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Ageing vessel design and optimization for continuous very-high-gravity ethanol fermentation processes
Process Biochemistry 47 (2012) 57–61
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Ageing vessel design and optimization for continuous very-high-gravity ethanol fermentation processes Chen-Guang Liu a , Yen-Han Lin b,∗ , Feng-Wu Bai a a b
School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116023, China Department of Chemical Engineering, University of Saskatchewan, Saskatoon, SK S7N5A9, Canada
a r t i c l e
i n f o
Article history: Received 8 June 2011 Received in revised form 12 September 2011 Accepted 8 October 2011 Available online 17 October 2011 Keywords: Ageing vessel design Very high gravity Ethanol Fermentation Yeast
a b s t r a c t A continuous very-high-gravity (VHG) ethanol fermentation process design, consisting of a chemostat vessel connected to several equal-sized ageing vessels configured in parallel, was developed. The objective of the developed process is to have complete glucose utilization during fermentation stage. The process design integrates the conservation of mass principle and the experimental data of collected residual glucose profiles measured under VHG conditions. An ageing vessel involves three consecutive time periods: filling, ageing and operating. The ageing time is biological relevance, and is affected by the initial glucose concentration, the ethanol concentration, and the yeast viability in an ageing vessel. The operating time period is adjustable; a short operating time means a high discharge rate in order to empty an ageing vessel. The filling time links to the selection of the number of equal-sized ageing vessels that are installed downstream to a chemostat device. The developed process features the use of equal sized fermenters for all chemostat and ageing vessels so that the vessel exchangeability and the flexibility of fermentation operation are increased. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction The application of very-high-gravity (VHG) fermentation technology to bio-ethanol production can significantly increase final ethanol concentrations and lower energy consumption during distillation stage of the process [1,2]. Additionally, the presence of high ethanol concentrations during the course of VHG fermentation can inhibit microbial contamination resulting from bacteria and other eukaryotes [3]. Ethanol tolerance studies revealed that some industrial yeast strains could survive under extremely high ethanol concentrations that were previously deemed not possible [4–6]. During VHG ethanol fermentation, an abrupt decline of yeast population is common, consequently resulting in incomplete glucose utilization and decreasing the fermentation rate [7]. A conventional chemostat device is suitable for low initial glucose concentrations. As the initial glucose concentration increases, there is unspent glucose found in the effluent stream. The higher the initial glucose concentration, the more residual glucose is underutilized. As a result, the annual ethanol productivity is reduced and the degree of difficulty of downstream separation of unspent glucose and ethanol increases as well. A new continuous VHG fermentation process is required to ensure complete usage of glucose
∗ Corresponding author. Tel.: +1 306 966 4764; fax: +1 306 966 4777. E-mail address: [email protected] (Y.-H. Lin). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.10.008
before discharging to distillation units from a chemostat that is operated under VHG conditions. Many of the reported continuous VHG ethanol fermentation processes were developed by trial-and-error approach [8–10]. Bayrock and Ingledew reported a fermentation train configuration where five chemostat devices were connected in series, and the dilution rates were varied in order to obtain zero glucose discharge at the last fermenter of the train [11]. Bai et al. designed a stirred tank reactor followed by three tubular reactors connected in series. The residual glucose concentration in the last reactor was 17.7 g/l when fed with 200 g glucose/l [8]. Such a design was improved by lowering the dilution rate and resulted in nearly complete glucose conversion [10]. The “in-series” fermentation configuration relies on the successful operation of the upstream reactors. When one reactor is contaminated, contamination spreads to down-stream reactors, thus resulting in a complete failure of the fermentation operation. Previously, we reported the development of a continuously like ethanol fermentation process where a chemostat device was connected to two ageing vessels [12], and compared it to a batch fermentation in terms of annual ethanol productivity. We concluded that the proposed process configuration is more efficient than the batch counterpart. A suitable range of dilution rates to operate such a process was present, and the selection criterion between a batch and the proposed fermentation process was also given. Due to the choice of two ageing vessel designs, the filling time for an ageing vessel varies along with the initial glucose concentration.
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C.-G. Liu et al. / Process Biochemistry 47 (2012) 57–61
In this article, we report the development of a new process design approach to shorten the filling time by incorporating multiple equal-sized ageing vessels downstream to a chemostat. The developed process can use any number of ageing vessels to meet their fermentation objectives. The new approach built upon the foundation of the mass conservation principle and required a residual glucose profile collected from batch fermentation in order to initiate the design procedure. Case studies were provided to illustrate the implementation of design steps. 2. Materials and methods 2.1. Strain, growth media, sample analysis, and operating conditions Strain, growth media, and sample analysis were previously reported [12]. Briefly, an industrial S. cerevisiae strain (Ethanol RedTM obtained from the Lesaffre Yeast Corp., Milwaukee, MI, USA) was pre-cultured overnight and cultivated in a jar fermenter with 1-liter working volume (model: Omni culture fermenter, New York, NY, USA). In addition to initial glucose concentrations (either ∼200, ∼250, or ∼300 g/l), the growth medium included trace mineral salts, vitamin cocktail, urea, yeast extract, and sodium glutamate. Aliquots of fermentation broth was collected every 6–8 h, and the violet red staining method was used to differentiate viable and dead cells [13]. As a result, cell viability was estimated. An HPLC equipped with an RI detector was used to quantify residual glucose and ethanol. The samples were properly diluted to fall into the linear region of the respective calibration curves. The dilution rate, working volume, agitation rate and temperature of the chemostat vessel (i.e., an Omni culture fermenter described above) was kept at 0.028 h−1 , 1 l, 150 rpm, and 30 ◦ C, respectively. Whereas, an ageing vessel was agitated at 50 rpm and the vessel temperature was maintained at 30 ◦ C.
proposed two model equations (Eqs. (2) and (3)) to simulate glucose consumption profiles during T1 and T2 period, respectively.
2.2. Data fitting The experimentally collected residual glucose profile was fitted to the proposed models (to be described in Section 3.1) by means of regression methods. The fitted results were then compared to experimental data to evaluate the goodness of fit. A simple R2 criterion was chosen for this purpose:
R
2
2 (m − mpred ) =1− 2 ¯ (m − m)
Fig. 1. Glucose consumption profile in an ageing vessel under different initial glucose concentrations. T1 , filling time; T2 , ageing time. Experimental data were excerpted from Fig. 1a in [12].
Sin − S∞ 1 + ˛T1
T1 period :
ST1 = S∞ +
T2 period :
ST2 = ST1 − kT2
(2) (3)
(1)
3. Results and discussion The general configuration of the proposed process consists of a chemostat vessel connected to two or more ageing vessels. The duration and operation of an ageing vessel can be divided into three time periods: filling time (T1 ), ageing time (T2 ) and operating time (T3 ). The filling time is the time required to fill the ageing vessel to the designed volume. The fermentation broth delivered from chemostat vessel contains unspent glucose and yeast. Hence, during the filling period, the fermentation operates as fedbatch mode. Once the desired volume in the ageing vessel is attained, the time required to completely convert glucose is called ageing time. During this period, the fermentation behaves as batch operation. The time further required to completely empty the ageing vessel and deliver the spent fermentation broth to the next process unit is called operating time.
3.1. Analysis of glucose consumption profile in an ageing vessel The operational scheduling for an ageing vessel depends on the pattern of the glucose consumption profile in an ageing vessel. Hence, it is essential to understand the pattern of the consumption profile and incorporate it into process design. According to Fig. 1, two observable consumption patterns are apparent; that is, a variable consumption pattern (during T1 period) followed by a constant consumption one (during T2 period). Fig. 1 also illustrates that a higher initial glucose concentration requires a longer T2 period. In order to design, size and configure proper ageing vessels, we
Given a set of experimentally collected residual glucose profiles and the experimentally determined Sin , ST1 , ST2 , T1 , and T2 (found from Fig. 1), regression methods were implemented to obtain S∞ , ˛, and k. As seen in Table 1, high initial glucose concentration results in slow overall specific glucose consumption rate for both periods (˛ and k) along with a high value of S∞ . These observations are attributed to the combined adverse effect of osmotic stress resulting from high initial glucose concentration and inhibitory stress resulting from the buildup of ethanol during fermentation [7]. In the T1 period, a high inlet residual glucose implies that high ethanol concentration is expected. As fermentation proceeds to T2 period, the impact of ethanol toxicity on yeast growth in an ageing vessel becomes significant; this is reflected on the k value as shown in Table 1. Notice that the k value for ∼300 g glucose/l case is three times smaller than that for ∼200 g glucose/l counterpart. A larger k value correlates to a shorter ageing time in an ageing vessel. For ∼300 g glucose/l case, a non-zero S∞ clearly indicates that an additional time period (defined as ageing time, T2 ) after filling time period (T1 ) is required in order to completely convert glucose to ethanol. In other words, ageing vessels should be installed downstream of the chemostat operation. A larger S∞ correlates to a longer T2 period. Although S∞ for both ∼200 g glucose/l and ∼250 g glucose/l cases are nearly zero, ageing vessels are still required but with shorter ageing times. The information listed in Table 1 was further attested to evaluate their goodness of fit. As shown in Fig. 1, the predicted glucose consumption profiles correlate to the experimental data with an R2 value greater than 0.99 for all three cases. Accordingly, the process design for ageing vessels and the process configuration for a chemostat vessel connected to two or more than two ageing vessels are presented below.
C.-G. Liu et al. / Process Biochemistry 47 (2012) 57–61
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Table 1 Experimental data and fitted model parameters for a two-ageing vessel configuration under three different initial glucose concentrations. Sin , residual glucose concentration entering ageing vessel; S∞ , residual glucose concentration when T1 approaches to infinite; ␣, overall specific glucose consumption rate during filling period in an ageing 2 , minimal filling time for a two-ageing vessel configuration. vessel; k, overall specific glucose consumption rate during ageing period; Tcri Sin (g/l)
Initial glucose concentration (g/l) 303 ± 4.24 255 ± 2.56 203 ± 2.94
159.05 93.412 21
S∞ (g/l) 57.49 0 0
3.2. Relationship of filling time (T1 ) and ageing time (T2 )
S∞ + (Sin − S∞ )/(1 + ˛T1 ) k
(4)
When a long filling time is encountered, corresponding to a low dilution rate during chemostat operation, a constant ageing time is expected, which is expressed by simplifying Eq. (4) and letting T2 equal to T2∞ T2∞ =
S∞ k
(5)
For a two-ageing vessel configuration, the operating time (T3 ) must satisfy T3 = T1 − T2 . In practice, T1 must always be greater than T2 . Under the extreme condition where T3 = 0, the minimal filling 2 ) is derived by letting T = T . time for two ageing vessels (Tcri 1 2 2 Tcri
=
(˛S∞ − k) ±
2
(˛S∞ − k) + 4˛kSin 2˛k
k (h−1 )
2 Tcri (h)
0.05003 0.06994 0.66667
1.047 1.114 3.187
75.266 28.208 2.482
along with the accumulated ethanol during the course of fermentation.
Complete glucose utilization is the ultimate objective when operating VHG fermentation before subsequent downstream processing. This means that ST2 in Eq. (3) must be set to zero and then combined with Eq. (2) to obtain T2 =
˛ (h−1 )
(6)
2 represents the minimal filling time required to operate the proTcri posed continuous VHG ethanol fermentation process connected 2 is listed in with two ageing vessels. The numerical value of Tcri Table 1 for each respective initial glucose concentration, and is illustrated in Fig. 2 where it is located on the abscissa for each respective initial glucose concentration condition. A higher initial 2 , and a non-linear proglucose concentration requires a longer Tcri 2 becomes obvious. This implies that yeast encounters portion of Tcri severe stresses resulting from high initial glucose concentration
3.3. Effect of filling time (T1 ) on operating time (T3 ) and discharge rate (F) When the residual glucose in an ageing vessel was completely utilized (i.e., ST2 = 0), a time period called operating time is required to discharge the spent fermentation broth into the subsequent separation units for further processing. Based on the material balance around an ageing vessel, the volumetric flow rate of the broth leaving an ageing vessel can be expressed by Eq. (7) or Eq. (8): F=
T1 DVC T3
(7)
F=
DVC 1 − (S∞ + ((Sin − S∞ )/(1 + ˛T1 )))/T1 /k
(8)
Note that the discharge rate (F) in Eq. (8) is the required minimal discharge rate of an ageing vessel. One needs to satisfy this requirement in order to discharge an ageing vessel at the specified T3 period. The relationships among T1 , T2 , T3 , and F are illustrated in Fig. 2. 2 , When a small T3 is specified; that is, T1 is in the vicinity of Tcri a drastic change of discharge rate is noticeable. It is an undesired condition, which leads to unstable operation of an ageing vessel. On the other hand, T3 increases linearly as T1 increases. Eventually, T3 intersects T2 , at which a nearly constant discharge rate is attainable. The corresponding T1 at this intersection point is regarded as the upper bound in the T1 region, and the corresponding discharge rate equals two folds of DVC . To avoid the fluctuation of discharge rate 2 , the lower-bounded T (T close to Tcri 1 1,low ) is subjectively defined by 2 and the upper-bounded T (T averaging Tcri 1 1,up ) where the equivalent discharge rate is approximately equal to three folds of DVC as shown in Eq. (9). T1,up = 2
S∞ + (Sin − S∞ )/(1 + ˛T1 ) k
(9)
Note that within the defined T1 region, T2 and F are relatively insensitive to the variation of T1 (Fig. 2). Comparatively, when the selected T1 is smaller than T1,low , a minute deviation in filling time will result in a radical variation in discharge rate. Or, when the selected T1 is greater than T1,up , the overall ageing period (i.e., T1 + T2 + T3 ) will be extended, consequently reducing the annual ethanol productivity. 3.4. Filling time (T1 ) and ageing vessel volume (VA ) When an adequate combination of T1 , T2 and T3 is determined, the next design step is to size the volume of an ageing vessel (VA ). The size of VA is affected by the dilution rate (D) during the chemostat operation, the volume of the chemostat vessel (VC ), and the T1 . Their relationship can be expressed as follows: VA = T1 DVC Fig. 2. Profiles of ageing time (T2 ), operating time (T3 ) and discharge rate (F) of two ageing vessels with respect to filling time (T1 ) under different initial glucose concentrations. The recommended T1 region for ∼300 g glucose/l is illustrated.
(10)
Under the condition of constant DVC , the size of VA is dominated by initial glucose concentration. In conjunction Fig. 2 with Eq. (10)
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Fig. 3. Three ageing vessel configuration. (a) Process schematic; (b) operational scheduling.
2 , the size of V can be where D is 0.028 h−1 and T1 is chosen at Tcri A three times larger than that of VC at ∼300 g glucose/l; or one tenth of that of VC at ∼200 g glucose/l. Accordingly, one can extrapolate that the higher the initial glucose concentration, the larger the ageing vessel is needed; therefore, a high initial capital investment on ageing vessels would be expected. Additionally, the use of various sizes of fermenters in a fermentation process will also increase the associate operating cost. One of the cost effective approaches to reduce capital investment and to increase the operating flexibility of the fermentation process is to design similar or equally sized chemostat vessels and ageing vessels, so that the vessel exchangeability and the vessel utilization can be maximized.
3.5. Design of multi equal-sized ageing vessels Given that a suitable overall ageing vessel period was determined, and the operational scheduling satisfied the condition of T1 = T2 + T3 , Eq. (11) is used for designing multiple equal-sized ageing vessels connected in parallel. For example, the fermentation process configuration and the corresponding operational scheduling for three equal-sized ageing vessels are illustrated in Fig. 3. This figure can be further extended to design a continuous VHG fermentation involving more than three equal-sized ageing vessels accordingly. T1 =
T2 + T3 N−1
(11)
The generalized representation to estimate the minimal filling time for multi-ageing vessel configuration shown in Eq. (12) was obtained by substituting Eq. (4) into Eq. (11) along with the condition that T3 must be zero. N Tcri
=
(S∞ ˛ − (N − 1)k) ±
Fig. 4. Profiles of ageing time (T2 ), operating time (T3 ) and discharge rate (F) for different number of ageing vessels (N) with respect to filling time (T1 ) under ∼300 g glucose/l condition. T2 , T3 , and F are calculated according to Eqs. (4), (11), and (7), respectively.
equal-sized ageing vessels are determined. As the number of ageing vessels increases, T1 decreases, whereas T2 remains the same since the ageing time is dependent on yeast activity under the influence of different glucose concentration and ethanol concentrations (Fig. 4). Examples will be given in the following section to illustrate the application of the above-described design procedure. 3.6. Case studies 3.6.1. Case 1: initial glucose concentration at ∼300 g/l A fermentation with initial glucose concentration of 303 ± 4.24 g/l was conducted, sampled every 6–8 h, and the residual glucose concentration were quantified. The measured profiles of residual glucose were fitted with Eqs. (2) and (3) to obtain S∞ , ˛, and k values (Table 1). By using the dilution rate (0.028 h−1 ) and the working volume of a chemostat vessel (1 liter), and by combining S∞ , ˛, k, and Eqs. (4)–(7), Fig. 2 is constructed, and the recommended T1 region is then identified (Table 2); or, simply followed the design instruction described in the third paragraph of Section 3.3 to determine T1 region (that is, 105.06–134.86 h in this case). In conjunction with Eq. (10) and dilution rate of 0.028 h−1 , the working volume of ageing vessel is 2.94–3.78 folds larger than that of a chemostat vessel. To design a fermentation process having equal-sized fermenters, the estimated T1 is 36 h (refer to Eq. (10)). This means that to reduce T1 period during continuous VHG fermentation, a multiple ageing vessel configuration is required. By referring to Table 2 that is generated by using Eq. (12), five ageing vessels connected in parallel to a single chemostat vessel are selected. Then, according to Eqs. (4), (11), and (7), T2 , T3 and F are determined as 89.54 h, 54.45 h, and 0.018 l/h, respectively.
2
(S∞ ˛ − (N − 1)k) + 4˛(N − 1)kSin 2˛(N − 1)k (12)
2 for a two equal-sized ageing vessels conFor example, Tcri figuration is estimated by letting N in Eq. (12) to be two, and Eq. (6) is obtained. In connection with Eq. (10), D and VC , multi
3.6.2. Case 2: initial glucose concentration at ∼250 g/l Similar to the design procedure described in the case study above, the recommended T1 region is 35.27–42.34 h (Table 2), and the working volume ratio of ageing vessel to chemostat vessel is 0.99–1.19. This means that when carrying out ethanol fermentation with ∼250 g glucose/l in the feed stream, three equal sizes of fermenters are required; one for the chemostat operation, and two
C.-G. Liu et al. / Process Biochemistry 47 (2012) 57–61
61
Table 2 Design parameters for multi equal-sized ageing vessels (with a dilution rate of 0.028 h−1 ). Number of ageing vessels
303 ± 4.24 (g/l) Recommended T1 region (h)
2 3 4 5 6 7
105.06–134.86 59.57–76.26 42.52–54.06 33.73–42.87 28.20–35.85 24.35–30.98
255 ± 2.56 (g/l) Recommended VA /VC 2.94–3.78 1.67–2.13 1.19–1.51 0.94–1.20 0.79–1.00 0.68–0.87
35.27–42.34 23.28–28.20 18.05–22.01 14.97–18.35 12.90–15.88 11.39–14.08
S∞
3.6.3. Case 3: initial glucose concentration at ∼200 g/l Under 203 ± 2.94 g glucose/l condition (Table 2), the highest recommended filling time is 3.72 h and the size of ageing vessel is one tenth of the chemostat vessel. A short filling time translates to a frequent switching between ageing vessels, incurring a high operating cost. A small ageing vessel implies that only a small amount of fermentation broth is available for the subsequent distillation unit. As a result, a high energy cost is expected. Referring to Fig. 1 under ∼200 g glucose/l condition, the residual glucose in an ageing vessel is nearly utilized during the course of filling period. This means that no ageing time is required in an ageing vessel during bio-ethanol production when the chemostat device is operated at a dilution rate of 0.028 h−1 with an initial glucose concentration of ∼200 g/l. The ageing vessel in the proposed fermentation configuration serves as a surge tank for temporary storage of spent fermentation broth before distillations. One can then conclude that the multi ageing vessel configuration is not suitable for a chemostat feeding with low initial glucose concentration.
ST1 ST2 T1 T1,up T1,low T2 T2∞ T3 VA VC 2 Tcri
4. Conclusions Ageing vessel design results in complete glucose utilization during fermentation stage of ethanol operation. The developed ageing vessel design procedure is based on the measured residual glucose profile in an ageing vessel. The profile consists of fedbatch consumption pattern during the filling time period followed by batch consumption pattern during the ageing time period. Two respective models were proposed and could predict patterns with high accuracy. Integrating predicted results with mass balance principle, several ageing vessel design criteria were derived for the selection of number, size, and operation of ageing vessels. Case studies were illustrated to assist the reader to familiarize with the design procedure. The continuous VHG fermentation design criteria can be easily adopted by the fuel alcohol producers where batch fermentation is their current operation. Appendix A. Nomenclature
m ¯ m mpred N
Recommended VA /VC
Recommended region (h)
for the ageing vessels. At a dilution rate of 0.028 h−1 , T1 becomes 36 h. The estimated T2 , T3 , and F are 23.83 h, 12.16 h, and 0.083 l/h, respectively.
D F k
203 ± 2.94 (g/l)
dilution rate (h−1 ) discharge rate of an ageing vessel (l/h) overall specific glucose consumption rate during T2 period (h−1 ) metabolite concentration (g/l) average metabolite concentration (g/l) predicted metabolite concentration (g/l) number of ageing vessel
Sin
N Tcri
˛
0.99–1.19 0.65–0.79 0.51–0.62 0.42–0.51 0.36–0.44 0.32–0.39
Recommended T1 region (h) 3.10–3.72 2.04–2.48 1.57–1.92 1.30–1.60 1.11–1.38 0.97–1.21
Recommended VA /VC 0.087–0.104 0.057–0.069 0.044–0.054 0.036–0.045 0.031–0.039 0.027–0.034
residual glucose concentration when T1 approaches to infinite (g/l) residual glucose concentration entering ageing vessel (g/l) residual glucose concentration after T1 period (g/l) residual glucose concentration after T2 period (g/l) feeding time for an ageing vessel (h) upper-bounded T1 (h) lower-bounded T1 (h) ageing time for an ageing vessel (h) constant ageing time derived from Eq. (5) (h) operating time for an ageing vessel (h) working volume for an ageing vessel (l) working volume for a chemostat vessel (l) minimal filling time for a two-ageing vessel configuration (h) minimal filling time for a N-ageing vessel configuration (h) overall specific glucose consumption rate during T1 period (h−1 )
References [1] Thomas KC, Hynes SH, Jones AM, Ingledew WM. Production of fuel alcohol from wheat by VHG technology – effect of sugar concentration and fermentation temperature. Appl Biochem Biotechnol 1993;43:211–26. [2] Thomas KC, Hynes SH, Ingledew WM. Practical and theoretical considerations in the production of high concentrations of alcohol by fermentation. Process Biochem 1996;31:321–31. [3] Bafrncová P, Smogroviˇcová D, Sláviková I, Pátková J, Dömény Z. Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae. Biotechnol Lett 1999;21:337–41. [4] Casey GP, Ingledew WMM. Ethanol tolerance in yeasts. Crit Rev Microbiol 1986;13:219–80. [5] Thomas KC, Ingeldew WM. Production of 21% (v/v) ethanol by fermentation of very high gravity (VHG) wheat mashes. J Ind Microbiol 1992;10:61–8. [6] Casey GP, Magnus CA, Ingledew WM. High-gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appl Environ Microbiol 1984;48:639–46. [7] Lin YH, Bayrock DP, Ingledew WM. Evaluation of Saccharomyces cerevisiae grown in a multistage chemostat environment under increasing levels of glucose. Biotechnol Lett 2002;24:449–53. [8] Bai FW, Chen LJ, Zhang Z, Anderson WA, Moo-Young M. Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions. J Biotechnol 2004;110:287–93. [9] Bai FW, Ge XM, Anderson WA, Moo-Young M. Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation. Biotechnol Bioeng 2009;102:113–21. [10] Xu TJ, Zhao XQ, Bai FW. Continuous ethanol production using self-flocculating yeast in a cascade of fermentors. Enzyme Microb Technol 2005;37:634–40. [11] Bayrock DP, Ingledew WM. Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology. J Ind Microbiol Biotechnol 2001;27:87–93. [12] Liu C-G, Lin Y-H, Bai F-W. Ageing vessel configuration for continuous redox potential-controlled very-high-gravity fermentation. J Biosci Bioeng 2011;111:61–6. [13] Smart KA, Chambers KM, Lambert I, Jenkins C, Smart CA. Use of methylene violet staining procedures to determine yeast viability and vitality. J Am Soc Brew Chem 1999;57:18–23.
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Ageing vessel design and optimization for continuous very-high-gravity ethanol fermentation processes Chen-Guang Liu a , Yen-Han Lin b,∗ , Feng-Wu Bai a a b
School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116023, China Department of Chemical Engineering, University of Saskatchewan, Saskatoon, SK S7N5A9, Canada
a r t i c l e
i n f o
Article history: Received 8 June 2011 Received in revised form 12 September 2011 Accepted 8 October 2011 Available online 17 October 2011 Keywords: Ageing vessel design Very high gravity Ethanol Fermentation Yeast
a b s t r a c t A continuous very-high-gravity (VHG) ethanol fermentation process design, consisting of a chemostat vessel connected to several equal-sized ageing vessels configured in parallel, was developed. The objective of the developed process is to have complete glucose utilization during fermentation stage. The process design integrates the conservation of mass principle and the experimental data of collected residual glucose profiles measured under VHG conditions. An ageing vessel involves three consecutive time periods: filling, ageing and operating. The ageing time is biological relevance, and is affected by the initial glucose concentration, the ethanol concentration, and the yeast viability in an ageing vessel. The operating time period is adjustable; a short operating time means a high discharge rate in order to empty an ageing vessel. The filling time links to the selection of the number of equal-sized ageing vessels that are installed downstream to a chemostat device. The developed process features the use of equal sized fermenters for all chemostat and ageing vessels so that the vessel exchangeability and the flexibility of fermentation operation are increased. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction The application of very-high-gravity (VHG) fermentation technology to bio-ethanol production can significantly increase final ethanol concentrations and lower energy consumption during distillation stage of the process [1,2]. Additionally, the presence of high ethanol concentrations during the course of VHG fermentation can inhibit microbial contamination resulting from bacteria and other eukaryotes [3]. Ethanol tolerance studies revealed that some industrial yeast strains could survive under extremely high ethanol concentrations that were previously deemed not possible [4–6]. During VHG ethanol fermentation, an abrupt decline of yeast population is common, consequently resulting in incomplete glucose utilization and decreasing the fermentation rate [7]. A conventional chemostat device is suitable for low initial glucose concentrations. As the initial glucose concentration increases, there is unspent glucose found in the effluent stream. The higher the initial glucose concentration, the more residual glucose is underutilized. As a result, the annual ethanol productivity is reduced and the degree of difficulty of downstream separation of unspent glucose and ethanol increases as well. A new continuous VHG fermentation process is required to ensure complete usage of glucose
∗ Corresponding author. Tel.: +1 306 966 4764; fax: +1 306 966 4777. E-mail address: [email protected] (Y.-H. Lin). 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.10.008
before discharging to distillation units from a chemostat that is operated under VHG conditions. Many of the reported continuous VHG ethanol fermentation processes were developed by trial-and-error approach [8–10]. Bayrock and Ingledew reported a fermentation train configuration where five chemostat devices were connected in series, and the dilution rates were varied in order to obtain zero glucose discharge at the last fermenter of the train [11]. Bai et al. designed a stirred tank reactor followed by three tubular reactors connected in series. The residual glucose concentration in the last reactor was 17.7 g/l when fed with 200 g glucose/l [8]. Such a design was improved by lowering the dilution rate and resulted in nearly complete glucose conversion [10]. The “in-series” fermentation configuration relies on the successful operation of the upstream reactors. When one reactor is contaminated, contamination spreads to down-stream reactors, thus resulting in a complete failure of the fermentation operation. Previously, we reported the development of a continuously like ethanol fermentation process where a chemostat device was connected to two ageing vessels [12], and compared it to a batch fermentation in terms of annual ethanol productivity. We concluded that the proposed process configuration is more efficient than the batch counterpart. A suitable range of dilution rates to operate such a process was present, and the selection criterion between a batch and the proposed fermentation process was also given. Due to the choice of two ageing vessel designs, the filling time for an ageing vessel varies along with the initial glucose concentration.
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C.-G. Liu et al. / Process Biochemistry 47 (2012) 57–61
In this article, we report the development of a new process design approach to shorten the filling time by incorporating multiple equal-sized ageing vessels downstream to a chemostat. The developed process can use any number of ageing vessels to meet their fermentation objectives. The new approach built upon the foundation of the mass conservation principle and required a residual glucose profile collected from batch fermentation in order to initiate the design procedure. Case studies were provided to illustrate the implementation of design steps. 2. Materials and methods 2.1. Strain, growth media, sample analysis, and operating conditions Strain, growth media, and sample analysis were previously reported [12]. Briefly, an industrial S. cerevisiae strain (Ethanol RedTM obtained from the Lesaffre Yeast Corp., Milwaukee, MI, USA) was pre-cultured overnight and cultivated in a jar fermenter with 1-liter working volume (model: Omni culture fermenter, New York, NY, USA). In addition to initial glucose concentrations (either ∼200, ∼250, or ∼300 g/l), the growth medium included trace mineral salts, vitamin cocktail, urea, yeast extract, and sodium glutamate. Aliquots of fermentation broth was collected every 6–8 h, and the violet red staining method was used to differentiate viable and dead cells [13]. As a result, cell viability was estimated. An HPLC equipped with an RI detector was used to quantify residual glucose and ethanol. The samples were properly diluted to fall into the linear region of the respective calibration curves. The dilution rate, working volume, agitation rate and temperature of the chemostat vessel (i.e., an Omni culture fermenter described above) was kept at 0.028 h−1 , 1 l, 150 rpm, and 30 ◦ C, respectively. Whereas, an ageing vessel was agitated at 50 rpm and the vessel temperature was maintained at 30 ◦ C.
proposed two model equations (Eqs. (2) and (3)) to simulate glucose consumption profiles during T1 and T2 period, respectively.
2.2. Data fitting The experimentally collected residual glucose profile was fitted to the proposed models (to be described in Section 3.1) by means of regression methods. The fitted results were then compared to experimental data to evaluate the goodness of fit. A simple R2 criterion was chosen for this purpose:
R
2
2 (m − mpred ) =1− 2 ¯ (m − m)
Fig. 1. Glucose consumption profile in an ageing vessel under different initial glucose concentrations. T1 , filling time; T2 , ageing time. Experimental data were excerpted from Fig. 1a in [12].
Sin − S∞ 1 + ˛T1
T1 period :
ST1 = S∞ +
T2 period :
ST2 = ST1 − kT2
(2) (3)
(1)
3. Results and discussion The general configuration of the proposed process consists of a chemostat vessel connected to two or more ageing vessels. The duration and operation of an ageing vessel can be divided into three time periods: filling time (T1 ), ageing time (T2 ) and operating time (T3 ). The filling time is the time required to fill the ageing vessel to the designed volume. The fermentation broth delivered from chemostat vessel contains unspent glucose and yeast. Hence, during the filling period, the fermentation operates as fedbatch mode. Once the desired volume in the ageing vessel is attained, the time required to completely convert glucose is called ageing time. During this period, the fermentation behaves as batch operation. The time further required to completely empty the ageing vessel and deliver the spent fermentation broth to the next process unit is called operating time.
3.1. Analysis of glucose consumption profile in an ageing vessel The operational scheduling for an ageing vessel depends on the pattern of the glucose consumption profile in an ageing vessel. Hence, it is essential to understand the pattern of the consumption profile and incorporate it into process design. According to Fig. 1, two observable consumption patterns are apparent; that is, a variable consumption pattern (during T1 period) followed by a constant consumption one (during T2 period). Fig. 1 also illustrates that a higher initial glucose concentration requires a longer T2 period. In order to design, size and configure proper ageing vessels, we
Given a set of experimentally collected residual glucose profiles and the experimentally determined Sin , ST1 , ST2 , T1 , and T2 (found from Fig. 1), regression methods were implemented to obtain S∞ , ˛, and k. As seen in Table 1, high initial glucose concentration results in slow overall specific glucose consumption rate for both periods (˛ and k) along with a high value of S∞ . These observations are attributed to the combined adverse effect of osmotic stress resulting from high initial glucose concentration and inhibitory stress resulting from the buildup of ethanol during fermentation [7]. In the T1 period, a high inlet residual glucose implies that high ethanol concentration is expected. As fermentation proceeds to T2 period, the impact of ethanol toxicity on yeast growth in an ageing vessel becomes significant; this is reflected on the k value as shown in Table 1. Notice that the k value for ∼300 g glucose/l case is three times smaller than that for ∼200 g glucose/l counterpart. A larger k value correlates to a shorter ageing time in an ageing vessel. For ∼300 g glucose/l case, a non-zero S∞ clearly indicates that an additional time period (defined as ageing time, T2 ) after filling time period (T1 ) is required in order to completely convert glucose to ethanol. In other words, ageing vessels should be installed downstream of the chemostat operation. A larger S∞ correlates to a longer T2 period. Although S∞ for both ∼200 g glucose/l and ∼250 g glucose/l cases are nearly zero, ageing vessels are still required but with shorter ageing times. The information listed in Table 1 was further attested to evaluate their goodness of fit. As shown in Fig. 1, the predicted glucose consumption profiles correlate to the experimental data with an R2 value greater than 0.99 for all three cases. Accordingly, the process design for ageing vessels and the process configuration for a chemostat vessel connected to two or more than two ageing vessels are presented below.
C.-G. Liu et al. / Process Biochemistry 47 (2012) 57–61
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Table 1 Experimental data and fitted model parameters for a two-ageing vessel configuration under three different initial glucose concentrations. Sin , residual glucose concentration entering ageing vessel; S∞ , residual glucose concentration when T1 approaches to infinite; ␣, overall specific glucose consumption rate during filling period in an ageing 2 , minimal filling time for a two-ageing vessel configuration. vessel; k, overall specific glucose consumption rate during ageing period; Tcri Sin (g/l)
Initial glucose concentration (g/l) 303 ± 4.24 255 ± 2.56 203 ± 2.94
159.05 93.412 21
S∞ (g/l) 57.49 0 0
3.2. Relationship of filling time (T1 ) and ageing time (T2 )
S∞ + (Sin − S∞ )/(1 + ˛T1 ) k
(4)
When a long filling time is encountered, corresponding to a low dilution rate during chemostat operation, a constant ageing time is expected, which is expressed by simplifying Eq. (4) and letting T2 equal to T2∞ T2∞ =
S∞ k
(5)
For a two-ageing vessel configuration, the operating time (T3 ) must satisfy T3 = T1 − T2 . In practice, T1 must always be greater than T2 . Under the extreme condition where T3 = 0, the minimal filling 2 ) is derived by letting T = T . time for two ageing vessels (Tcri 1 2 2 Tcri
=
(˛S∞ − k) ±
2
(˛S∞ − k) + 4˛kSin 2˛k
k (h−1 )
2 Tcri (h)
0.05003 0.06994 0.66667
1.047 1.114 3.187
75.266 28.208 2.482
along with the accumulated ethanol during the course of fermentation.
Complete glucose utilization is the ultimate objective when operating VHG fermentation before subsequent downstream processing. This means that ST2 in Eq. (3) must be set to zero and then combined with Eq. (2) to obtain T2 =
˛ (h−1 )
(6)
2 represents the minimal filling time required to operate the proTcri posed continuous VHG ethanol fermentation process connected 2 is listed in with two ageing vessels. The numerical value of Tcri Table 1 for each respective initial glucose concentration, and is illustrated in Fig. 2 where it is located on the abscissa for each respective initial glucose concentration condition. A higher initial 2 , and a non-linear proglucose concentration requires a longer Tcri 2 becomes obvious. This implies that yeast encounters portion of Tcri severe stresses resulting from high initial glucose concentration
3.3. Effect of filling time (T1 ) on operating time (T3 ) and discharge rate (F) When the residual glucose in an ageing vessel was completely utilized (i.e., ST2 = 0), a time period called operating time is required to discharge the spent fermentation broth into the subsequent separation units for further processing. Based on the material balance around an ageing vessel, the volumetric flow rate of the broth leaving an ageing vessel can be expressed by Eq. (7) or Eq. (8): F=
T1 DVC T3
(7)
F=
DVC 1 − (S∞ + ((Sin − S∞ )/(1 + ˛T1 )))/T1 /k
(8)
Note that the discharge rate (F) in Eq. (8) is the required minimal discharge rate of an ageing vessel. One needs to satisfy this requirement in order to discharge an ageing vessel at the specified T3 period. The relationships among T1 , T2 , T3 , and F are illustrated in Fig. 2. 2 , When a small T3 is specified; that is, T1 is in the vicinity of Tcri a drastic change of discharge rate is noticeable. It is an undesired condition, which leads to unstable operation of an ageing vessel. On the other hand, T3 increases linearly as T1 increases. Eventually, T3 intersects T2 , at which a nearly constant discharge rate is attainable. The corresponding T1 at this intersection point is regarded as the upper bound in the T1 region, and the corresponding discharge rate equals two folds of DVC . To avoid the fluctuation of discharge rate 2 , the lower-bounded T (T close to Tcri 1 1,low ) is subjectively defined by 2 and the upper-bounded T (T averaging Tcri 1 1,up ) where the equivalent discharge rate is approximately equal to three folds of DVC as shown in Eq. (9). T1,up = 2
S∞ + (Sin − S∞ )/(1 + ˛T1 ) k
(9)
Note that within the defined T1 region, T2 and F are relatively insensitive to the variation of T1 (Fig. 2). Comparatively, when the selected T1 is smaller than T1,low , a minute deviation in filling time will result in a radical variation in discharge rate. Or, when the selected T1 is greater than T1,up , the overall ageing period (i.e., T1 + T2 + T3 ) will be extended, consequently reducing the annual ethanol productivity. 3.4. Filling time (T1 ) and ageing vessel volume (VA ) When an adequate combination of T1 , T2 and T3 is determined, the next design step is to size the volume of an ageing vessel (VA ). The size of VA is affected by the dilution rate (D) during the chemostat operation, the volume of the chemostat vessel (VC ), and the T1 . Their relationship can be expressed as follows: VA = T1 DVC Fig. 2. Profiles of ageing time (T2 ), operating time (T3 ) and discharge rate (F) of two ageing vessels with respect to filling time (T1 ) under different initial glucose concentrations. The recommended T1 region for ∼300 g glucose/l is illustrated.
(10)
Under the condition of constant DVC , the size of VA is dominated by initial glucose concentration. In conjunction Fig. 2 with Eq. (10)
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C.-G. Liu et al. / Process Biochemistry 47 (2012) 57–61
Fig. 3. Three ageing vessel configuration. (a) Process schematic; (b) operational scheduling.
2 , the size of V can be where D is 0.028 h−1 and T1 is chosen at Tcri A three times larger than that of VC at ∼300 g glucose/l; or one tenth of that of VC at ∼200 g glucose/l. Accordingly, one can extrapolate that the higher the initial glucose concentration, the larger the ageing vessel is needed; therefore, a high initial capital investment on ageing vessels would be expected. Additionally, the use of various sizes of fermenters in a fermentation process will also increase the associate operating cost. One of the cost effective approaches to reduce capital investment and to increase the operating flexibility of the fermentation process is to design similar or equally sized chemostat vessels and ageing vessels, so that the vessel exchangeability and the vessel utilization can be maximized.
3.5. Design of multi equal-sized ageing vessels Given that a suitable overall ageing vessel period was determined, and the operational scheduling satisfied the condition of T1 = T2 + T3 , Eq. (11) is used for designing multiple equal-sized ageing vessels connected in parallel. For example, the fermentation process configuration and the corresponding operational scheduling for three equal-sized ageing vessels are illustrated in Fig. 3. This figure can be further extended to design a continuous VHG fermentation involving more than three equal-sized ageing vessels accordingly. T1 =
T2 + T3 N−1
(11)
The generalized representation to estimate the minimal filling time for multi-ageing vessel configuration shown in Eq. (12) was obtained by substituting Eq. (4) into Eq. (11) along with the condition that T3 must be zero. N Tcri
=
(S∞ ˛ − (N − 1)k) ±
Fig. 4. Profiles of ageing time (T2 ), operating time (T3 ) and discharge rate (F) for different number of ageing vessels (N) with respect to filling time (T1 ) under ∼300 g glucose/l condition. T2 , T3 , and F are calculated according to Eqs. (4), (11), and (7), respectively.
equal-sized ageing vessels are determined. As the number of ageing vessels increases, T1 decreases, whereas T2 remains the same since the ageing time is dependent on yeast activity under the influence of different glucose concentration and ethanol concentrations (Fig. 4). Examples will be given in the following section to illustrate the application of the above-described design procedure. 3.6. Case studies 3.6.1. Case 1: initial glucose concentration at ∼300 g/l A fermentation with initial glucose concentration of 303 ± 4.24 g/l was conducted, sampled every 6–8 h, and the residual glucose concentration were quantified. The measured profiles of residual glucose were fitted with Eqs. (2) and (3) to obtain S∞ , ˛, and k values (Table 1). By using the dilution rate (0.028 h−1 ) and the working volume of a chemostat vessel (1 liter), and by combining S∞ , ˛, k, and Eqs. (4)–(7), Fig. 2 is constructed, and the recommended T1 region is then identified (Table 2); or, simply followed the design instruction described in the third paragraph of Section 3.3 to determine T1 region (that is, 105.06–134.86 h in this case). In conjunction with Eq. (10) and dilution rate of 0.028 h−1 , the working volume of ageing vessel is 2.94–3.78 folds larger than that of a chemostat vessel. To design a fermentation process having equal-sized fermenters, the estimated T1 is 36 h (refer to Eq. (10)). This means that to reduce T1 period during continuous VHG fermentation, a multiple ageing vessel configuration is required. By referring to Table 2 that is generated by using Eq. (12), five ageing vessels connected in parallel to a single chemostat vessel are selected. Then, according to Eqs. (4), (11), and (7), T2 , T3 and F are determined as 89.54 h, 54.45 h, and 0.018 l/h, respectively.
2
(S∞ ˛ − (N − 1)k) + 4˛(N − 1)kSin 2˛(N − 1)k (12)
2 for a two equal-sized ageing vessels conFor example, Tcri figuration is estimated by letting N in Eq. (12) to be two, and Eq. (6) is obtained. In connection with Eq. (10), D and VC , multi
3.6.2. Case 2: initial glucose concentration at ∼250 g/l Similar to the design procedure described in the case study above, the recommended T1 region is 35.27–42.34 h (Table 2), and the working volume ratio of ageing vessel to chemostat vessel is 0.99–1.19. This means that when carrying out ethanol fermentation with ∼250 g glucose/l in the feed stream, three equal sizes of fermenters are required; one for the chemostat operation, and two
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Table 2 Design parameters for multi equal-sized ageing vessels (with a dilution rate of 0.028 h−1 ). Number of ageing vessels
303 ± 4.24 (g/l) Recommended T1 region (h)
2 3 4 5 6 7
105.06–134.86 59.57–76.26 42.52–54.06 33.73–42.87 28.20–35.85 24.35–30.98
255 ± 2.56 (g/l) Recommended VA /VC 2.94–3.78 1.67–2.13 1.19–1.51 0.94–1.20 0.79–1.00 0.68–0.87
35.27–42.34 23.28–28.20 18.05–22.01 14.97–18.35 12.90–15.88 11.39–14.08
S∞
3.6.3. Case 3: initial glucose concentration at ∼200 g/l Under 203 ± 2.94 g glucose/l condition (Table 2), the highest recommended filling time is 3.72 h and the size of ageing vessel is one tenth of the chemostat vessel. A short filling time translates to a frequent switching between ageing vessels, incurring a high operating cost. A small ageing vessel implies that only a small amount of fermentation broth is available for the subsequent distillation unit. As a result, a high energy cost is expected. Referring to Fig. 1 under ∼200 g glucose/l condition, the residual glucose in an ageing vessel is nearly utilized during the course of filling period. This means that no ageing time is required in an ageing vessel during bio-ethanol production when the chemostat device is operated at a dilution rate of 0.028 h−1 with an initial glucose concentration of ∼200 g/l. The ageing vessel in the proposed fermentation configuration serves as a surge tank for temporary storage of spent fermentation broth before distillations. One can then conclude that the multi ageing vessel configuration is not suitable for a chemostat feeding with low initial glucose concentration.
ST1 ST2 T1 T1,up T1,low T2 T2∞ T3 VA VC 2 Tcri
4. Conclusions Ageing vessel design results in complete glucose utilization during fermentation stage of ethanol operation. The developed ageing vessel design procedure is based on the measured residual glucose profile in an ageing vessel. The profile consists of fedbatch consumption pattern during the filling time period followed by batch consumption pattern during the ageing time period. Two respective models were proposed and could predict patterns with high accuracy. Integrating predicted results with mass balance principle, several ageing vessel design criteria were derived for the selection of number, size, and operation of ageing vessels. Case studies were illustrated to assist the reader to familiarize with the design procedure. The continuous VHG fermentation design criteria can be easily adopted by the fuel alcohol producers where batch fermentation is their current operation. Appendix A. Nomenclature
m ¯ m mpred N
Recommended VA /VC
Recommended region (h)
for the ageing vessels. At a dilution rate of 0.028 h−1 , T1 becomes 36 h. The estimated T2 , T3 , and F are 23.83 h, 12.16 h, and 0.083 l/h, respectively.
D F k
203 ± 2.94 (g/l)
dilution rate (h−1 ) discharge rate of an ageing vessel (l/h) overall specific glucose consumption rate during T2 period (h−1 ) metabolite concentration (g/l) average metabolite concentration (g/l) predicted metabolite concentration (g/l) number of ageing vessel
Sin
N Tcri
˛
0.99–1.19 0.65–0.79 0.51–0.62 0.42–0.51 0.36–0.44 0.32–0.39
Recommended T1 region (h) 3.10–3.72 2.04–2.48 1.57–1.92 1.30–1.60 1.11–1.38 0.97–1.21
Recommended VA /VC 0.087–0.104 0.057–0.069 0.044–0.054 0.036–0.045 0.031–0.039 0.027–0.034
residual glucose concentration when T1 approaches to infinite (g/l) residual glucose concentration entering ageing vessel (g/l) residual glucose concentration after T1 period (g/l) residual glucose concentration after T2 period (g/l) feeding time for an ageing vessel (h) upper-bounded T1 (h) lower-bounded T1 (h) ageing time for an ageing vessel (h) constant ageing time derived from Eq. (5) (h) operating time for an ageing vessel (h) working volume for an ageing vessel (l) working volume for a chemostat vessel (l) minimal filling time for a two-ageing vessel configuration (h) minimal filling time for a N-ageing vessel configuration (h) overall specific glucose consumption rate during T1 period (h−1 )
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