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Continuous ethanol production using self-flocculating yeast in a cascade of fermentors
Enzyme and Microbial Technology 37 (2005) 634–640
Continuous ethanol production using self-flocculating yeast in a cascade of fermentors T.J. Xu, X.Q. Zhao, F.W. Bai ∗ Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, PR China Received 28 February 2005; received in revised form 11 April 2005; accepted 12 April 2005
Abstract Continuous ethanol fermentation using a self-flocculating yeast strain SPSC01 was examined in a four-stage tanks in series fermentation system with a total working volume of 4000 ml. The first tank was designated for seed cultivation and the others for ethanol fermentation. Two-stage enzymatic hydrolysate of corn powder containing total sugar of 120 g l−1 , supplemented with 2.0 g l−1 (NH4 )2 HPO4 and K2 HPO4 , respectively, was used as seed culture substrate and fed into the seed fermentor at the dilution rate of 0.017 h−1 . Meanwhile, the hydrolysate containing total sugar of 220 g l−1 , supplemented with 0.5 g l−1 (NH4 )2 HPO4 and 0.15 g l−1 K2 HPO4 was used as ethanol fermentation substrate and fed into the second tank at the dilution rates of 0.017, 0.025, 0.033, 0.040 and 0.050 h−1 , respectively. Steady states were observed when the fermentation system was operated at the dilution rates of 0.017, 0.025, 0.033 and 0.050 h−1 . However, when the fermentation system was operated at the dilution rate of 0.040 h−1 , unsteady states and oscillations that characterized by big fluctuations of residual sugar, ethanol and biomass were detected. Compared with freely suspended yeast cell ethanol fermentation system, this self-flocculating yeast strain could partly self-immobilize within the tanks. The effluent containing 95.6 g l−1 ethanol, 1.5 g l−1 reducing sugar and 2.5 g l−1 total sugar was steadily produced when the fermentation system was operated at the dilution rate of 0.033 h−1 . The ethanol productivity of 3.44 g l−1 h−1 was achieved, which almost doubled that of freely suspended yeast cell system when the same levels of ethanol and residual sugars were reached. The conversion yield of ethanol to total sugar in the media was calculated to be 0.465, equivalent to 91.1% of its theoretical value of 0.511. © 2005 Elsevier Inc. All rights reserved. Keywords: Self-flocculating yeast; Ethanol fermentation; Multi-stage tanks in series; Steady-state; Unsteady-state; Oscillation
1. Introduction The gradual depletion of crude oil and the biological environmental deterioration resulted from the over consumption of petroleum-derived transportation fuels have garnered great attentions again, which makes it urgent to develop alternatives that are both renewable and environmentally friendly. Bioethanol, produced from renewable biomass such as sugar and starch at present and maybe lignocellulosic materials in the future, is believed to be one of these alternatives. Currently, freely suspended yeast cells are widely used in ethanol fermentation plants. Free yeast cells leave fermentors with effluent during continuous operations. Therefore, high yeast cell densities cannot be achieved within fermen∗
Corresponding author. Tel.: +86 411 8470 6308; fax: +86 411 8470 6329. E-mail address: [email protected] (F.W. Bai).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.04.005
tors and ethanol productivities are inevitably low unless yeast cells are separated by centrifugation and recycled thereafter. High capital investment for centrifuges and energy costs for operating this equipment greatly hinder the applications of centrifuges in ethanol fermentation plants, especially in developing countries where energy cost is relatively high and large centrifuges need to be imported. Flocculating of yeast cells, usually spontaneously, has been widely investigated and used for separating yeast cells from beer in brewery industry [1]. Ethanol fermentations using self-flocculating yeast strains that aimed at increasing yeast cell densities within fermentors and improved ethanol productivities of fermentation systems were also reported. Different fermentor configurations, including air-lift fermentor [2], single packed column fermentors [3–5] and two-stage packed column fermentors in series coupled with settlers [6] and without settlers [7], CO2 suspended-bed fermentor
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
Nomenclature D P Pm P1 P2
q
Sr Srm St S0,1 S0,2 t V1 V2 V Xin Xout Y
dilution rate of fermentation system, h−1 ethanol in the effluent overflowed from the tanks, g l−1 ethanol in the effluent overflowed from the last tank, g l−1 ethanol in the seed culture, 46.0 g l−1 ethanol in the effluent overflowed from the last tank and produced from Medium B, g l−1 , 1 V1 which was calculated by P2 = Pm V V−P 2 average ethanol productivity of fermentation system, g l−1 h−1 , which can be calculated by q = DP residual reducing sugar in the effluent overflowed from the tanks, g l−1 residual reducing sugar in the effluent overflowed from the last tank, g l−1 residual total sugar in the effluent overflowed from the last tank, g l−1 total sugar in Medium A, g l−1 total sugar in Medium B, g l−1 average fermentation time, equivalent to D−1 , h volumetric flowrate of Medium A fed into the seed fermentor, ml h−1 volumetric flowrate of Medium B fed into the second tank, ml h−1 volumetric flowrate of effluent overflowed from the last tank, V ∼ = V1 + V2 , ml h−1 biomass concentration within the tanks, g (d.w.) l−1 biomass concentration in the effluent overflowed from the tanks, g (d.w.) l−1 ethanol conversion yield based on sugar concentration in the medium, which can be calculated by Y = V1 S0, 1VP +V2 S0, 2
coupled with a buffer and a separation tank [8] or only with a separation tank for CO2 to be separated and recycled [9], were developed, correspondingly. However, most currently available self-flocculating yeast strains are not desirable for industrial applications because of their poor ethanol tolerances, narrow sugar utilization spectrum and low conversion yields. Meanwhile, the column fermentors previously developed for self-flocculating yeast strains are quite different from tanks widely used in industry and can not be scaled up to their industrial scales for ethanol production, in which fermentors with working volume of hundreds, even thousands, of cubic meters were required. In this study, a fermentation system composed of fourstage tanks in series and with a total working volume of 4000 ml was established, and continuous ethanol fermentation using a self-flocculating yeast strain bred by proto-
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plast fusion technology from an industrial ethanol fermentation strain, Saccharomyces cerevisiae and a self-flocculating strain, Schizosaccharomyces pombe, was investigated. The aim of this work was to examine the feasibility of such a self-flocculating yeast cell ethanol fermentation system to be applied in industrial ethanol production and evaluate its advantages over conventional ethanol fermentation technologies using freely suspended yeast cells.
2. Materials and methods 2.1. Microorganism and its pre-culture The strain SPSC01, a self-flocculating fusant bred by the Department of Bioscience and Bioengineering, Dalian University of Technology, PR China from S. cerevisiae and S. pombe, was kindly provided by our laboratory technician, Mrs. Ning Li and is also available for research at Chinese General Microbiological Culture Collection Center (CGMCC) with a deposited number of 0587. This selfflocculating fusant has been examined to be stable and has identical ethanol fermentation performances with its parent strain, S. cerevisiae K2 (CGMCC 2.607), an industrial ethanol fermentation strain widely used in China. Pre-culture of this self-flocculating yeast strain was carried out in 250 ml Erlenmeyer flasks containing 100 ml sterilized medium composed of (g l−1 ) glucose, 30; yeast extract, 5 and peptone, 3. After inoculation, the rotary shaker speed and temperature were set at 150 rpm and 30 ◦ C. The self-flocculating of yeast cells occurred after the cultivation was initiated and the flocs in size of millimeter scale could be observed within several hours. Normally, overnight culture could be used to inoculate fermentors. 2.2. Media for seed tank culture and ethanol fermentation Corn powder that had been de-germed, dry milled, and passed through 20 mesh screen was donated by a local distiller and was hydrolyzed by two-stage enzymatic hydrolysis in a standard stirring tank with a working volume of 15 l. The corn powder mixed with tap water at the ratio of 1:2. ␣-Amylase (commercial ␣-amylase, 20,000 u ml−1 , donated by Novozymes, China) was added into the slurry at 0.05% (v/w) of corn powder when the slurry was heated to about 55 ◦ C by steam injected into the tank’s jacket. The slurry was continuously heated to 95 ◦ C and liquefied for 90 min. When liquefaction was completed, the mash was cooled to 65 ◦ C by pumping tap water into the jacket. pH was adjusted to 4.5 by adding H2 SO4 . Glucoamylase (commercial glucoamylase, 100,000 u ml−1 , donated by Novozymes China) was added into the mash at 0.15% (v/w) of corn powder. After overnight (10–12 h) saccharification, the dextrose equivalent (DE) value, percentage of glucose to total sugar in the mash, could reach 90 or higher, which was required
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T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
by self-flocculating yeast cell ethanol fermentation system. The residue in the mash was removed by filtration and the liquor hydrolysate harvested was used for both seed culture and ethanol fermentation. The hydrolysate diluted by tap water to total sugar of 120 g l−1 , supplemented with 2.0 g l−1 (NH4 )2 HPO4 and K2 HPO4 , respectively, named Medium A, was used for seed culture. Meanwhile, the hydrolysate diluted by tap water to total sugar of 220 g l−1 , supplemented with 0.5 g l−1 (NH4 )2 HPO4 and 0.15 g l−1 K2 HPO4 , named Medium B, was used for ethanol fermentation. These medium compositions are commonly used by distillers in northeast China where corn is used as raw material for ethanol production. One thousand milliliters flasks were used as the medium storage tanks for Medium A, in which 600 ml Medium A was contained. The flasks were sterilized at 121 ◦ C for 15 min, then cooled to room temperature by immerging into tap water sink. Three thousand milliliters flasks were used as the medium storage tanks for Medium B. The empty flasks were sterilized at 121 ◦ C for 15 min. Then, 2500 ml Medium B was filled up and sterilized at 110 ◦ C for 15 min. After sterilization, the flasks were immerged in tap water to cool the contents to room temperature. Glucoamylase that had been filtered through a 0.2 mm micromembrane to remove potentially contaminated microbes was added into Medium B at 0.05% (v/v) to compensate the activity loss of glucoamylase resulted from sterilization so that residual sugar in the final effluent could reach its minimum and ethanol conversion yield based on the sugar in the medium could reach the level required by industry. 2.3. Fermentation system As ethanol fermentation is a well-known product inhibition process, tanks in series fermentation systems are widely used in industry. A small laboratory fermentation system composed of four-stage tanks in series, as illustrated in Fig. 1, was established to simulate industrial situations. Four double jacket glass tanks (∼110 mm in both diameter and height), each with a working volume of 1000 ml, were stirred by magnetic stirring (φ 8 mm × 10 mm stirrers). The stirring speed was controlled at 600–800 rpm to guarantee the yeast flocs being suspended homogeneously. The first tank was designated for seed culture, and the remained for ethanol fermentation. Tanks contained 850 ml Medium A were sterilized at 121 ◦ C for 15 min, then cooled to room temperature and inoculated independently, each with 50 ml yeast flocs prepared through flask cultivation. After inoculation, batch cultivation was initiated. The temperature for the self-flocculating yeast cultivation was controlled at 30 ± 0.5 ◦ C by pumping thermowater into the tanks’ jackets. pH was controlled at 4.2 ± 0.2 by automatically adding ammonia water into the tanks. Air was aerated into the
Fig. 1. Process diagram for continuous ethanol production using selfflocculating yeast in a cascade of fermentors: (1) air compressor, (2) air flowmeters, (3) filters, (4) substrate storage tanks, (5) peristaltic pumps, (6) tank for yeast seed culture, (7) tanks for ethanol fermentation, each with a working volume of 1000 ml, (8) temperature controlling units, (9) pH controlling units, (10) thermostat water inlets, (11) thermostat water outlets.
tanks at the flowrate of 0.05 vvm at this stage to stimulate yeast cell growth. When the residual sugars within the tanks were detected to be lower than 1.0 g l−1 , the first tank was switched from batch to continuous operation by feeding Medium A at the dilution rate of 0.017 h−1 . The seed culture overflowed from the first tank flowed into the second tank. Meanwhile, continuous ethanol fermentations were also switched on by feeding Medium B into the second tank at the dilution rates of 0.017, 0.025, 0.033, 0.040 and 0.050 h−1 (based on the total working volume of the three tanks), respectively. After ethanol fermentation was initiated, the temperature of the fermentation system was controlled at 33 ± 0.5 ◦ C. Aerations for the second, third and fourth tanks were interrupted during ethanol fermentation. 2.4. Analytical methods After system equilibrium, samples were taken daily. Ethanol was analyzed by gas chromatography (Agilent 6890A, USA. Solid phase: cross-linked polyethylene glycol; carrier gas: nitrogen; 90 ◦ C isothermol capillary column; injection temperature 160 ◦ C; flame ionization detector temperature 230 ◦ C; Agilent ChemStation Data Analysis System) and isopropanol was used as an internal standard. Both reducing and total sugar concentrations were analyzed by Fehling titration, but the samples for total sugar analysis should be completely hydrolyzed by HCl before Fehling titration (Chinese National Standard for Sugar Analysis GB/T 6194-86). A dry weight method was used to measure yeast cell biomass concentrations, whereby 3× 1 ml samples were centrifuged, washed three times by de-ionized water, dried at 85 ◦ C for 24 h and weighted. Yeast cell viability loss was qualitatively evaluated by routine methylene blue stain technique.
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
3. Results and discussion 3.1. Fermentation system running states For the seed tank, steady state was observed when it was operated at the dilution rate of 0.017 h−1 . Over two month running, its reducing sugar, total sugar and ethanol concentrations were maintained at the average levels of 3.0, 5.5 and 46.1 g l−1 with very small fluctuations, completely within the normal analytical errors. For the fermentation system, five different dilution rates were selected based on our expertise and suggestions from industry. Fermentation results were examined over two month continuous running after the system reached its equilibrium, which was judged by comparing the sugar concentration in the media fed into the system with the residual sugar level and ethanol produced in the effluent overflowed from the last tank. Fig. 2 illustrated the fermentation profiles and the average fermentation results were summarized in Table 1. The lowest dilution rate was 0.017 h−1 , and its corresponding average fermentation time was about 60 h, almost the
Fig. 2. Fermentation parameters vs. run time at different dilution rates. (0–7 days, D = 0.017 h−1 ; 8–17 days, D = 0.033 h−1 ; 18–27 days, D = 0.050 h−1 ; 28–48 days, D = 0.040 h−1 ; 49–62 days, D = 0.025 h−1 ).
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same as that required by starch material ethanol fermentation plants in which freely suspended yeast cells are used. Steady states were observed for residual sugars, ethanol and biomass over a period of one week. The average residual sugar levels of 0.5 g l−1 for reducing sugar and 1.5 g l−1 for total sugar indicated that ethanol fermentation potential was not fully exploited at this dilution rate. The lower ethanol concentration of 75.0 g l−1 was contributed to the strong dilution effect of the seed culture on the fermentation broth. When the dilution rate was increased to 0.033 h−1 , the residual sugar levels increased slightly, to 1.5 g l−1 for reducing sugar and 2.5 g l−1 for total sugar, which did not exceed the criterions of 2.5 g l−1 for reducing sugar and 3.5 g l−1 for total sugar required by industry, but the ethanol productivity of the fermentation system increased to 3.44 from 1.78 g l−1 h−1 . The average ethanol concentration also increased to 95.6 g l−1 or 12.1% (v/v), higher than currently industrial acceptable level of 11.5% (v/v). The fermentation system was still at its steady state. In order to fully exploit this fermentation system’s ethanol fermentation capacity, we continuously increased the dilution rate to 0.05 h−1 . The ethanol productivity increased about 16% compared with that for the dilution rate of 0.033 h−1 and the fermentation system was still at its steady states, but the residual sugar concentrations increased rapidly, to 27.0 g l−1 for reducing sugar and 30.0 g l−1 for total sugar, too high to be tolerated by industry. Therefore, we decreased the dilution rate to 0.04 h−1 and expected to reach a promise between ethanol productivity and residual sugar levels. However, we were surprised when unsteady behavior was observed for residual sugars, ethanol, and biomass concentrations for all three tanks, as illustrated in Fig. 2. As long as 20 days were maintained at this special dilution rate so that some basic information related to these unsteady states could be collected. The trough and peak values for reducing sugar were 49.0 and 114.0 g l−1 for the second tank, 21.0 and 78.0 g l−1 for the third tank, 6.8 and 32.0 g l−1 for the fourth tank. The fluctuation amplitudes for reducing sugar were calculated to be about ±40, ±55 and ±65% based on their average levels of 83.2, 50.6 and 19.2 g l−1 for the second, third and fourth tanks, respectively. Similar fluctuation profiles also presented for ethanol and biomass. Furthermore, these fluctuations were observed to be symmetrical oscillations up and down their average levels and seemed to be sustainable. After 20 days’ running, the dilution rate was decreased to 0.025 h−1 . We expected that the steady states observed previously to be restored and the experimental data fully validated our predictions. Parameter oscillations were reported previously when we worked on freely suspended yeast cell ethanol fermentation system. The lag response of S. cerevisiae to its high ethanol stress was believed to be one of the main factors that incited these oscillations, which was validated by comparing the data collected from ethanol fermentations using ethanologen bacterium, Zymomonas mobilis and the yeast, S. cerevisiae [10]. In this study, the dilution rate, one of the most
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
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Table 1 Self-flocculating yeast cell ethanol fermentation results D (h−1 )
Pm (g l−1 )
Srm (g l−1 )
St (g l−1 )
P2 (g l−1 )
q (g l−1 h−1 )
0.017 0.025 0.033 0.040 0.050
75.0 93.0 95.6 91.2 76.1
0.50 1.00 1.50 9.10 27.0
1.50 2.25 2.50 11.0 30.0
104.0 103.7 104.1 97.6 79.5
1.78 2.59 3.44 3.90 3.98
important operating parameters, also incited oscillations for this self-flocculating yeast cell ethanol fermentation system. During continuous cultures, the specific growth rates must be balanced by the dilution rates at their steady states, therefore, the impact of the dilution rate on the unsteady states and oscillations of this ethanol fermentation system probably is the specific grow rate of this self-flocculating yeast strain other than the dilution rate. Unsteady states and oscillations are expected to occur when the dilution rate coincides with the special specific growth rate that incites oscillations. 3.2. Yeast cell self-immobilization One of the biggest advantages using self-flocculating yeast strains is that higher yeast cell densities can be achieved within the designed fermentors. The average biomass concentrations within the tanks and in the effluent overflowed from the tanks were summarized and compared in Table 2. Table 2 illustrates that the biomass concentrations within the tanks were much higher than those in the effluent overflowed from the tanks. The biomass concentrations in the effluent overflowed from the tanks were estimated to be only 25% of those within the tanks, which indicated the yeast cells spontaneously immobilized. Compared with yeast cell immobilization technologies developed previously [11], no any inert carriers were consumed for this yeast cell selfimmobilization system, which made this technology potentially more economically competitive. Yeast cell viability was quantitatively evaluated by routine methylene blue stain technique and it was found that the percentage of viable cells within the tanks was higher than that overflowed from the tanks, which indicated viability lost cells could be automatically eliminated. Ethanol, a well-known primary metabolite of yeast cells, its formation is tightly coupled with the growth of yeast cells during ethanol fermentation. Therefore, the over produced yeast cells must be removed in time from immobilized yeast cell systems in order to maintain the fermentation systems to be run for a long period of time, and this requirement was satisfied spontaneously for our self-flocculating yeast ethanol fermentation system. Yeast cell concentrations detected both within the tanks and in the effluent overflowed from the tanks decreased slightly as the effluent flowed through the tanks, which indicated the lysis of yeast cells happened, especially within the third and fourth tanks in which high ethanol concentrations
and poor nutrition conditions presented. Microscope observations supported this conclusion. 3.3. Impact of glucoamylase added on residual sugars and contamination prevention It is a normal practice in industry to feed the fresh mash or hydrolysate directly into fermentors without sterilization so that the activity of glucoamylase added for saccharification can be partly maintained and residual sugar in the final effluent can reach its minimum, normally below 2.5 g l−1 for reducing sugar and 3.5 g l−1 for total sugar. When unsterilized hydrolysate was directly fed into our ethanol fermentation system, contamination occurred and conversion yield of ethanol to sugar was decreased to 0.43–0.44, only 84–86% of its theoretical value of 0.511. In ethanol fermentation industry, the fresh mash is fed into fermentors immediately after it is cooled to fermentation temperature of 35 ◦ C from its saccharification temperature of 65 ◦ C. Moreover, lower pH and anaerobic environments within fermentors are unfavorable for contaminated microorganisms to growth. Therefore, contamination can be effectively prevented and conversion yield of ethanol to sugar can reach as high as 90–92% of its theoretical value. For our laboratory scale fermentation system, it is impractical to prepare hydrolysate continuously as industry does. We strictly controlled sterilization temperature and time when Medium B was sterilized to avoid sugar loss and other by-product formation. After sterilization, the hydrolysate was immediately cooled to room temperature and glucoamylase filtered by 0.2 mm micromembrane to remove potentially contaminated microbes was added at 0.05% (v/v) to compensate the activity loss of glucoamylase resulted from sterilization. It has been proven this strategy was very effective. The residual sugars in the final effluent were decreased to 1.5 g l−1 for reducing sugar and 2.5 g l−1 for total sugar compared with 4.5 g l−1 for reducing sugar and 8.4 g l−1 for total sugar when sterilized hydrolysate without glucoamylase was fed. The conversion yield of ethanol to sugar was calculated to be as high as 91.1% of its theoretical value, which is acceptable in industry. 3.4. Advantages over conventional ethanol fermentation technologies Table 3 illustrates the comparison of the ethanol fermentation performances between this self-flocculating yeast strain and K2.
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
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Table 2 Comparisons of yeast cell concentrations (g (d.w.) l−1 ) D (h−1 )
0.017 0.025 0.033 0.040 0.050
Second tank
Third tank
Fourth tank
Xin
Xout
Xout /Xin
Xin
Xout
Xout /Xin
Xin
Xout
Xout /Xin
14.8 13.8 12.4 11.0 9.1
3.6 3.4 3.2 3.1 2.3
0.243 0.246 0.258 0.282 0.252
13.2 12.9 11.2 10.5 7.4
3.4 3.2 2.9 2.8 2.2
0.258 0.248 0.258 0.266 0.297
12.1 11.7 10.6 9.1 6.5
3.2 2.8 2.7 2.6 1.8
0.264 0.239 0.255 0.286 0.277
It can be seen that when the same levels of ethanol and residual sugars in the effluent were reached, the average ethanol fermentation time required by self-flocculating yeast strain was only 50% of that required by K2, and the average ethanol productivity doubled, correspondingly, because of higher yeast cell concentrations within the fermentors. This indicates fermentor capital investment for large-scale plant construction can be saved significantly. The synchronous increases of yeast cell concentrations and ethanol productivity also indicated yeast cell viability loss resulted from selfflocculating was minor. Currently, yeast cell recovery in ethanol fermentation industry is realized by centrifugation. The excellent settling performance of this self-flocculating yeast strain made it quite practical to recover yeast cells by settlement instead of centrifugation. This engineering advantage has been fully exploited in our pilot plant at BBCA, Anhui Province, East China, in which 36 t ethanol was produced daily using this self-flocculating yeast strain. Convention yield is the most important factor that the distillers care about because over 60% of ethanol production cost in China is coming from raw material consumption. From the data showed in Table 3, the yields based on sugar concentrations in the media were almost same for SPSC01 and K2, which further guarantees this self-flocculating yeast strain to be suitable for large scale industry application. An ethanol fermentation plant with a fuel ethanol production capacity of 200,000 t per year is under construction at BBCA, Anhui Province, East China, and expected to be put into operation at the end of 2006.
4. Conclusions Continuous ethanol fermentation using self-flocculating yeast strain SPSC01 in a multi-stage tanks in series fermentation system was experimentally proven to be practical. Yeast cells partly self-immobilized within the tanks. Steady states were observed for most experimental running, but unsteady states and oscillations of residual sugar, ethanol and biomass concentrations occurred for one designated dilution rate, in our case, 0.04 h−1 . For self-flocculating yeast cell ethanol fermentation, its ethanol productivity almost doubled that of freely suspended yeast cell ethanol fermentation system when the same levels of residual sugar and ethanol concentrations were reached, which indicates tank capital investment for large scale plant construction can be saved significantly. Moreover, self-flocculating yeast cells can be easily recovered from the effluent by settlement instead of by centrifugation that is widely used in freely suspended yeast ethanol fermentation system, centrifuge capital investment and running energy can be also saved.
Acknowledgements Funding for this research was provided from the HiTech R&D Program of China (863) with a Grant no. 2002AA647060 and National Key Scientific Research Program of China with a Grant no. 2004BA713B08.
References Table 3 Comparisons of the ethanol fermentation performances of SPSC01 with K2 Fermentation results
K2a
SPSC01
Pm (g l−1 ) P2 (g l−1 ) Srm (g l−1 ) St (g l−1 ) Y Xin (g (d.w.) l−1 ) D (h−1 ) t (h) q (g l−1 h−1 ) Yeast recovery method
95.2 103.5 2.5 3.5 0.462 3.0–5.0 0.017 60 1.76 Settlement
95.6 104.1 1.5 2.5 0.466 7.0–14.0 0.033 30 3.44 Centrifugation
a Five hundred milliliters seed fermentor was used for K2 fermentation system to guarantee the ratio of seed culture to ethanol fermentation broth to be identical with that for SPSC01 fermentation system.
[1] Verstrepen KJ, Derdelinckx G, Verachtert H. Yeast flocculation: what brewers should know. Appl Microbiol Biotechnol 2003;61:197–205. [2] Bu’lock JD, Comberbach DM, Ghommidh C. A study of continuous ethanol production using a highly flocculent yeast in the gas lift tower fermentor. Chem Eng J 1984;29:9–24. [3] Gong CS, Chen LF. Ethanol production by a self-aggregating mutant of Saccharomyces uvarum. Biotechnol Bioeng Symp 1984;14:257–68. [4] Jones ST, Korus RA, Admassu W, Heimsch RC. Ethanol fermentation in a continuous tower fermentor. Biotechnol Bioeng 1984;16:742–7. [5] Admassu W, Korus RA. Ethanol fermentation with a flocculating yeast. Chem Eng J 1985;31:B1–8. [6] Kuriyama H, Ishibashi H, Miyagawa H, Kobayashi H, Mikami E. Optimization of two-stage continuous ethanol fermentation using flocculating yeast. Biotechnol Lett 1993;15:415–20.
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[10] Bai FW, Chen LJ, Anderson WA, Moo-Young M. Parameter oscillations in a very high gravity medium continuous ethanol fermentation and their attenuation on a multi-stage packed column bioreactor system. Biotechnol Bioeng 2004;88:558–66. [11] Nagashima M, Azuma M, Noguchi S, Inuzuka K, Samejima H. Continuous ethanol fermentation using immobilized yeast cells. Biotechnol Bioeng 1984;16:992–7.
Continuous ethanol production using self-flocculating yeast in a cascade of fermentors T.J. Xu, X.Q. Zhao, F.W. Bai ∗ Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, PR China Received 28 February 2005; received in revised form 11 April 2005; accepted 12 April 2005
Abstract Continuous ethanol fermentation using a self-flocculating yeast strain SPSC01 was examined in a four-stage tanks in series fermentation system with a total working volume of 4000 ml. The first tank was designated for seed cultivation and the others for ethanol fermentation. Two-stage enzymatic hydrolysate of corn powder containing total sugar of 120 g l−1 , supplemented with 2.0 g l−1 (NH4 )2 HPO4 and K2 HPO4 , respectively, was used as seed culture substrate and fed into the seed fermentor at the dilution rate of 0.017 h−1 . Meanwhile, the hydrolysate containing total sugar of 220 g l−1 , supplemented with 0.5 g l−1 (NH4 )2 HPO4 and 0.15 g l−1 K2 HPO4 was used as ethanol fermentation substrate and fed into the second tank at the dilution rates of 0.017, 0.025, 0.033, 0.040 and 0.050 h−1 , respectively. Steady states were observed when the fermentation system was operated at the dilution rates of 0.017, 0.025, 0.033 and 0.050 h−1 . However, when the fermentation system was operated at the dilution rate of 0.040 h−1 , unsteady states and oscillations that characterized by big fluctuations of residual sugar, ethanol and biomass were detected. Compared with freely suspended yeast cell ethanol fermentation system, this self-flocculating yeast strain could partly self-immobilize within the tanks. The effluent containing 95.6 g l−1 ethanol, 1.5 g l−1 reducing sugar and 2.5 g l−1 total sugar was steadily produced when the fermentation system was operated at the dilution rate of 0.033 h−1 . The ethanol productivity of 3.44 g l−1 h−1 was achieved, which almost doubled that of freely suspended yeast cell system when the same levels of ethanol and residual sugars were reached. The conversion yield of ethanol to total sugar in the media was calculated to be 0.465, equivalent to 91.1% of its theoretical value of 0.511. © 2005 Elsevier Inc. All rights reserved. Keywords: Self-flocculating yeast; Ethanol fermentation; Multi-stage tanks in series; Steady-state; Unsteady-state; Oscillation
1. Introduction The gradual depletion of crude oil and the biological environmental deterioration resulted from the over consumption of petroleum-derived transportation fuels have garnered great attentions again, which makes it urgent to develop alternatives that are both renewable and environmentally friendly. Bioethanol, produced from renewable biomass such as sugar and starch at present and maybe lignocellulosic materials in the future, is believed to be one of these alternatives. Currently, freely suspended yeast cells are widely used in ethanol fermentation plants. Free yeast cells leave fermentors with effluent during continuous operations. Therefore, high yeast cell densities cannot be achieved within fermen∗
Corresponding author. Tel.: +86 411 8470 6308; fax: +86 411 8470 6329. E-mail address: [email protected] (F.W. Bai).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.04.005
tors and ethanol productivities are inevitably low unless yeast cells are separated by centrifugation and recycled thereafter. High capital investment for centrifuges and energy costs for operating this equipment greatly hinder the applications of centrifuges in ethanol fermentation plants, especially in developing countries where energy cost is relatively high and large centrifuges need to be imported. Flocculating of yeast cells, usually spontaneously, has been widely investigated and used for separating yeast cells from beer in brewery industry [1]. Ethanol fermentations using self-flocculating yeast strains that aimed at increasing yeast cell densities within fermentors and improved ethanol productivities of fermentation systems were also reported. Different fermentor configurations, including air-lift fermentor [2], single packed column fermentors [3–5] and two-stage packed column fermentors in series coupled with settlers [6] and without settlers [7], CO2 suspended-bed fermentor
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
Nomenclature D P Pm P1 P2
q
Sr Srm St S0,1 S0,2 t V1 V2 V Xin Xout Y
dilution rate of fermentation system, h−1 ethanol in the effluent overflowed from the tanks, g l−1 ethanol in the effluent overflowed from the last tank, g l−1 ethanol in the seed culture, 46.0 g l−1 ethanol in the effluent overflowed from the last tank and produced from Medium B, g l−1 , 1 V1 which was calculated by P2 = Pm V V−P 2 average ethanol productivity of fermentation system, g l−1 h−1 , which can be calculated by q = DP residual reducing sugar in the effluent overflowed from the tanks, g l−1 residual reducing sugar in the effluent overflowed from the last tank, g l−1 residual total sugar in the effluent overflowed from the last tank, g l−1 total sugar in Medium A, g l−1 total sugar in Medium B, g l−1 average fermentation time, equivalent to D−1 , h volumetric flowrate of Medium A fed into the seed fermentor, ml h−1 volumetric flowrate of Medium B fed into the second tank, ml h−1 volumetric flowrate of effluent overflowed from the last tank, V ∼ = V1 + V2 , ml h−1 biomass concentration within the tanks, g (d.w.) l−1 biomass concentration in the effluent overflowed from the tanks, g (d.w.) l−1 ethanol conversion yield based on sugar concentration in the medium, which can be calculated by Y = V1 S0, 1VP +V2 S0, 2
coupled with a buffer and a separation tank [8] or only with a separation tank for CO2 to be separated and recycled [9], were developed, correspondingly. However, most currently available self-flocculating yeast strains are not desirable for industrial applications because of their poor ethanol tolerances, narrow sugar utilization spectrum and low conversion yields. Meanwhile, the column fermentors previously developed for self-flocculating yeast strains are quite different from tanks widely used in industry and can not be scaled up to their industrial scales for ethanol production, in which fermentors with working volume of hundreds, even thousands, of cubic meters were required. In this study, a fermentation system composed of fourstage tanks in series and with a total working volume of 4000 ml was established, and continuous ethanol fermentation using a self-flocculating yeast strain bred by proto-
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plast fusion technology from an industrial ethanol fermentation strain, Saccharomyces cerevisiae and a self-flocculating strain, Schizosaccharomyces pombe, was investigated. The aim of this work was to examine the feasibility of such a self-flocculating yeast cell ethanol fermentation system to be applied in industrial ethanol production and evaluate its advantages over conventional ethanol fermentation technologies using freely suspended yeast cells.
2. Materials and methods 2.1. Microorganism and its pre-culture The strain SPSC01, a self-flocculating fusant bred by the Department of Bioscience and Bioengineering, Dalian University of Technology, PR China from S. cerevisiae and S. pombe, was kindly provided by our laboratory technician, Mrs. Ning Li and is also available for research at Chinese General Microbiological Culture Collection Center (CGMCC) with a deposited number of 0587. This selfflocculating fusant has been examined to be stable and has identical ethanol fermentation performances with its parent strain, S. cerevisiae K2 (CGMCC 2.607), an industrial ethanol fermentation strain widely used in China. Pre-culture of this self-flocculating yeast strain was carried out in 250 ml Erlenmeyer flasks containing 100 ml sterilized medium composed of (g l−1 ) glucose, 30; yeast extract, 5 and peptone, 3. After inoculation, the rotary shaker speed and temperature were set at 150 rpm and 30 ◦ C. The self-flocculating of yeast cells occurred after the cultivation was initiated and the flocs in size of millimeter scale could be observed within several hours. Normally, overnight culture could be used to inoculate fermentors. 2.2. Media for seed tank culture and ethanol fermentation Corn powder that had been de-germed, dry milled, and passed through 20 mesh screen was donated by a local distiller and was hydrolyzed by two-stage enzymatic hydrolysis in a standard stirring tank with a working volume of 15 l. The corn powder mixed with tap water at the ratio of 1:2. ␣-Amylase (commercial ␣-amylase, 20,000 u ml−1 , donated by Novozymes, China) was added into the slurry at 0.05% (v/w) of corn powder when the slurry was heated to about 55 ◦ C by steam injected into the tank’s jacket. The slurry was continuously heated to 95 ◦ C and liquefied for 90 min. When liquefaction was completed, the mash was cooled to 65 ◦ C by pumping tap water into the jacket. pH was adjusted to 4.5 by adding H2 SO4 . Glucoamylase (commercial glucoamylase, 100,000 u ml−1 , donated by Novozymes China) was added into the mash at 0.15% (v/w) of corn powder. After overnight (10–12 h) saccharification, the dextrose equivalent (DE) value, percentage of glucose to total sugar in the mash, could reach 90 or higher, which was required
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by self-flocculating yeast cell ethanol fermentation system. The residue in the mash was removed by filtration and the liquor hydrolysate harvested was used for both seed culture and ethanol fermentation. The hydrolysate diluted by tap water to total sugar of 120 g l−1 , supplemented with 2.0 g l−1 (NH4 )2 HPO4 and K2 HPO4 , respectively, named Medium A, was used for seed culture. Meanwhile, the hydrolysate diluted by tap water to total sugar of 220 g l−1 , supplemented with 0.5 g l−1 (NH4 )2 HPO4 and 0.15 g l−1 K2 HPO4 , named Medium B, was used for ethanol fermentation. These medium compositions are commonly used by distillers in northeast China where corn is used as raw material for ethanol production. One thousand milliliters flasks were used as the medium storage tanks for Medium A, in which 600 ml Medium A was contained. The flasks were sterilized at 121 ◦ C for 15 min, then cooled to room temperature by immerging into tap water sink. Three thousand milliliters flasks were used as the medium storage tanks for Medium B. The empty flasks were sterilized at 121 ◦ C for 15 min. Then, 2500 ml Medium B was filled up and sterilized at 110 ◦ C for 15 min. After sterilization, the flasks were immerged in tap water to cool the contents to room temperature. Glucoamylase that had been filtered through a 0.2 mm micromembrane to remove potentially contaminated microbes was added into Medium B at 0.05% (v/v) to compensate the activity loss of glucoamylase resulted from sterilization so that residual sugar in the final effluent could reach its minimum and ethanol conversion yield based on the sugar in the medium could reach the level required by industry. 2.3. Fermentation system As ethanol fermentation is a well-known product inhibition process, tanks in series fermentation systems are widely used in industry. A small laboratory fermentation system composed of four-stage tanks in series, as illustrated in Fig. 1, was established to simulate industrial situations. Four double jacket glass tanks (∼110 mm in both diameter and height), each with a working volume of 1000 ml, were stirred by magnetic stirring (φ 8 mm × 10 mm stirrers). The stirring speed was controlled at 600–800 rpm to guarantee the yeast flocs being suspended homogeneously. The first tank was designated for seed culture, and the remained for ethanol fermentation. Tanks contained 850 ml Medium A were sterilized at 121 ◦ C for 15 min, then cooled to room temperature and inoculated independently, each with 50 ml yeast flocs prepared through flask cultivation. After inoculation, batch cultivation was initiated. The temperature for the self-flocculating yeast cultivation was controlled at 30 ± 0.5 ◦ C by pumping thermowater into the tanks’ jackets. pH was controlled at 4.2 ± 0.2 by automatically adding ammonia water into the tanks. Air was aerated into the
Fig. 1. Process diagram for continuous ethanol production using selfflocculating yeast in a cascade of fermentors: (1) air compressor, (2) air flowmeters, (3) filters, (4) substrate storage tanks, (5) peristaltic pumps, (6) tank for yeast seed culture, (7) tanks for ethanol fermentation, each with a working volume of 1000 ml, (8) temperature controlling units, (9) pH controlling units, (10) thermostat water inlets, (11) thermostat water outlets.
tanks at the flowrate of 0.05 vvm at this stage to stimulate yeast cell growth. When the residual sugars within the tanks were detected to be lower than 1.0 g l−1 , the first tank was switched from batch to continuous operation by feeding Medium A at the dilution rate of 0.017 h−1 . The seed culture overflowed from the first tank flowed into the second tank. Meanwhile, continuous ethanol fermentations were also switched on by feeding Medium B into the second tank at the dilution rates of 0.017, 0.025, 0.033, 0.040 and 0.050 h−1 (based on the total working volume of the three tanks), respectively. After ethanol fermentation was initiated, the temperature of the fermentation system was controlled at 33 ± 0.5 ◦ C. Aerations for the second, third and fourth tanks were interrupted during ethanol fermentation. 2.4. Analytical methods After system equilibrium, samples were taken daily. Ethanol was analyzed by gas chromatography (Agilent 6890A, USA. Solid phase: cross-linked polyethylene glycol; carrier gas: nitrogen; 90 ◦ C isothermol capillary column; injection temperature 160 ◦ C; flame ionization detector temperature 230 ◦ C; Agilent ChemStation Data Analysis System) and isopropanol was used as an internal standard. Both reducing and total sugar concentrations were analyzed by Fehling titration, but the samples for total sugar analysis should be completely hydrolyzed by HCl before Fehling titration (Chinese National Standard for Sugar Analysis GB/T 6194-86). A dry weight method was used to measure yeast cell biomass concentrations, whereby 3× 1 ml samples were centrifuged, washed three times by de-ionized water, dried at 85 ◦ C for 24 h and weighted. Yeast cell viability loss was qualitatively evaluated by routine methylene blue stain technique.
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
3. Results and discussion 3.1. Fermentation system running states For the seed tank, steady state was observed when it was operated at the dilution rate of 0.017 h−1 . Over two month running, its reducing sugar, total sugar and ethanol concentrations were maintained at the average levels of 3.0, 5.5 and 46.1 g l−1 with very small fluctuations, completely within the normal analytical errors. For the fermentation system, five different dilution rates were selected based on our expertise and suggestions from industry. Fermentation results were examined over two month continuous running after the system reached its equilibrium, which was judged by comparing the sugar concentration in the media fed into the system with the residual sugar level and ethanol produced in the effluent overflowed from the last tank. Fig. 2 illustrated the fermentation profiles and the average fermentation results were summarized in Table 1. The lowest dilution rate was 0.017 h−1 , and its corresponding average fermentation time was about 60 h, almost the
Fig. 2. Fermentation parameters vs. run time at different dilution rates. (0–7 days, D = 0.017 h−1 ; 8–17 days, D = 0.033 h−1 ; 18–27 days, D = 0.050 h−1 ; 28–48 days, D = 0.040 h−1 ; 49–62 days, D = 0.025 h−1 ).
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same as that required by starch material ethanol fermentation plants in which freely suspended yeast cells are used. Steady states were observed for residual sugars, ethanol and biomass over a period of one week. The average residual sugar levels of 0.5 g l−1 for reducing sugar and 1.5 g l−1 for total sugar indicated that ethanol fermentation potential was not fully exploited at this dilution rate. The lower ethanol concentration of 75.0 g l−1 was contributed to the strong dilution effect of the seed culture on the fermentation broth. When the dilution rate was increased to 0.033 h−1 , the residual sugar levels increased slightly, to 1.5 g l−1 for reducing sugar and 2.5 g l−1 for total sugar, which did not exceed the criterions of 2.5 g l−1 for reducing sugar and 3.5 g l−1 for total sugar required by industry, but the ethanol productivity of the fermentation system increased to 3.44 from 1.78 g l−1 h−1 . The average ethanol concentration also increased to 95.6 g l−1 or 12.1% (v/v), higher than currently industrial acceptable level of 11.5% (v/v). The fermentation system was still at its steady state. In order to fully exploit this fermentation system’s ethanol fermentation capacity, we continuously increased the dilution rate to 0.05 h−1 . The ethanol productivity increased about 16% compared with that for the dilution rate of 0.033 h−1 and the fermentation system was still at its steady states, but the residual sugar concentrations increased rapidly, to 27.0 g l−1 for reducing sugar and 30.0 g l−1 for total sugar, too high to be tolerated by industry. Therefore, we decreased the dilution rate to 0.04 h−1 and expected to reach a promise between ethanol productivity and residual sugar levels. However, we were surprised when unsteady behavior was observed for residual sugars, ethanol, and biomass concentrations for all three tanks, as illustrated in Fig. 2. As long as 20 days were maintained at this special dilution rate so that some basic information related to these unsteady states could be collected. The trough and peak values for reducing sugar were 49.0 and 114.0 g l−1 for the second tank, 21.0 and 78.0 g l−1 for the third tank, 6.8 and 32.0 g l−1 for the fourth tank. The fluctuation amplitudes for reducing sugar were calculated to be about ±40, ±55 and ±65% based on their average levels of 83.2, 50.6 and 19.2 g l−1 for the second, third and fourth tanks, respectively. Similar fluctuation profiles also presented for ethanol and biomass. Furthermore, these fluctuations were observed to be symmetrical oscillations up and down their average levels and seemed to be sustainable. After 20 days’ running, the dilution rate was decreased to 0.025 h−1 . We expected that the steady states observed previously to be restored and the experimental data fully validated our predictions. Parameter oscillations were reported previously when we worked on freely suspended yeast cell ethanol fermentation system. The lag response of S. cerevisiae to its high ethanol stress was believed to be one of the main factors that incited these oscillations, which was validated by comparing the data collected from ethanol fermentations using ethanologen bacterium, Zymomonas mobilis and the yeast, S. cerevisiae [10]. In this study, the dilution rate, one of the most
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
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Table 1 Self-flocculating yeast cell ethanol fermentation results D (h−1 )
Pm (g l−1 )
Srm (g l−1 )
St (g l−1 )
P2 (g l−1 )
q (g l−1 h−1 )
0.017 0.025 0.033 0.040 0.050
75.0 93.0 95.6 91.2 76.1
0.50 1.00 1.50 9.10 27.0
1.50 2.25 2.50 11.0 30.0
104.0 103.7 104.1 97.6 79.5
1.78 2.59 3.44 3.90 3.98
important operating parameters, also incited oscillations for this self-flocculating yeast cell ethanol fermentation system. During continuous cultures, the specific growth rates must be balanced by the dilution rates at their steady states, therefore, the impact of the dilution rate on the unsteady states and oscillations of this ethanol fermentation system probably is the specific grow rate of this self-flocculating yeast strain other than the dilution rate. Unsteady states and oscillations are expected to occur when the dilution rate coincides with the special specific growth rate that incites oscillations. 3.2. Yeast cell self-immobilization One of the biggest advantages using self-flocculating yeast strains is that higher yeast cell densities can be achieved within the designed fermentors. The average biomass concentrations within the tanks and in the effluent overflowed from the tanks were summarized and compared in Table 2. Table 2 illustrates that the biomass concentrations within the tanks were much higher than those in the effluent overflowed from the tanks. The biomass concentrations in the effluent overflowed from the tanks were estimated to be only 25% of those within the tanks, which indicated the yeast cells spontaneously immobilized. Compared with yeast cell immobilization technologies developed previously [11], no any inert carriers were consumed for this yeast cell selfimmobilization system, which made this technology potentially more economically competitive. Yeast cell viability was quantitatively evaluated by routine methylene blue stain technique and it was found that the percentage of viable cells within the tanks was higher than that overflowed from the tanks, which indicated viability lost cells could be automatically eliminated. Ethanol, a well-known primary metabolite of yeast cells, its formation is tightly coupled with the growth of yeast cells during ethanol fermentation. Therefore, the over produced yeast cells must be removed in time from immobilized yeast cell systems in order to maintain the fermentation systems to be run for a long period of time, and this requirement was satisfied spontaneously for our self-flocculating yeast ethanol fermentation system. Yeast cell concentrations detected both within the tanks and in the effluent overflowed from the tanks decreased slightly as the effluent flowed through the tanks, which indicated the lysis of yeast cells happened, especially within the third and fourth tanks in which high ethanol concentrations
and poor nutrition conditions presented. Microscope observations supported this conclusion. 3.3. Impact of glucoamylase added on residual sugars and contamination prevention It is a normal practice in industry to feed the fresh mash or hydrolysate directly into fermentors without sterilization so that the activity of glucoamylase added for saccharification can be partly maintained and residual sugar in the final effluent can reach its minimum, normally below 2.5 g l−1 for reducing sugar and 3.5 g l−1 for total sugar. When unsterilized hydrolysate was directly fed into our ethanol fermentation system, contamination occurred and conversion yield of ethanol to sugar was decreased to 0.43–0.44, only 84–86% of its theoretical value of 0.511. In ethanol fermentation industry, the fresh mash is fed into fermentors immediately after it is cooled to fermentation temperature of 35 ◦ C from its saccharification temperature of 65 ◦ C. Moreover, lower pH and anaerobic environments within fermentors are unfavorable for contaminated microorganisms to growth. Therefore, contamination can be effectively prevented and conversion yield of ethanol to sugar can reach as high as 90–92% of its theoretical value. For our laboratory scale fermentation system, it is impractical to prepare hydrolysate continuously as industry does. We strictly controlled sterilization temperature and time when Medium B was sterilized to avoid sugar loss and other by-product formation. After sterilization, the hydrolysate was immediately cooled to room temperature and glucoamylase filtered by 0.2 mm micromembrane to remove potentially contaminated microbes was added at 0.05% (v/v) to compensate the activity loss of glucoamylase resulted from sterilization. It has been proven this strategy was very effective. The residual sugars in the final effluent were decreased to 1.5 g l−1 for reducing sugar and 2.5 g l−1 for total sugar compared with 4.5 g l−1 for reducing sugar and 8.4 g l−1 for total sugar when sterilized hydrolysate without glucoamylase was fed. The conversion yield of ethanol to sugar was calculated to be as high as 91.1% of its theoretical value, which is acceptable in industry. 3.4. Advantages over conventional ethanol fermentation technologies Table 3 illustrates the comparison of the ethanol fermentation performances between this self-flocculating yeast strain and K2.
T.J. Xu et al. / Enzyme and Microbial Technology 37 (2005) 634–640
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Table 2 Comparisons of yeast cell concentrations (g (d.w.) l−1 ) D (h−1 )
0.017 0.025 0.033 0.040 0.050
Second tank
Third tank
Fourth tank
Xin
Xout
Xout /Xin
Xin
Xout
Xout /Xin
Xin
Xout
Xout /Xin
14.8 13.8 12.4 11.0 9.1
3.6 3.4 3.2 3.1 2.3
0.243 0.246 0.258 0.282 0.252
13.2 12.9 11.2 10.5 7.4
3.4 3.2 2.9 2.8 2.2
0.258 0.248 0.258 0.266 0.297
12.1 11.7 10.6 9.1 6.5
3.2 2.8 2.7 2.6 1.8
0.264 0.239 0.255 0.286 0.277
It can be seen that when the same levels of ethanol and residual sugars in the effluent were reached, the average ethanol fermentation time required by self-flocculating yeast strain was only 50% of that required by K2, and the average ethanol productivity doubled, correspondingly, because of higher yeast cell concentrations within the fermentors. This indicates fermentor capital investment for large-scale plant construction can be saved significantly. The synchronous increases of yeast cell concentrations and ethanol productivity also indicated yeast cell viability loss resulted from selfflocculating was minor. Currently, yeast cell recovery in ethanol fermentation industry is realized by centrifugation. The excellent settling performance of this self-flocculating yeast strain made it quite practical to recover yeast cells by settlement instead of centrifugation. This engineering advantage has been fully exploited in our pilot plant at BBCA, Anhui Province, East China, in which 36 t ethanol was produced daily using this self-flocculating yeast strain. Convention yield is the most important factor that the distillers care about because over 60% of ethanol production cost in China is coming from raw material consumption. From the data showed in Table 3, the yields based on sugar concentrations in the media were almost same for SPSC01 and K2, which further guarantees this self-flocculating yeast strain to be suitable for large scale industry application. An ethanol fermentation plant with a fuel ethanol production capacity of 200,000 t per year is under construction at BBCA, Anhui Province, East China, and expected to be put into operation at the end of 2006.
4. Conclusions Continuous ethanol fermentation using self-flocculating yeast strain SPSC01 in a multi-stage tanks in series fermentation system was experimentally proven to be practical. Yeast cells partly self-immobilized within the tanks. Steady states were observed for most experimental running, but unsteady states and oscillations of residual sugar, ethanol and biomass concentrations occurred for one designated dilution rate, in our case, 0.04 h−1 . For self-flocculating yeast cell ethanol fermentation, its ethanol productivity almost doubled that of freely suspended yeast cell ethanol fermentation system when the same levels of residual sugar and ethanol concentrations were reached, which indicates tank capital investment for large scale plant construction can be saved significantly. Moreover, self-flocculating yeast cells can be easily recovered from the effluent by settlement instead of by centrifugation that is widely used in freely suspended yeast ethanol fermentation system, centrifuge capital investment and running energy can be also saved.
Acknowledgements Funding for this research was provided from the HiTech R&D Program of China (863) with a Grant no. 2002AA647060 and National Key Scientific Research Program of China with a Grant no. 2004BA713B08.
References Table 3 Comparisons of the ethanol fermentation performances of SPSC01 with K2 Fermentation results
K2a
SPSC01
Pm (g l−1 ) P2 (g l−1 ) Srm (g l−1 ) St (g l−1 ) Y Xin (g (d.w.) l−1 ) D (h−1 ) t (h) q (g l−1 h−1 ) Yeast recovery method
95.2 103.5 2.5 3.5 0.462 3.0–5.0 0.017 60 1.76 Settlement
95.6 104.1 1.5 2.5 0.466 7.0–14.0 0.033 30 3.44 Centrifugation
a Five hundred milliliters seed fermentor was used for K2 fermentation system to guarantee the ratio of seed culture to ethanol fermentation broth to be identical with that for SPSC01 fermentation system.
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