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Up-Flow Attached-Bed Bioreactor for Continuous Ethanol Fermentation Hung-Chang Chenf Development Center for Biotechnology, 81 Chang Hsing Street, Taipei, Taiwan, Republic of China Ethanol fermentation was carried out in an up-flow attached-bed (UFAB) continuous fermentor using different microorganisms, substrates, and attachment supports. When Saccharomyces fragilis was attached to cellulose acetate to ferment deproteinized and concentrated acid whey, ethanol productivity as high as 6.9 g/(L.h), 10 times that of a batch reactor, was obtained in a continuous UFAB bioreactor. When Zymomonas mobilis was attached to vermiculite in a continuous UFAB bioreactor to ferment a 10% glucose feed, ethanol productivity as high as 105.7 g/(L.h) was achieved with a dilution rate of 3.6 h-l and an ethanol concentration of 29.0 g/L.
Introduction Interest in economical production of ethanol from renewable biomass as fuel substitute has stimulated the development of many continuous and rapid fermentation technologies (Table I). The development of such a system relies on overcoming two problems associated with ethanol production, namely, the low cell concentration and the inhibitory effect of ethanol, which have resulted in very low ethanol production rates [l-2 g/(L.h)] in batch fermentors (I). Stirred tankfermentors with cell recycling (2) have been employed to achieve cell concentrations as high as 50 g/L on the dry weight basis. Consequently, high alcohol productivity of 29 g/(L.h) has been achieved. However, a continuous centrifuge capable of aseptically recycling cells would become an expensive necessity. Margaritis and Wilke (3)replaced the cell recycle method by putting a rotating membrane within the fermentor that retained cells and allowed the fermented beer to permeate out. Although high cell concentration (30.9 g/L) and a substantial improvement in fermentation rate [27.3 g/(L*h)]were achieved, this system is not likely to become a competitive industrial process due to the complex nature of its operation. In order to eliminate ethanol inhibition, ethanol was continuously removed from the fermenting beer by maintaining the fermentor under vacuum. The vacuum fermentor in conjunction with cell recycle (2) permitted theuseof a concentrated sugar solution (33.4%) as feed and productivity as high as 82 g/ (Lmh)was obtained. However, one major disadvantages of this approach is that nonvolatile metabolic end products accumulate in the fermentor and inhibit the fermentation. In addition, vacuum fermentation is not economical for industrial application. Similar approaches (4-6) have been applied to Zymomonus mobilis, and much higher productivities were obtained compared to yeast fermentations. The difficulty of cell separation has been overcome partially in the tower fermentor (7),using internal sedimentation, which is a characteristic of fluidized bed systems. However, tower fermentors are limited to flocculating microorganisms. Also, a constant supply of microorganisms has to be provided in the inlet, thus requiring recycling of cells. In contrast to the fermentor containing suspended microbial flocs, microbial mass ~
+ Current address:
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Department of Food Engineering, Dai Yeh Institute of Technology, 112 Shanjeau Rd., Daitauen, Chang Hwa, Taiwan 51505, Republic of China. 875&7938/Q1/3007-0311$02.50/0
might be incorporated into a matrix of gelatin (8) or alginate gel (9),which removes the two limitations observed in the tower fermentor. However, in these systems mass transfer resistance is introduced, and this may reduce fermentation rate and consequently ethanol productivity. Sitton and his colleagues studied the performance of a fixed-film fermentor. They showed that the fixed-film fermentor allowed a high dilution rate of 1.75 h-l, which was 6 times the washout rate of a continuous stirred tank reactor (CSTR) (10). The fixed-film reactor was demonstrated to enable the accumulation of up to 10 times the mass per unit reactor volume over the suspended microbial systems. However, this large mass presented two problems. First, clogging will eventually occur due to the accumulation of thick biomass in the static void spaces. Second,the total biomass that was active in the thick film is limited due to substrate diffusion. In view of the limitations encountered in the ethanol production bioreactors described above, we proposed that an ideal ethanol fermentation process should utilize attached microbial film on a large surface area per unit volume to minimize diffusion limitation and that the process should be nonclogging. One method of achieving these characteristics is to exploit the well-known biological phenomenon of microbial adhesion to interfaces in an upflow bioreactor containing small and inert particles that encourages microbial attachment. This paper describes the continuous production of ethanol in such a UFAB (up-flow attached-bed) bioreactor.
Materials and Methods Two organisms were used in this study. One was Saccharomyces fragilis, a lactose-fermenting yeast obtained from the stock cultures of Food Science Department at Cornel1University; the other was a fast ethanol-producing bacterium, Zymomonas mobilis ATCC 10988. Two different types of media were fermented by Saccharomyces and Zymomonas to demonstrate that the UFAB process is general and applicable to any system. The yeast fermentation medium has the following ingredients (in grams per liter): cottage cheese whey deproteinized and concentrated to lactose equivalent to 100; KH2PO4, 2.5; (NH4)2S04,5.0; yeast extract, 1.0. The bacteria fermentation medium has the following ingredients (in grams per liter): glucose (cerelose), 100;yeast extract, 5; KH2POr, 2.5; (NH&S04, 1.0; MgS04~7Hz0, 0.5. Six materials-namely, cellulose acetate, mixed-type resin,
0 1991 American Chemical Society and American Institute of Chemical Engineers
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Table I. Commrative Ethanol Productivities of Different Continuoue Systems dilution organism system feed, g/L rate, h-' cells, g/L 100 0.17 S. cereuisioe ATCC 4126 CSTR 12 recycle 100 0.68 50 rotor 104 0.58 30.9 vacuum 334 50 recycle w/ vacuum 334 0.23 124 30 S. cereuisioe ATCC 24858 CSTR 0.18 fixed film 30 1.75 150 2.mobilis ATCC 10988 CSTR 2.5 0.20 recycle 100 2.70 38.0 recycle w/ vacuum 200 1.0 25 immobilized 50 0.05
diatomite, bentonite, kaolin, and vermiculite-were used for the attachment of microorganisms. The continuous UFAB ethanol fermentation was conducted in a tapered reactor shown in Figure 1. The total fermentor working volume of 600 mL (500 mL for main fermentor and 100 mL for recycling unit) was maintained by the overflow ports. One-third of the fermentor working volume was first filled with attachment material and the rest was filled with fermentation medium. The system was then inoculated with shaker-grown culture and its temperature was controlled at 30 and 32 "C for Saccharomyces and Zymomonas, respectively. After 1 day of batch growth, continuous feeding began. Unless specificallymentioned, the system was slowly fed at the beginning. The feeding rate was then shifted up gradually after a t least a feeding period of 3-4 residence times, which is a rule of thumb for the establishment of steady state. The residual sugar concentration and ethanol concentration from the effluent were determined by the phenol-sulfuric acid method and gas chromatography (II), respectively.
Results and Discussion Among the six materials tested in this study, cellulose acetate and verniiculitewere the best attachment materials for Saccharomyces fragilis (Figure 2) and Zymomonas mobilis (Figure 3), respectively. There are two possible mechanisms involved in the attachment of yeast cells to cellulose acetate. The first is the reduction of total free energy by cell contact with or adhering to particle surface (12). The relevant thermodynamic potential for cell contact or adhesion is the Helmholtz free energy, defined as F = (rps- rp- rJA, where rP, r,,, and rs are interfacial tensions of particle-surface, particle (yeast cells), and surface (cellulose acetate), respectively, and A is interfacial area. If F is negative, adhesion takes place, whereas a positive F implies no adhesion. Cellulose acetate has a very high critical surface tension value (13). Thus, it may be argued that the high energy surface of cellulose acetate has induced the yeast cell adhesion and consequently reduces the total free energy of the system. The second possible mechanism is the interaction between polysaccharide chains of cell wall and cellulose acetate. Microbial polysaccharides may interact with plant polysaccharides in two ways (14): (1) by mutual exclusion of incompatible molecules to give an increased local concentration of both polymers and (2) by energetically favorable association of structurally similar chain segments. Cellulose acetate is a chain of glucose and acetylated glucose linked by 8-l,4-glycosidic bonds, and the major component of yeast cell wall is mannan phosphate, which is a chain containing mannose phosphate units also linked by 8-1,Cglycosidic bonds. Thus, these two polysaccharides may be energetically favorably associated due to structure similarity.
EtOH, g/L 41 43 47 160 160 9.8 9.1 55 44.5 85 6.6
productivity,
g/(L.h) 7.0 29.0 27.3 60 82 1.75 15.9 11
120 85 132
ref 1 2
3 2 2 10 10
5 5 4 6
On the other hand, the strong attachment of Zymomonus mobilis to vermiculite but neither to kaolin nor to bentonite may result from the charge differences of these clay minerals. According to Russell (15),kaolin and vermiculite have net negative charges of 10-50 and 100150 mequiv/100 g, respectively. A charged surface will attract counterions from the surrounding aqueous phase. This process is opposed by the thermal motion of the counterions tending to distribute them evenly throughout the aqueous phase. The effects of electrostatic attraction and thermal motion on the counterions lead to the formation of a region, often referred to as the Gouy-Chapman diffuse electrical double layer, next to the charged surface where the concentrations of counterions are higher than in the rest of the bulk phase. Therefore, the bacterial cells will experience a repulsive force when their diffuse double layers overlap with the double layers of clay minerals. However, this barrier can be eliminated in a solution high in electrolyte concentration, such as culture medium, and the net result is a strong attraction between cells and clay particles (16). Thus, stronger interaction with bacterial cells will occur in highly charged vermiculite than in less charged bentonite or kaolin. Also, the response of the microorganismsto the environment may play an important role in the attachment process. These responses are (1) motility, which can overcome the repulsive force to make contact with the surface, and (2) the synthesis or secretion of polymers and/or enzymes required for polymeric bridging of the bacteria and surfaces. Zymomonas is known to produce levan from sucrose medium (17) but forms no capsules (18). Nevertheless, some Zymomonas strains are motile by 1-4 polar flagella. The possession of mobility may offer the bacteria an advantage to overcome the repulsive force and reach the particle-liquid interfaces, which tend to accumulate higher concentrations of nutrients such as sugars, amino acids, and inorganic ions than the rest of the aqueous phase. The results of continuous UFAB ethanol fermentation of a cottage cheese whey medium by S. fragilis attached to cellulose acetate are summarized in Table 11. Fermentor productivity increased with increased dilution rate due to faster growth of the microorganism a t the higher dilution rate. However, ethanol concentration decreased with increasing dilution rate, which was also reflected by increasing residual lactose concentration. In a CSTR the maximum productivity can only be obtained theoretically at a dilution rate close to the maximum specific growth rate. Thus, without attachment of yeast cells to cellulose acetate, the cell would have been washed out a t a dilution rate higher than the maximum specific growth rate. The highest productivity obtained was 6.9 g/(L.h) at dilution rate of 1.1 h-l, which was almost 4 times the maximum specific growth rate, i.e., 0.28 h-l, obtainable in batch culture (15) by using same medium composition. One
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Figure 1. Diagram of experimental apparatus used for continuous UFAB ethanol fermentation. Table 11. Summary Results of Continuous UFAB Fermentation with S. fmgilisUsing Cottage Cheese Whey as Substrate rad dilution, lactose, ethanol, productivity, conversion, yield, h-' g/L g/L g/ (L-h) ?6 75 ~
0.06 0.10 0.19 0.30 0.50 0.90 1.25 Y
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Figure 2. Attachment of S.fragilis to celluloseacetate (400X).
23.6 28.4 58.3 72.3 76.2 81.8 88.1
76.4 71.6 41.7 27.7 23.8 18.2 11.9
90.7 85.4 84.6 85.0 82.5 81.9 79.8
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disadvantage of using cellulose acetate was the poor settling velocity of this powder. Substantial amounts of this material with attached yeast cells floated out of the fermentor with rising carbon dioxide. The results of continuous UFAB ethanol fermentation of a defined glucose medium by 2. mobilis attached to vermiculite are shown in Table 111. The first eight samples collected at the dilution rate of 0.125 h-l were without pH control. The pH of the effluent during this period of operation dropped from the initial 5.0 to a range of 3.54.2. Such low pHs were considered to have a significant
2.2 3.1 3.4 3.7 5.0 6.9 6.1
Table 111. Summary Results of Continuous UFAB Fermentation with 2.mobilis ATCC 10988 Using Glucose as Substrate
0.125" 0.125 0.200 0.331 0.551 1.000 1.333 2.000 3.636 9.090
Figure 3. Attachment of 2. mobilis to vermiculite (400X).
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41.9 47.4 42.2 46.1 41.6 37.7 36.9 34.9 29.0 8.4
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86.6 97.8 89.1 94.3 90.6 85.0 86.2 82.3 64.2 19.7
90.5 88.5 87.3 90.3 93.2 '30.3 90.7 90.4 86.0 81.2
Samples collected without pH control.
detrimental effect on growth and fermentation rates. Hence, a two-sided pH control was aseptically set up (low point set at 4.6 and high point at 5.0) on the eighth day of the continuous operation. After pH was controlled, the concentration of ethanol in the product stream increased and fluctuated less. The dilution rate was subsequeqtly increased. As expected, the ethanol concentration decreased and the residual glucose concentration increased when the dilution rate increased. The fermentor productivity increased almost linearly up to a dilution rate of 3.6 h-l, where the maximum productivity of 105.7 g/(L-h) was obtained in this study. The possible reason for the declined productivity beyond a dilution rate of 9.1 h-l was that the system was operated at a situation where the shearing force of the fluid exceeded the adhesion force and cells were washed out consequently. Nonetheless, a complete washout was not observed in a period 4 times this hydraulic retention time, as evidenced
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by the carbon dioxide still being generated from the bed material. Thus, a UFAB system in contrast to a CSTR system can be operated as close as possible to the dilution rate, which gives highest productivity with less threat of complete washout. Figure 4 shows a time course plot of increasing ethanol concentration and decreasing residual glucose concentration at a dilution rate of 2.0 h-l. The experiment was initiated from one day of growth a t 0.067 h-l dilution rate. The dilution rate was then increased suddenly 30-fold to observe the ability of the system to endure a shock loading of substrate. After 1h of operation, the broth in the fermentor was almost clear because most of the unattached single cells and microbial flocs were lost in the effluent. However, the ethanol concentration after 3 h of operation was recovered to 16.7 g/(L.h) and it increased almost linearly up to 38.8 g/(L.h) within another 4 h. The maximum productivity of 78.8 g/ (L-h) was virtually identical with the steady-state productivity obtained at the same dilution rate shown in the previous study. This system has the capability of reaching steady state in 1416 residence times when the dilution rate was abruptly switched up 30-fold. The rapid equilibrating capability would be extremely helpful to a high-productivity continuous fermentation in case a process upset such as contamination occurs and the system has to be shut down and restarted again.
Conclusions A comparison of this system to other previously discussed high-productivity systems indicates this system has many advantages. The productivity of this system is comparable to that of a membrane filtration fermentor and flocculent reactor and higher than a vacuum fermentor for Zymomonas. A membrane filtration fermentor is technologically prohibited by industrial substrate containing membrane-clogging impurities. A flocculent reactor is low in ethanol concentration, and a vacuum fermentor is economically not feasible. By exploiting a natural attachment process for cell immobilization, the UFAB bioreactor tends to eliminate microbial loss of viability overtime, which is frequently encountered in gel-
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immobilized cell systems. In addition, the UFAB system provided a high surface area to volume ratio for efficient use of fermentor volume by using small and discrete particles and reduced mass transfer resistance by using a tapered, fluidized bed design. These distinct features make the UFAB bioreactor both technically and economically sound for continuous ethanol fermentation. The attachment mechanisms of Saccharomyces and Zymomonas discussed in this article are somewhat speculative, and further studies of these attachment processes are required.
Literature Cited (1) Cysewski, G. R.; Wilke, C. R. Fermentation kinetics and
processeconomics for the production of ethanol. Report LBL-
4480; Lawrence Berkeley Laboratory: Berkeley, CA, 1976. (2) Cysewski, G. R.; Wilke, C. R. Rapid ethanol fermentation using vacuum and cell recyle. Biotechnol. Bioeng. 1977, 19, 1125. (3) Magaritis, A.; Wilke, C. R. The rotofermentor 11. Application to ethanol fermentation. Biotechnol. Bioeng. 1978,20, 727. (4) Lee, J. H.; Woodard, J. I.; Pagan, R. J.; Rogers, P. L. Vacuum
fermentation for ethanol production using strains of Zymomonas mobilis. Biotechnol. Lett. 1981, 3, 177. (5) Lee, K. J.; Lefebver, M.; Pagan, R. J.; Rogers, P. L. High productivity ethanol fermentation with Zymomonas mobilis using continuous cell recycle. BiotechnoL Lett. 1980,2,487. (6) Arcuri, E. J.; Worden, R. M.; Shumate, S. E., I1 Ethanol production by immobilizedcells of Zymomonas mobilis. Biotechnol. Lett. 1980,2, 499. (7) Greenshields,R. N.; Smith, E. L. Tower fermentation systems and their applications. Chem. Eng. (London) 1971,249,182. (8) Siva Raman, H.; Seetarama, R. B.; Pundle, A. V.; Siva Raman, C. Continuous ethanol production by yeast cells immobilized in open pore gelatin matrix. Biotechnol. Lett. 1982, 4 (61, 359. (9) HSU,W. P.; Bernstein, L. A new type of bioreactor employing immobilizedyeast. J.Am. SOC.Brew. Chem. 1985,43 (2), 100. (10) Sitton, 0. C.; Magruder, B. C.; Book, N. L.; Gaddy, J. L.
in Biotechnology in Energy Production and Conservation. Proceedings of the Symposium, Gatlinburg, Tennessee, 1978; Wiley: New York, 1979. (11) Chen, H. C.; Zall, R. R. Continuous fermentation of whey into alcohol using an attached-film expanded bed reactor. Process Biochem. 1982,17 (Jan/Feb), 20. (12) Zisman, W. A. Relation of the equilibrium contact angle to liquid and solid composition. Ado. Chem. Ser. 1964, 43, 1. (13) Baier, R. L. Surface properties influencing biological adhesion. In Adhesion in Biological Systems;Academic Press: New York, 1970. (14) Fletcher, M.; Latham, M. J.; Lynch, J. M.; Rutter, P. R. The characteristics of interfaces and their roles in microbial attachment. In Microbial Adhesion to Surfaces;Ellis Horwood: Chichester, England, 1980. (15) Russell, E. W. In Soil Conditions and Plant Growth, 10 ed., Longmans: London, 1973. (16) Rutter, P. L. The adhesion of microorganisms to surfaces: physico-chemical aspects. In Microbial Adhesion to Surfaces; Ellis Horwood: Chichester, England, 1980. (17) Skotnicki, M. L.; Warr, R. G.; Goodman, A. E.; Rogers, P. L. In Genetics of Industrial Microorganisms; Proceedings of the Fourth International Symposium,Kyoto, Japan, June 6-11, 1982;Ikeda,Y.,Beppu,T.,Eds.; KodanshaLtd.: Tokyo,Japan, 1983; pp 361-365.- (18) Swings, J.; De Ley, J. In Bergey's Manual of Systematic Bacteriolom: Kriee. N. R.. Ed.: Williams & Wilkins: Baltimore/Lon&n, 19G; Vol. I, pp'576-580. Accepted April 9, 1991. Registry No. EtOH, 64-17-5.
