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Continuous Ethanol Fermentation Coupled with Recycling of Yeast Flocs
CHINESE JOURNAL OF BIOTECHNOLOGY Volume 22, Issue 5, September 2006 Online English edition of the Chinese language journal Cite this article as: Chin J Biotech, 2006, 22(5), 816−820.
RESEARCH PAPER
Continuous Ethanol Fermentation Coupled with Recycling of Yeast Flocs WANG Bo, GE Xu-Meng, LI Ning, BAI Feng-Wu* Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, China
Abstract: A continuous ethanol fermentation system composed of three-stage tanks in series coupled with two sedimentation tanks was established. A self-flocculating yeast strain developed by protoplast fusion from Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage enzymatic hydrolysate of corn powder containing 220 g/L residual reducing sugar, supplemented with 1.5 g/L (NH4)2HPO4 and 2.5 g/L KH2PO4, was used as the ethanol fermentation substrate and fed into the first fermentor at the dilution rate of 0.057 h−1. The yeast flocs separated by sedimentation were recycled into the first fermentor as two models: activation-recycle and direct-recycle. The quasi-steady states were obtained for both operation models after the fermentation systems experienced short periods of transitions. Activation process helped enhance the performance of ethanol fermentation at high dilution rates. The broth containing more than 101 g/L ethanol, 3.2 g/L residual reducing sugar, and 7.7 g/L residual total sugar was produced. The ethanol productivity was calculated to be 5.77 g L−1 h−1, which increased by more than 70 % compared with that achieved in the same tank in series system without recycling of yeast cells. Key Words:
fuel ethanol; self-flocculating yeast cells; recycling of yeast; activation; productivity
Bioethanol, as a renewable and environmentally friendly energy source, produced from renewable biomass, is becoming increasingly popular and quite important for mitigating the current depletion of crude oil and biological environmental deterioration. However, China’s conventional ethanol fermentation industry is low on technique and poor in economic conditions, which seriously restricts the development of the fuel ethanol industry. At present, besides the empoldering new technique and the founding of the industrialization demonstration project, reconstructing the conventional ethanol fermentation technique with the existing equipment systems to adapt to the development of the fuel ethanol industry in China is very vital. Compared with the current widely adopted technique for the production of ethanol, which is the opposite of the clear liquid fermentation technique, one of the advantages of the
clear liquid fermentation technique is that the raw material draff is separated in the hydrolysate of corn powder and eliminated in the fermentation system, and thus the recycling of yeast flocs and the by-product in the process of production of ethanol is realized at the same time. Therefore, it could improve ethanol productivity by increasing yeast cell densities within the fermentor and lead to enhanced economic benefits by the production of by-product of yeast. Currently, freely suspended yeast cells are widely used in ethanol fermentation plants both in China and abroad. Free yeast cells leave behind effluents in the fermentor during continuous operations and the cells have to be separated by centrifugation and recycled or by-produced thereafter. High capital investment for centrifuges and energy costs for operating and maintaining equipments greatly hinder the application of centrifuges in the reconstruction of China’s ethanol fermentation plants.
Received: April 21, 2006; Accepted: May 23, 2006. * Corresponding author. Tel: +86-411-84706308; Fax: +86-411-84706329; E-mail: [email protected] This work was supported by the grant from the National Natural Sciences Foundation of China (No. 20576017). Copyright © 2006, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
Compared with the fermentation technique using freely suspended yeast cells, which using the self-flocculating yeast strain could conveniently separate yeast cells from the effluent by spontaneous flocculation of the yeast cells. As this process does not involve high energy costs and huge investments for equipments, it has obvious economic advantages[1]. However, most previously developed self-flocculating yeast techniques are designed for suspended bioreactors that are quite different from the tanks that are widely used in Chinese industries and cannot be used for the reconstruction of ethanol fermentation plants currently functioning in China[2–5]. Xu Tiejun et al were the first to study the continuous ethanol fermentation process using a self-flocculating yeast in four-stage magnetic-stirred tanks in series fermentation system, in which one tank was for seed cultivation and others were for ethanol fermentation. An understanding of whether this setup could take the advantage of the easy settlement of flocculating yeast cells to realize both the recycling of cells and control the formation of by-product for enhancing the process of production of ethanol requires further experimental research and verification. In this study, a cultured yeast strain, SPSC01, which has both excellent ethanol fermentation abilities as well as self-flocculating nature, was used. A continuous ethanol fermentation system composed of three stage tanks in series, simulating the tanks currently used for ethanol fermentation in the industry, coupled with two sedimentation tanks was established. This set up is the first to study the technique ethanol fermentation coupled with recycling of the self-flocculating yeast SPSC01. Furthermore, this study compares the performance of this technique with that of other techniques to examine the feasibility of applying this technique for the improvement of the fermentation process and for the reconstruction of existing ethanol fermentation plants.
1
Materials and methods
1.1 Microorganism Self-flocculating yeast, SPSC01, was cultured by the authors of this study. 1.2 Material and reagents Material: Corn powder, degermed and dry milled, was donated by Jin Yu ethanol fermentation plant, Heilongjiang, China. Reagents: α-amylase (commercial α-amylase, 20 000 u/mL was donated by Novozymes, China); KH2PO4 was produced by the Shenyang Chemistry Reagent Factory (China). 1.3 Hydrolysate and media Flask seed media (glucose, 30 g/L; yeast extract, 5 g/L; peptone, 3 g/L) were sterilized at 121 °C for 20 min. Preparation of hydrolysate: Corn powder was hydrolyzed by two-stage enzymatic hydrolysis in a standard stirring tank
with a working volume of 25 L. The degermed corn powder was mixed with tap water at about 60 °C in the ratio 1:2.5. α-amylase was added to the slurry at 0.06 % (V/W) of corn powder and the slurry was then heated to about 85–95 °C by steam injected into the tank’s jacket and liquefied for 60 min. When liquefaction was completed, the mash was cooled to 60–65 °C by pumping tap water into the jacket. Glucoamylase (commercial glucoamylase, 100 000 u/mL, donated by Novozymes, China) was added to the mash at 0.12 % (V/W) of corn powder. After overnight (10–12 h) saccharification, the residue in the mash was removed by filtration. Increasing culture medium: the above hydrolysate was diluted by adding tap water to reducing sugar of 100 g/L, supplemented with 2.0 g/L (NH4)2HPO4 and K2HPO4, respectively, and sterilized at 121 °C for 15 min. Fermentation culture medium: the above hydrolysate was diluted by adding tap water to total sugar of 220 g/L, supplemented with 1.5 g/L (NH4)2HPO4 and 2.5 g/L K2HPO4, respectively, and sterilized at 121 °C for 15 min. Activation culture medium (glucose, 20 g/L; (NH4)2HPO4 2.5 g/L; KH2PO4 2.5 g/L; pH 2.0) was sterilized at 121 °C for 15 min (As acidification treatment is widely used in the recovery of yeast cells by ethanol fermentation plants, this study simulates the industry process to maintain the culture medium at pH 2.0). 1.4 Analytical methods 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 the internal standard. Glucose concentration was analyzed using a biosenser; the concentrations of both reducing and total sugar were analyzed by Fehling titration. But it was necessary that the samples for total sugar analysis 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. Samples were filtrated on a preparing filter paper, which was dried and weighed, and then washed thrice by deionized water, and then dried at 85 °C for 24 h and weighed. 1.5 Cultivation and continuous fermentation Aeration was interrupted for each tank at the beginning of inoculation. The temperature was controlled at (28.5±0.5) °C, pH was controlled at (4.2±0.2) by the automatic addition of ammonia water into the tanks. When the residual sugars within the tanks were found to be lower than 1.0 g/L, the increasing culture medium was fed into each tank; when the biomass within tanks reached 10–15 g/L dry weight, the tanks were treated in a series and the fermentation culture medium was fed into the first tank at the settled dilution rate and
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
overflowed in turn into the second and third tanks. Conversely, the water from the thermostat water tank was pumped from the third tank to the second and then to the first to maintain the temperature in the first tank at (30.5±0.5) °C and that in the second and the third tanks at 31–32 °C, pH within each tank was controlled at 4.2±0.2. The yeast cells within the effluent, which overflowed from the third tank into the sedimentation tank, settled at the bottom of the sedimentation tank. Activation-recycling: about 200 mL yeast slurry from the bottom of the first-stage sedimentation tank was added to a 1 000-mL Erlenmeyer flask containing 500 mL activation culture medium by peristaltic pump every 24 h. The speed and temperature of the rotar shaker were set at 150 r/min and 28 °C. After 8 h of activation, the yeast slurry was added to the second-stage sedimentation tank (Fig. 1-15) and allowed to settle for an hour, and then the fermentation culture medium peristaltic pump was stopped (Fig. 1-11) and started (Fig. 1-14) to recycle the yeast slurry in the second-stage sedimentation tank into the first tank, which was done by
recycling, restarting fermentation culture medium peristaltic pump (Fig. 1-11), and aerating each tank for 10 min. Direct-recycling: with every 24 h, fermentation culture medium peristaltic pump was stopped (Fig. 1-11) and peristaltic pump was started (Fig. 1-14) to recycle the yeast slurry in the second-stage sedimentation tank into the first tank, which was done by recycling, restarting fermentation culture medium peristaltic pump (Fig. 1-11), and aerating each tank for 10 min. 1.6 Experimental equipments and techniques In this study, three double-jacket glass fermentation tanks, each with a working volume of 1 100 mL and two sedimentation tanks with tapered base and working volume of 1 000 mL, respectively, were established to simulate industrial conditions. Stainless steel baffles and machine-stirring dashers were set in the fermentation tanks. The techniques and processes of all the experimental equipments are shown in Fig. 1.
Fig. 1 Process diagram of continuous ethanol fermentation coupled with recycling of self-flocculating yeast cells 1: air compressor; 2: air-flow meters; 3: needle valves; 4: pH probes and controllers; 5: temperature probes and controllers; 6: electromagnetic valve; 7: thermostat water tank; 8: thermostat water inlet; 9: thermostat water outlet; 10: storage tank for medium; 11: peristaltic pumps for medium input; 12: stirred tank fermentors; 13: storage tanks for broth; 14: peristaltic pumps for yeast slurry recycle; 15: sedimentation tanks; 16: yeast sludge; 17: supernatant; 18: activation tank.
2
Results and discussion
2.1 Operating stages of fermentation system Fig. 2 shows the fermentation profiles of biomass, residual
sugar, and ethanol under both models of activation-recycling and direct-recycling.
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
Fig. 2 Time courses of biomass, residual sugar, and ethanol during fermentation (a, b, and c represent biomass, residual sugar, and ethanol, respectively) Activation-recycle model: tank 1 (─■─), tank 2 (─●─), tank 3 (─▲─). Direct-recycle model: tank 1 (─□─), tank 2 (─○─), tank 3 (─∆─).
discharge rates from the system are concerned, the system reached similar steady states. From day 18, the activation operation was halted, and the direct-recycling of yeast slurry was initiated. The residual reducing sugar concentration markedly increased, the ethanol concentration decreased accordingly; however, over a time period and through self-equilibrium regulation, the residual sugar, ethanol, and biomass concentration in each tank stabilized. The fermentation parameters of ethanol and other compounds were all under the fermentation levels of the activation-recycling model. This indicated that the activation treatment exerts remarkable function in enhancing the fermentation performance of the yeast recycling technique. 2.2 The evaluation and comparison of fermentation performance The average fermentation results of this study and current industry production and published reports are summarized and compared in Table 1.
From Fig. 2, within the initial three days of yeast-slurry recycling, the biomass oscillation at certain fluctuation amplitudes was observed for all the three tanks; as the recycling times increased, the biomass concentration gradually increased (Fig. 2a), residual reducing sugar concentration gradually decreased (Fig. 2b), ethanol concentration gradually increased (Fig. 2c) in each fermentation tank, and the variational tide remained calm. After 10 d, a similar steady state was observed. The reason for the above-mentioned phenomenon is that the yeast flocs did not settle completely and overflowed into the sedimentation tank with effluent, namely certain yeast cells overflowed into the storage tanks for broth (Fig. 1-13) with up-lucidness effluent. In the initial stages of fermentation, yeast flocs’ recycling increased the biomass concentration by gradual accumulation, along with the decrease in residual sugar concentration and the increase in ethanol concentration, and then resulted in the decrease in the specific growth rates of yeasts. As far as the balance between the yeast growth rates in the system and the yeast
Table 1 Comparison of several different ethanol fermentation technologies Process strategies Fermentation results
Tanks in series system for
Freely suspended yeast cell
Tanks in series system for
Suspended-bed system for
system a
flocs a
flocs a
P, g/L
88–96
96–104
96–104
99–103
P, g/L
2.5
2.0
2.0
3.2
flocs coupled with yeast-cell recycling
ST, g/L
5.0
3.5
3.5
7.7
X, g/L
3–5
7–14
40–60
25–30
t, h
50–60
25–35
20–25
17–18
q, g L−1 h−1
1.65
3.32
4.44
5.77
[6]
a: data were from the article written by Xu et al ; P: ethanol concentration; SR: residual reductive sugar concentration; ST: residual total sugar concentration; X: biomass concentration; t: average ethanol fermentation time; q: ethanol productivity.
Table 1 shows that the average ethanol concentration in this study reached 101 g/L, the residual reducing sugar and total sugar were 3.2 and 7.7 g/L, respectively, compared with the
other three fermentation strategies. When the same levels of ethanol and residual sugars in the effluent were attained, the average time required for ethanol fermentation evidently
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
beginning from the dissociating state of micron-sized to millimeter-sized particles to the self-flocculation of yeast flocs and even after self-flocculation, the influence of internal diffusion on the particles were severe. When compared with suspended-bed system for flocs, the cut speed in the stirred tanks was quite high, so that the diameter of the particles were small thereby reducing the influence of internal diffusion on the particles. Therefore, the biomasses in the stirred tanks were far lower than those in the suspended-bed system, but the ethanol productivity in the stirred tanks is nearly equal to or even above the suspended-bed system. When compared with nonrecycling assembled fermentation system by Xu Tiejun et al, this study excludes seed culture process, but includes sedimentation, activation, and recycling processes. Table 2 shows the comparison of the sugar consumption, ethanol formation, and yield of ethanol from sugar in both strategies.
reduced, and the average ethanol productivity correspondingly increased: compared with conventional freely suspended yeast-strain system, the average time required for ethanol fermentation reduced by 1/3, and ethanol productivity increased by 2.5 times; compared with nonrecycling strategy by design of Xu Tiejun et al, the average time required for ethanol fermentation reduced by 42 % and ethanol productivity increased by 70 %; compared with the suspendedbed system for flocs, the average time required for ethanol fermentation reduced by 22 % and ethanol productivity increased by 30 %. From Table 1, it can be seen that by enhancing biomass concentration, ethanol productivity can be increased; however, the increase is not in direct proportion. For example, when compared with conventional freely suspended yeast strain system, the biomass concentration in self-flocculation yeast strain strategy increased almost 10 times, but ethanol productivity increased only 2 times. This indicates that
Table 2 Comparison of the sugar consumption, ethanol formation, and yield with the reference[6] Fermentation results
Tanks in series system for flocs coupled with seed culturea
Tanks in series system for flocs coupled with activation and recycling of yeast cells
Seed culture
Fermentors
Total
Activation
Fermentors
S0, g/L
100
220
–
–
220
Total –
Fin, L/d
0.41
2.38
2.79
0.20
4.51
4.71
GS, g/d
41
524
565
10
994
1004
VP, L/d
–
–
2.79
–
–
4.66
Pf, g/L
36.8
95.6
95.6
0
101.16
101.16
GP, g/d
15
252
267
0
477
477
YP/S
0.37
0.48
0.47
0
0.48
0.48
a: data were obtained from the article written by Xu et al[6]; S0: feeding sugar concentration; Fin: volume of medium input per day; GS: mass of feeding sugar consumed per day; VP: volume of broth obtained per day; Pf: ethanol concentration in the final broth; GP: mass of ethanol produced per day; YP/S: yield of ethanol from sugar.
From Table 2, it can be seen that for the seed culture strategy, the sugar consumption in the seed culture unit is 7 % of the total sugar consumption, but the yield of ethanol from sugar is only 0.37. This results in the decrease in total yield in the whole technique; on the contrary, for the activationrecycling strategy in this study, the sugar consumption in the activation unit is only 1 % of the total sugar consumption and does not produce evident influence. The separated yeasts from the sedimentation tank in this study were recycled to a certain degree during the entire operation and were not produced. In the practical application, a part of yeast cells will be inserted into the sedimentation tank on a certain scale based on settled biomass concentration target in the fermentation tanks to enhance economic benefit, and the others for recycling to increase the ethanol productivity and also adapting the ratio of output to recycling on the market demand. In addition, the technique in this study based on a small laboratory size that is still required to further
validate in the equipments of industrial scales.
3
Conclusions
(1) This study deals with the strategy of ethanol fermentation coupled with recycling of self-flocculating yeast SPSC01 in a continuous ethanol fermentation system composed of three stages in series stirred fermentator tanks and coupled with two sedimentation tanks initially. (2) Similar steady states were reached under both activation-recycling and direct-recycling strategies, and the activation treatment exerts remarkable function in enhancing the fermentation performance of yeast recycling technique. (3) The continuous ethanol fermentation coupled with recycling of yeast flocs in three stages in series-stirred fermentator-tank system evidently enhanced the ethanol productivity at the premises of other fermentation performances.
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
Acknowledgements
[4]
Hoshino K, Tanikuchi M, Marumoto H, et al. Continuous ethanol production from raw starch using a reversibly
Funds for this research were provided by the National Natural Science Foundation of China (No. 20576017).
soluble-auto precipitating amylase and flocculating yeast cells. J Ferment Bioeng, 1990, 69: 228–233. [5]
Yan Z, Zi LH, Li N, Wang F, Bai FW. Continuous ethanol fermentation using self-flocculating yeast in multi-stage
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1980
RESEARCH PAPER
Continuous Ethanol Fermentation Coupled with Recycling of Yeast Flocs WANG Bo, GE Xu-Meng, LI Ning, BAI Feng-Wu* Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, China
Abstract: A continuous ethanol fermentation system composed of three-stage tanks in series coupled with two sedimentation tanks was established. A self-flocculating yeast strain developed by protoplast fusion from Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage enzymatic hydrolysate of corn powder containing 220 g/L residual reducing sugar, supplemented with 1.5 g/L (NH4)2HPO4 and 2.5 g/L KH2PO4, was used as the ethanol fermentation substrate and fed into the first fermentor at the dilution rate of 0.057 h−1. The yeast flocs separated by sedimentation were recycled into the first fermentor as two models: activation-recycle and direct-recycle. The quasi-steady states were obtained for both operation models after the fermentation systems experienced short periods of transitions. Activation process helped enhance the performance of ethanol fermentation at high dilution rates. The broth containing more than 101 g/L ethanol, 3.2 g/L residual reducing sugar, and 7.7 g/L residual total sugar was produced. The ethanol productivity was calculated to be 5.77 g L−1 h−1, which increased by more than 70 % compared with that achieved in the same tank in series system without recycling of yeast cells. Key Words:
fuel ethanol; self-flocculating yeast cells; recycling of yeast; activation; productivity
Bioethanol, as a renewable and environmentally friendly energy source, produced from renewable biomass, is becoming increasingly popular and quite important for mitigating the current depletion of crude oil and biological environmental deterioration. However, China’s conventional ethanol fermentation industry is low on technique and poor in economic conditions, which seriously restricts the development of the fuel ethanol industry. At present, besides the empoldering new technique and the founding of the industrialization demonstration project, reconstructing the conventional ethanol fermentation technique with the existing equipment systems to adapt to the development of the fuel ethanol industry in China is very vital. Compared with the current widely adopted technique for the production of ethanol, which is the opposite of the clear liquid fermentation technique, one of the advantages of the
clear liquid fermentation technique is that the raw material draff is separated in the hydrolysate of corn powder and eliminated in the fermentation system, and thus the recycling of yeast flocs and the by-product in the process of production of ethanol is realized at the same time. Therefore, it could improve ethanol productivity by increasing yeast cell densities within the fermentor and lead to enhanced economic benefits by the production of by-product of yeast. Currently, freely suspended yeast cells are widely used in ethanol fermentation plants both in China and abroad. Free yeast cells leave behind effluents in the fermentor during continuous operations and the cells have to be separated by centrifugation and recycled or by-produced thereafter. High capital investment for centrifuges and energy costs for operating and maintaining equipments greatly hinder the application of centrifuges in the reconstruction of China’s ethanol fermentation plants.
Received: April 21, 2006; Accepted: May 23, 2006. * Corresponding author. Tel: +86-411-84706308; Fax: +86-411-84706329; E-mail: [email protected] This work was supported by the grant from the National Natural Sciences Foundation of China (No. 20576017). Copyright © 2006, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
Compared with the fermentation technique using freely suspended yeast cells, which using the self-flocculating yeast strain could conveniently separate yeast cells from the effluent by spontaneous flocculation of the yeast cells. As this process does not involve high energy costs and huge investments for equipments, it has obvious economic advantages[1]. However, most previously developed self-flocculating yeast techniques are designed for suspended bioreactors that are quite different from the tanks that are widely used in Chinese industries and cannot be used for the reconstruction of ethanol fermentation plants currently functioning in China[2–5]. Xu Tiejun et al were the first to study the continuous ethanol fermentation process using a self-flocculating yeast in four-stage magnetic-stirred tanks in series fermentation system, in which one tank was for seed cultivation and others were for ethanol fermentation. An understanding of whether this setup could take the advantage of the easy settlement of flocculating yeast cells to realize both the recycling of cells and control the formation of by-product for enhancing the process of production of ethanol requires further experimental research and verification. In this study, a cultured yeast strain, SPSC01, which has both excellent ethanol fermentation abilities as well as self-flocculating nature, was used. A continuous ethanol fermentation system composed of three stage tanks in series, simulating the tanks currently used for ethanol fermentation in the industry, coupled with two sedimentation tanks was established. This set up is the first to study the technique ethanol fermentation coupled with recycling of the self-flocculating yeast SPSC01. Furthermore, this study compares the performance of this technique with that of other techniques to examine the feasibility of applying this technique for the improvement of the fermentation process and for the reconstruction of existing ethanol fermentation plants.
1
Materials and methods
1.1 Microorganism Self-flocculating yeast, SPSC01, was cultured by the authors of this study. 1.2 Material and reagents Material: Corn powder, degermed and dry milled, was donated by Jin Yu ethanol fermentation plant, Heilongjiang, China. Reagents: α-amylase (commercial α-amylase, 20 000 u/mL was donated by Novozymes, China); KH2PO4 was produced by the Shenyang Chemistry Reagent Factory (China). 1.3 Hydrolysate and media Flask seed media (glucose, 30 g/L; yeast extract, 5 g/L; peptone, 3 g/L) were sterilized at 121 °C for 20 min. Preparation of hydrolysate: Corn powder was hydrolyzed by two-stage enzymatic hydrolysis in a standard stirring tank
with a working volume of 25 L. The degermed corn powder was mixed with tap water at about 60 °C in the ratio 1:2.5. α-amylase was added to the slurry at 0.06 % (V/W) of corn powder and the slurry was then heated to about 85–95 °C by steam injected into the tank’s jacket and liquefied for 60 min. When liquefaction was completed, the mash was cooled to 60–65 °C by pumping tap water into the jacket. Glucoamylase (commercial glucoamylase, 100 000 u/mL, donated by Novozymes, China) was added to the mash at 0.12 % (V/W) of corn powder. After overnight (10–12 h) saccharification, the residue in the mash was removed by filtration. Increasing culture medium: the above hydrolysate was diluted by adding tap water to reducing sugar of 100 g/L, supplemented with 2.0 g/L (NH4)2HPO4 and K2HPO4, respectively, and sterilized at 121 °C for 15 min. Fermentation culture medium: the above hydrolysate was diluted by adding tap water to total sugar of 220 g/L, supplemented with 1.5 g/L (NH4)2HPO4 and 2.5 g/L K2HPO4, respectively, and sterilized at 121 °C for 15 min. Activation culture medium (glucose, 20 g/L; (NH4)2HPO4 2.5 g/L; KH2PO4 2.5 g/L; pH 2.0) was sterilized at 121 °C for 15 min (As acidification treatment is widely used in the recovery of yeast cells by ethanol fermentation plants, this study simulates the industry process to maintain the culture medium at pH 2.0). 1.4 Analytical methods 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 the internal standard. Glucose concentration was analyzed using a biosenser; the concentrations of both reducing and total sugar were analyzed by Fehling titration. But it was necessary that the samples for total sugar analysis 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. Samples were filtrated on a preparing filter paper, which was dried and weighed, and then washed thrice by deionized water, and then dried at 85 °C for 24 h and weighed. 1.5 Cultivation and continuous fermentation Aeration was interrupted for each tank at the beginning of inoculation. The temperature was controlled at (28.5±0.5) °C, pH was controlled at (4.2±0.2) by the automatic addition of ammonia water into the tanks. When the residual sugars within the tanks were found to be lower than 1.0 g/L, the increasing culture medium was fed into each tank; when the biomass within tanks reached 10–15 g/L dry weight, the tanks were treated in a series and the fermentation culture medium was fed into the first tank at the settled dilution rate and
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
overflowed in turn into the second and third tanks. Conversely, the water from the thermostat water tank was pumped from the third tank to the second and then to the first to maintain the temperature in the first tank at (30.5±0.5) °C and that in the second and the third tanks at 31–32 °C, pH within each tank was controlled at 4.2±0.2. The yeast cells within the effluent, which overflowed from the third tank into the sedimentation tank, settled at the bottom of the sedimentation tank. Activation-recycling: about 200 mL yeast slurry from the bottom of the first-stage sedimentation tank was added to a 1 000-mL Erlenmeyer flask containing 500 mL activation culture medium by peristaltic pump every 24 h. The speed and temperature of the rotar shaker were set at 150 r/min and 28 °C. After 8 h of activation, the yeast slurry was added to the second-stage sedimentation tank (Fig. 1-15) and allowed to settle for an hour, and then the fermentation culture medium peristaltic pump was stopped (Fig. 1-11) and started (Fig. 1-14) to recycle the yeast slurry in the second-stage sedimentation tank into the first tank, which was done by
recycling, restarting fermentation culture medium peristaltic pump (Fig. 1-11), and aerating each tank for 10 min. Direct-recycling: with every 24 h, fermentation culture medium peristaltic pump was stopped (Fig. 1-11) and peristaltic pump was started (Fig. 1-14) to recycle the yeast slurry in the second-stage sedimentation tank into the first tank, which was done by recycling, restarting fermentation culture medium peristaltic pump (Fig. 1-11), and aerating each tank for 10 min. 1.6 Experimental equipments and techniques In this study, three double-jacket glass fermentation tanks, each with a working volume of 1 100 mL and two sedimentation tanks with tapered base and working volume of 1 000 mL, respectively, were established to simulate industrial conditions. Stainless steel baffles and machine-stirring dashers were set in the fermentation tanks. The techniques and processes of all the experimental equipments are shown in Fig. 1.
Fig. 1 Process diagram of continuous ethanol fermentation coupled with recycling of self-flocculating yeast cells 1: air compressor; 2: air-flow meters; 3: needle valves; 4: pH probes and controllers; 5: temperature probes and controllers; 6: electromagnetic valve; 7: thermostat water tank; 8: thermostat water inlet; 9: thermostat water outlet; 10: storage tank for medium; 11: peristaltic pumps for medium input; 12: stirred tank fermentors; 13: storage tanks for broth; 14: peristaltic pumps for yeast slurry recycle; 15: sedimentation tanks; 16: yeast sludge; 17: supernatant; 18: activation tank.
2
Results and discussion
2.1 Operating stages of fermentation system Fig. 2 shows the fermentation profiles of biomass, residual
sugar, and ethanol under both models of activation-recycling and direct-recycling.
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
Fig. 2 Time courses of biomass, residual sugar, and ethanol during fermentation (a, b, and c represent biomass, residual sugar, and ethanol, respectively) Activation-recycle model: tank 1 (─■─), tank 2 (─●─), tank 3 (─▲─). Direct-recycle model: tank 1 (─□─), tank 2 (─○─), tank 3 (─∆─).
discharge rates from the system are concerned, the system reached similar steady states. From day 18, the activation operation was halted, and the direct-recycling of yeast slurry was initiated. The residual reducing sugar concentration markedly increased, the ethanol concentration decreased accordingly; however, over a time period and through self-equilibrium regulation, the residual sugar, ethanol, and biomass concentration in each tank stabilized. The fermentation parameters of ethanol and other compounds were all under the fermentation levels of the activation-recycling model. This indicated that the activation treatment exerts remarkable function in enhancing the fermentation performance of the yeast recycling technique. 2.2 The evaluation and comparison of fermentation performance The average fermentation results of this study and current industry production and published reports are summarized and compared in Table 1.
From Fig. 2, within the initial three days of yeast-slurry recycling, the biomass oscillation at certain fluctuation amplitudes was observed for all the three tanks; as the recycling times increased, the biomass concentration gradually increased (Fig. 2a), residual reducing sugar concentration gradually decreased (Fig. 2b), ethanol concentration gradually increased (Fig. 2c) in each fermentation tank, and the variational tide remained calm. After 10 d, a similar steady state was observed. The reason for the above-mentioned phenomenon is that the yeast flocs did not settle completely and overflowed into the sedimentation tank with effluent, namely certain yeast cells overflowed into the storage tanks for broth (Fig. 1-13) with up-lucidness effluent. In the initial stages of fermentation, yeast flocs’ recycling increased the biomass concentration by gradual accumulation, along with the decrease in residual sugar concentration and the increase in ethanol concentration, and then resulted in the decrease in the specific growth rates of yeasts. As far as the balance between the yeast growth rates in the system and the yeast
Table 1 Comparison of several different ethanol fermentation technologies Process strategies Fermentation results
Tanks in series system for
Freely suspended yeast cell
Tanks in series system for
Suspended-bed system for
system a
flocs a
flocs a
P, g/L
88–96
96–104
96–104
99–103
P, g/L
2.5
2.0
2.0
3.2
flocs coupled with yeast-cell recycling
ST, g/L
5.0
3.5
3.5
7.7
X, g/L
3–5
7–14
40–60
25–30
t, h
50–60
25–35
20–25
17–18
q, g L−1 h−1
1.65
3.32
4.44
5.77
[6]
a: data were from the article written by Xu et al ; P: ethanol concentration; SR: residual reductive sugar concentration; ST: residual total sugar concentration; X: biomass concentration; t: average ethanol fermentation time; q: ethanol productivity.
Table 1 shows that the average ethanol concentration in this study reached 101 g/L, the residual reducing sugar and total sugar were 3.2 and 7.7 g/L, respectively, compared with the
other three fermentation strategies. When the same levels of ethanol and residual sugars in the effluent were attained, the average time required for ethanol fermentation evidently
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
beginning from the dissociating state of micron-sized to millimeter-sized particles to the self-flocculation of yeast flocs and even after self-flocculation, the influence of internal diffusion on the particles were severe. When compared with suspended-bed system for flocs, the cut speed in the stirred tanks was quite high, so that the diameter of the particles were small thereby reducing the influence of internal diffusion on the particles. Therefore, the biomasses in the stirred tanks were far lower than those in the suspended-bed system, but the ethanol productivity in the stirred tanks is nearly equal to or even above the suspended-bed system. When compared with nonrecycling assembled fermentation system by Xu Tiejun et al, this study excludes seed culture process, but includes sedimentation, activation, and recycling processes. Table 2 shows the comparison of the sugar consumption, ethanol formation, and yield of ethanol from sugar in both strategies.
reduced, and the average ethanol productivity correspondingly increased: compared with conventional freely suspended yeast-strain system, the average time required for ethanol fermentation reduced by 1/3, and ethanol productivity increased by 2.5 times; compared with nonrecycling strategy by design of Xu Tiejun et al, the average time required for ethanol fermentation reduced by 42 % and ethanol productivity increased by 70 %; compared with the suspendedbed system for flocs, the average time required for ethanol fermentation reduced by 22 % and ethanol productivity increased by 30 %. From Table 1, it can be seen that by enhancing biomass concentration, ethanol productivity can be increased; however, the increase is not in direct proportion. For example, when compared with conventional freely suspended yeast strain system, the biomass concentration in self-flocculation yeast strain strategy increased almost 10 times, but ethanol productivity increased only 2 times. This indicates that
Table 2 Comparison of the sugar consumption, ethanol formation, and yield with the reference[6] Fermentation results
Tanks in series system for flocs coupled with seed culturea
Tanks in series system for flocs coupled with activation and recycling of yeast cells
Seed culture
Fermentors
Total
Activation
Fermentors
S0, g/L
100
220
–
–
220
Total –
Fin, L/d
0.41
2.38
2.79
0.20
4.51
4.71
GS, g/d
41
524
565
10
994
1004
VP, L/d
–
–
2.79
–
–
4.66
Pf, g/L
36.8
95.6
95.6
0
101.16
101.16
GP, g/d
15
252
267
0
477
477
YP/S
0.37
0.48
0.47
0
0.48
0.48
a: data were obtained from the article written by Xu et al[6]; S0: feeding sugar concentration; Fin: volume of medium input per day; GS: mass of feeding sugar consumed per day; VP: volume of broth obtained per day; Pf: ethanol concentration in the final broth; GP: mass of ethanol produced per day; YP/S: yield of ethanol from sugar.
From Table 2, it can be seen that for the seed culture strategy, the sugar consumption in the seed culture unit is 7 % of the total sugar consumption, but the yield of ethanol from sugar is only 0.37. This results in the decrease in total yield in the whole technique; on the contrary, for the activationrecycling strategy in this study, the sugar consumption in the activation unit is only 1 % of the total sugar consumption and does not produce evident influence. The separated yeasts from the sedimentation tank in this study were recycled to a certain degree during the entire operation and were not produced. In the practical application, a part of yeast cells will be inserted into the sedimentation tank on a certain scale based on settled biomass concentration target in the fermentation tanks to enhance economic benefit, and the others for recycling to increase the ethanol productivity and also adapting the ratio of output to recycling on the market demand. In addition, the technique in this study based on a small laboratory size that is still required to further
validate in the equipments of industrial scales.
3
Conclusions
(1) This study deals with the strategy of ethanol fermentation coupled with recycling of self-flocculating yeast SPSC01 in a continuous ethanol fermentation system composed of three stages in series stirred fermentator tanks and coupled with two sedimentation tanks initially. (2) Similar steady states were reached under both activation-recycling and direct-recycling strategies, and the activation treatment exerts remarkable function in enhancing the fermentation performance of yeast recycling technique. (3) The continuous ethanol fermentation coupled with recycling of yeast flocs in three stages in series-stirred fermentator-tank system evidently enhanced the ethanol productivity at the premises of other fermentation performances.
WANG Bo et al. / Chinese Journal of Biotechnology, 2006, 22(5): 816–820
Acknowledgements
[4]
Hoshino K, Tanikuchi M, Marumoto H, et al. Continuous ethanol production from raw starch using a reversibly
Funds for this research were provided by the National Natural Science Foundation of China (No. 20576017).
soluble-auto precipitating amylase and flocculating yeast cells. J Ferment Bioeng, 1990, 69: 228–233. [5]
Yan Z, Zi LH, Li N, Wang F, Bai FW. Continuous ethanol fermentation using self-flocculating yeast in multi-stage
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