration or off-gas carbon dioxide evolution rate as the measured variable to incorporate the feedback control strategy for SCF
∗ Corresponding author. Fax: +1 306 966 4777. E-mail address: [email protected] (Y.-H. Lin). 1 These authors contributed equally. 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.12.018
processes under aerobic conditions. The reason for the inoperability of a DO-driven SCF process is mainly on the basis of sensor design and have been explained in detail elsewhere [3]. Prior research in the area of SCF processes has been discussed for various bacteria and yeasts other than Saccharomyces cerevisiae [4]. Most of these studies, where the DO concentration has been used as a control variable to activate self-cycling operation, were concerned with carbohydrate fermentation lasting for a maximum of 40–50 h with cycle times in the range of 2.5–4 h [3–5]. Sauvageau et al. [4] also proposed SCF as a physical fractionation process, in addition to methods like chemical induction and starvation, to bring about synchronization in cell populations. In contrast to chemical induction and starvation, physical fractionation processes can work with high cell population densities and maintain the synchronization beyond many generations [4,6]. According to Sheppard and Dawson [7], in a synchronized cell population individual cells are said to undergo division simultaneously and follow a particular rhythm or a periodic oscillation in contrast to normal cell populations where individual cells follow through different stages in their life cycle in their natural rhythm. Though previous research [4,7] proposed SCF as a means to synchronize cell populations, no concrete proof of the fact was provided through a standard like flow cytometric analysis. Instead, the most common observations in SCF processes were system measured parameters, including biomass, glucose and ethanol concentration, being forced to oscillate. Similar results were also reported by Her et al. and Bai et al. [8,9]. Ethanol fermentation by S. cerevisiae is facultative to nearly anaerobic fermentation process wherein the DO level is so low that
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a DO electrode becomes insensitive to changes in DO concentration in the fermenter. In contrast, redox potential reflects the momentary metabolic status of oxidization or reduction of yeast cells in a fermentation process. Both NADH, which serves as the electron donor, and DO, which serves as electron acceptor resulting from agitation or sparging, are the major contributors to the change of redox potential [10]. Due to the design of redox potential sensor, it offers a longer and more reliable measurement than a DO sensor. Very-high-gravity (VHG) ethanol fermentation is characterized by osmotic stresses and ethanol toxicity that are induced by high initial glucose concentration and high final ethanol concentration respectively. This eventually results in lower cell activity. Previous studies have credited a longer lag phase and incomplete glucose utilization to osmotic stress and ethanol toxicity respectively. The need for a synchronized cell population in ethanolic fermentation processes has been reinforced by different authors [7,11]. Once the toxic ethanol concentration threshold is exceeded, an abrupt reduction of cell viability occurs [10]. High ethanol toxicity and low cell viability would reduce the cells’ activities, resulting in sluggish fermentation. We hypothesized that the repeated batch operation of a fermentation system through SCF can result in increased ethanol tolerance in S. cerevisiae, thereby increasing glucose utilization and ethanol production under VHG conditions. To test the hypothesis, VHG fermentation was carried out utilizing two different feed glucose concentrations, ∼200 and ∼250 g/l, and the redox potential was used as a control variable to activate the system. 2. Materials and methods 2.1. Strain and growth media The active dry yeast (Ethanol RedTM ) was obtained from Lesaffre Yeast Corp (Milwaukee, MI, USA). The dry yeast was rehydrated with 50 ml sterilized water, and cultured in YPD agar (10 g/l yeast extract, 10 g/l peptone, 20 g/l dextrose, and 20 g/l agar). Two sub-culture steps were performed to purify yeast strains for later use. Yeast extract was obtained from HiMedia Laboratories (Mumbai, India). All other chemicals were of reagent grade or higher purity. The growth medium consisted of yeast extract (1%, w/v), sodium glutamate (0.1%, w/v), MgSO4 ·7H2 O (2 mM), KH2 PO4 (3.67 mM), urea (16 mM), (NH4 )2 SO4 (1 mM), and mineral salts, including 24 M H3 BO3 , 1.5 M Na2 MoO4 , 20 M MnSO4 ·H2 O, 10 M CuSO4 , 1.8 M KI, 100 M FeCl3 ·6H2 O, 82 M CaCl2 ·2H2 O, and 1000 M ZnSO4 ·7H2 O. The growth medium was steam sterilized at 121 ◦ C for 15 min. 2.2. Fermentation and redox potential measurement Fermentation was carried out in jar fermenters (Model: Omni Culture, New York, NY, USA) with 1-l working volumes, and the agitation rate was kept at 150 rpm for all runs. Each fermenter was equipped with an autoclavable redox potential electrode that was custom-made and ordered through Cole-Palmer Inc. (12 mm × 250 mm, Vernon Hills, IL, USA). Redox potentials were acquired by using LabView (Version 8.5, National Instrument, Austin, TX, USA), and a PID control algorithm was implemented to control the redox potential at a desired level by adjusting the redox potential-controlled scheme (to be described in Section 3). The yeast culture was cultivated in a shake flask until its mid-exponential phase, and then inoculated into a fermenter. The inoculum level was 5% of the working volume, and the pitching rate was adjusted to ca. 107 viable yeast cells per ml for all experiments. Each experiment was performed in duplicate. 2.3. Determination of self-cycling period Under 200 ± 3.3 g glucose/l condition, as the slope of the redox potential changed from negative or zero to positive, one half of the fermentation broth was withdrawn and an equal volume of fresh media was replenished. The self-cycling period was estimated by measuring time elapsed between peaks of two contiguous cycles. In the 250 ± 5.3 g glucose/l case, a manually adjusted period of 36 h was pre-determined according to Liu et al. [12] and the cycle of withdrawal and replenishment was repeated as described above. 2.4. Sample analysis A 5-ml fermentation broth aliquot was withdrawn every 6–8 h. In some cycles, samples were collected every 2 h. One portion of the aliquot was used to enumerate yeast cell population under a microscope. Viable and dead cells were differentiated
by using the methylene-violet staining procedure [13]. The viability was then calculated by taking the number of viable cells divided by the total number of both viable and dead cells and multiplied it by 100. The remaining portion of the aliquot was centrifuged (9000 × g, 4 ◦ C) for 5 min. Glucose and ethanol in the supernatant were quantified by high performance liquid chromatography (HPLC) (Model: HP 1100 series, Agilent Technologies, Mississauga, ON, Canada) employing a refractive index (RI) detector (Model: 1200 series from Agilent Technologies). An ion exclusion column (Model: ORH-801, Transgenomic, Inc., Omaha, NE, USA) was used to separate the metabolites. The mobile phase consisted of 40 mM H2 SO4 (HPLC grade) at a flow rate of 0.25 ml/min. Column and RI detector temperatures were maintained at 65 and 35 ◦ C, respectively.
3. Results and discussion Based on the slope change of the redox potential profile, yeast cells were subjected to SCF operation, resulting in a self-regulated repeated batch fermentation for the ∼200 g glucose/l case. In contrast, under the ∼250 g glucose/l condition, the effects of ethanol toxicity forced the self-cycling period to be manually selected as 36 h in order to lower residual glucose concentration between oscillating cycles. 3.1. Reproducibility of redox potential-driven repeated batch fermentation The experiments were initiated in batch mode followed by SCF operation. The SCF operation was designed to activate when the redox potential profile began changing its slope from negative or zero to positive at each cycle. Once activated, one half of the working volume in the fermenter was withdrawn, followed by the addition of an equal volume of fresh media. The SCF operation was then repeated until the media in the feed container was exhausted. If redox potential became lower than the set point value, i.e. −100 mV, an appropriate amount of filter-sterilized air, determined by the PID control algorithm, was sparged into the fermenter to maintain redox potential at the desired level. As illustrated in Fig. 1, under 200 ± 3.3 g glucose/l condition, the first cycle took 27.5 h to lower redox potential to a level at which no residual glucose was detected and SCF operation was activated. The self-cycling period, the time period between two consecutive cycles, dropped from ∼18 h and finally stabilized at 14.21 ± 0.4 h. After 265 h of fermentation, constant concentrations of biomass and ethanol were obtained as 4.02 ± 0.4 and 88.39 ± 1.0 g/l, respectively. The feature of the current operation is that there was no residual glucose and the slope of redox potential profile changed from negative or zero to positive at the end of each cycle (Fig. 2). It also can be seen that higher biomass concentration also indicates more viable cells, which results in a shorter self-cycling period. It was postulated that after four cycles of SCF operation, yeast cells adapted to the fermentation environment and the inhibitory effect of ethanol on yeast propagation is reduced. In contrast, under 250 ± 5.3 g glucose/l condition, ∼55 g/l residual glucose remained at the point at which the slope of the redox potential profile began changing from negative to positive (Fig. 1). In order to lower the amount of residual glucose, we decided to manually activate SCF operation every 36 h, which was equivalent to a dilution rate of 0.028 h−1 . The selection of this dilution rate was based on one of our previous VHG fermentation works [12] where the initial glucose concentration was 250 ± 2.56 g/l. After 370 h of fermentation, constant harvest concentrations of biomass, ethanol, and glucose at the each end of cycle were recorded as 3.1 ± 0.1, 15.19 ± 1.3, and 106.76 ± 0.7 g/l, respectively. Due to the operating characteristics of SCF, one half of the fermenter broth was harvested at the beginning of each cycle and the same volume of fresh media was refilled. The lag time was eliminated and the osmotic stresses were alleviated since a significant portion of the acclimatized cells was left in the fermenter at the
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Fig. 1. Profiles of glucose and ethanol concentrations, redox potential, and biomass concentration under 200 ± 3.3 and 250 ± 5.3 g glucose/l condition. The maximum ethanol concentrations are 88.4 ± 1.0 and 106.76 ± 0.7 for the ∼200 and ∼250 g glucose/l cases respectively.
end of each cycle. Cell viabilities are also indicative of accumulating ethanol concentration; however, once ethanol concentrations were above 85 g/l, a rapid decline in the cell viabilities was observed. It should not exclude that the effect of depletion of dissolved oxygen in the fermentation broth may also contribute to the abrupt reduction of yeast population, thereby lowering fermentation rate. 3.2. Mode of redox potential-driven repeated batch fermentation Section 3.1 describes that depending on the initial glucose concentration, two fermentation modes were obtained: self-cycling and manually adjusted. The ethanol fermentation by means of S. cerevisiae is regarded as a primary fermentation (i.e. ethanol is one of the primary metabolites directly derived from glucose). In other words, a higher initial glucose concentration would result in a higher final ethanol concentration. However, when ethanol concentration in a fermenter exceeds the ethanol toxic concentration, 85–90 g glucose/l in this study, a drastic reduction in yeast
viability becomes apparent [10]. As a result, incomplete fermentation (i.e. unspent glucose after the termination of fermentation) or sluggish fermentation (i.e. no residual glucose after a lengthy period of fermentation) is commonly observed. It is estimated that up to 10% of total glucose is being utilized for the formations of biomass and metabolic by-products, and the attainable ethanol yield is between 90 and 93% during ethanol fermentation [14]. Stoichiometrically, one mole of glucose is converted to two moles of ethanol. For the case of 200 and 250 g/l initial glucose concentrations, the theoretical ethanol concentrations are 91.98 (=200 × 0.9 × 0.511) and 108.08 g/l (=235 × 0.9 × 0.511) respectively. Note that for ∼250 g glucose/l condition, there were ∼15 g glucose/l remained in the fermenter. With reference to Fig. 1 (case of ∼200 g glucose/l), although the final ethanol concentration of 88.39 ± 1.0 g/l measured at the end of each cycle was within the ethanol toxic concentration range (i.e., 85–90 g/l), the corresponding biomass showed a continually increasing trend for the first four cycles, and the yeast viability
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Fig. 2. Illustration of redox potential and glucose concentration profiles occurring at the 10th cycle for 200 ± 3.3 g glucose/l case and the 5th cycle for 250 ± 5.3 g glucose/l case. This depicts the distinct differences existing among 200 and 250 g glucose/l in an SCF operation and the need to implement manual broth removal and refilling for the 250 g glucose/l case.
increased from 80% and finally stabilized at 97.2 ± 1.1% (Fig. 3). This indicated that the ethanol toxicity imposed minor to unnoticeable influence on yeast propagation under current conditions. It is postulated that yeast cells adapt to the toxic ethanol environment in the fermenter. As a result, SCF operation mode was engaged, resulting in repeated batch fermentation. In contrast, under ∼250 g/l initial glucose concentration, 50–65 g/l unspent glucose remained in the fermenter when the redox potential reached its lowest level (Figs. 1 and 2). This particular level occurred at the 19th hour of each cycle, at which point the biomass and ethanol concentrations were ∼3.1, and ∼85 g/l, respectively, and the yeast viability was 85 ± 5.1% (Fig. 3). Since there were 20–26% of initial glucose remained in the fermenter, a manually adjusted period of 36 h (reciprocal of 0.028 h−1 as derived from Liu et al. [12], in which a similar initial glucose concentration was used) was selected in an attempt to lower the residual glucose. As fermentation proceeded, a constant biomass concentration
Fig. 3. Cell viability profiles for the case of 200 ± 3.3 and 250 ± 5.3 g glucose/l during redox potential-driven repeated batch fermentation. While the initial viabilities drop in a similar pattern for both 200 and 250 cases, for the 200 g glucose/l case the viabilities eventually rise to 97.2 ± 1.1% indicating an acclimatization effect while for 250 g glucose/l case, the viabilities remained at 85 ± 5.1% indicating that cells do not get acclimatized to the high ethanol concentration environment.
(3.1 ± 0.1 g/l) between the 19th and the 36th hour was recorded (Fig. 1); however, the corresponding yeast viability decreased significantly to 75 ± 1.4%. The ethanol concentration at the end of each cycle (measured at the 36th hour) was 106.76 ± 0.7 g/l, and the residual glucose concentration was 15 ± 1.3 g/l. This indicated that yeast was at its stationary to death phase. Under ∼200 g glucose/l condition, it took ∼14 h for the redox potential in the fermenter to start rising from the lowest level; whereas about 19 h was required for the case of ∼250 g glucose/l. Such a time delay was related to the osmotic stress attributable to high initial glucose concentration [10]. The activation of SCF operation under ∼200 g glucose/l condition could be correlated to complete glucose utilization because the oxidizing power outperforms the reducing power in the fermenter. Consequently, an increase of redox potential from its lowest level triggers the SCF operation. In contrast, for the case of ∼250 g glucose/l, the residual glucose (50–65 g/l) in conjunction with the ethanol concentration (85 ± 1.4 g/l measured at the 19th hour, falls in the ethanol toxicity range, 85–90 g/l) retarded yeast growth in the fermenter. As a result, an increasing redox potential profile reflects insufficient provision for reducing power (contributed by yeast cells) in order to counteract oxidizing power (contributed by DO supplied through agitation or sparging). Because over 20% of unused glucose imposes difficulty for the subsequent downstream processing, it is necessary to increase glucose consumption. The simplest approach would be extending the duration of fermentation, as demonstrated in this article. Hence, the SCF operation was discarded; instead, a forced repeated batch operation was implemented for 250 g/l feeding glucose condition. 3.3. Measurement of yeast synchronization A quantifier to measure whether the culture in a fermenter is under synchronization operation is necessary. Blumenthal and Zahler [15] formulated a synchrony index (F) to represent the degree of synchronization during cell division: F=
Nt − 2t/g N0
(1)
In the formula, Nt , N0 , t, and g stand for cell concentration at time t, cell concentration at time 0, culture time, and doubling time of the cultivated cell respectively. When F equals to one, it represents a perfect synchronization (i.e., t approaches to zero). When F equals to zero, an exponential growth is established (i.e., t approaches to g). Given that the maximum specific growth rate of S. cerevisiae was assumed to be 0.454 h−1 , the equivalent doubling time was then calculated to be 1.53 h (=0.693/0.454). During ethanol fermentation, t is greater than g, making Eq. (1) inapplicable. Sauvageau et al. [4] modified Eq. (1) by reversing the definition of t and g, and letting the ratio of Nt to N0 be two under SCF operation. They concluded that synchronization was achieved if F value was greater than or equal to 0.6. However, the details of selecting 0.6 as a threshold F value were not provided. Though, the definition of Eq. (1) by Sauvageau et al. ensured that the F value was always positive, it did not explain the rationale behind using a constant value of two for the ratio of Nt to N0 . As a matter of fact, such a ratio was less than two based on measured biomass concentration (Fig. 1). Moreover, since the term synchronization corresponds to intracellular activity, cell synchronization as it is defined through Eq. (1) by Sauvageau et al. [4] alone cannot be used to quantify cell synchronization. So, we chose to use the term repeated batch operation for both ∼200 and ∼250 g glucose/l cases as opposed to synchronization operation. In order to distinguish between the two cases, we further differentiate the terms self-regulated and forced repeated batch operation. For ∼200 g/l feed glucose case, there was no manual interference with regards to the self-cycling operation, so the phenomenon was
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Table 1 Comparison of annual ethanol productivity for batch, redox potential-driven repeated batch (also called self-cycling shown below), and continuous fermentation under ∼200 g glucose/l feed concentration. Vw = 100 m3 , Tw = 7920 h, redox potential = −100 mV. Down time, Td (h)
Fermentation mode
Batch or cycle time, Tf (h)
Batcha Self-cycling Continuousb
24 6 14.25 0.25 6 h in ageing vessel with 0.028 h−1 dilution rate (D)
a b
Ethanol concentration, C (g/l in single batch or cycle)
Annual productivity, Py (tons)
89.3 ± 0.2 88.4 ± 1.0 96.2 ± 2.6 (ageing vessel)
2358 2414 2133
Adapted from Lin et al. [10]. Adapted from Liu et al. [12].
termed as self-regulated repeated batch operation. In the ∼250 g/l feed glucose case, the cycle times were chosen manually in order to reduce residual glucose, so the observed phenomenon was termed as forced repeated batch operation. 3.4. Applicability of redox potential-driven repeated batch fermentation In order to determine the effect of redox potential-driven repeated batch fermentation on ethanol production under VHG conditions, a comparison of ethanol productivities among batch [10], redox potential-driven repeated batch (also called selfcycling), and continuous fermentation [12] operations was performed. The formulae used to calculate ethanol productivities are shown in Eqs. (2)–(4), and the calculated results are compiled in Table 1. Batch :
Py =
Tw × Vw × C Tf + Td
(2)
Self-cycling :
Tw Vw Py = ×C × Tf + Td 2
(3)
Continuous :
Py = D × C × Vw × Tw
(4)
Table 1 shows that redox potential-driven repeated batch fermentation (current work) increases ethanol productivity by 2.4% and 13.2% with respect to batch and continuous fermentation operation. One also observes that the increased productivity is partly related to shorter filling time (about 0.25 h) between cycles during repeated batch operation; whereas, it typically takes 6 h of down time between batch operation. 4. Conclusions Redox potential-driven SCF operation as a means to maintain repeated batch operation was studied for VHG ethanol fermentation processes with feed glucose concentrations of ∼200 and ∼250 g/l. Apart from the differences in residual glucose levels, the cell viability differences between initial glucose concentrations were idiosyncratic and indicative of the toxic effects of ethanol on yeast. It was concluded that (1) the redox potential-driven repeated batch fermentation operated under ∼200 g glucose/l condition improved ethanol tolerance of yeast cells and maintained high cell activities towards the end of each cycle, while such an operation failed to improve the cells’ maximum ethanol tolerance under ∼250 g glucose/l condition; (2) redox potential was a reliable measure of progress of ethanolic fermentation and it could be used as a control measure to trigger a self-cycling process. Elimination of lag phase through the reported fermentation operation has also shown to improve the annual ethanol productivity from its batch and continuous counterparts.
Appendix A. Nomenclature
C D F g Nt N0 Py t Td Tf Tw Vw
final ethanol concentration, g/l dilution rate, h−1 synchrony index cycle time, h biomass concentration at the end of cycle, g/l biomass concentration in the beginning of cycle, g/l annual ethanol productivity, tons/year doubling time, h down time, h fermentation time, h annual operating time, h working volume, m3
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