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J. Gen. Appl. Microbiol.

, 41, 461-473 (1995)





AKIO NISHIWAKI* AND JOHN R. BOURNE' Department of Materials Chemistry and Bioengineering, Oyama National College of Technology, Oyama 323, Japan ' Technisch-Chemisches Laboratorium ETH , CH-8092 Zurich, Switzerland (Received October 17, 1994; Accepted September 19, 1995)

The steady-state performance of a multistage fermentor with cell filtering through a membrane and recycling of concentrated cells is studied numerically for continuous production of acetic acid. The tanks-in-series model is used to describe the flow behaviour of culture broth in the multistage fermentor. Kinetic expressions and parameter values are taken from the literature. The effects of the total number of stages, the dilution rate, the bleed ratio, the recycle ratio and the feed concentrations as operating conditions on fermentor characteristics such as the concentrations of total and viable cells as well as substrate and product in each stage, the cell viability, the acetic acid productivity and the substrate conversion were examined under the conditions of equal tank volumes and a recycle ratio equal to or greater than one. An increase in the number of stages and decreases in the bleed and recycle ratios and in the feed concentration of product enhance the productivity. The relations between the maximum productivity and dilution rate and between the corresponding four components and the optimum dilution the corresponding optimum outlet concentrations of the rate are presented as figures. rate feed

Equations for the average cell viability and the critical dilution causing cell washout are also obtained. Furthermore, the minimum

concentration of substrate for its complete consumption is mentioned. Compared to a single tank fermentor, staging and cell recycle increase substrate utilisation and acetic acid productivity. The computed performance of the fermentor configuration proposed in this study is sufficiently now promising for the continuous be checked experimentally. production of acetic acid that it should

* Address Bioengineering,

reprint Oyama

requests National


Dr . Akio

Nishiwaki, 771

Department Nakakuki,


Materials 323,

Chemistry Japan.



of Technology, 461


the latter method is not yet familiar for the case of acetic acid production. thus resulting in a remarkable difference between these two processes. a membrane filter module is attached to a single continuous stirred tank fermentor with a semi-closed loop for cell recycling. an analysis of the reactor performance of multistage systems is required for rational design and operations. cell growth and death. PERFORMANCE EQUATIONS The continuous steady-state operation of a multistage fermentor with cell filtering and recycling for acetic acid production is considered. Ghose and Tyagi (1) showed that productivity was increased by using two stirred tanks in series in place of a single tank fermentor. 1. The use of a tanks-in-series or a multistage column fermentor instead of the single tank in the above system may be expected to bring an overall increase in acetic acid productivity. multistage fermentor-separator operations will become more important as a practical method to enhance acetic acid productivity. in acetic acid fermentation is quite different from that in ethanol fermentation. Park and Toda (4) investigated high cell-density fermentation of acetic acid by using a membrane recycle bioreactor system and reported that a theoretical maximum acetic acid productivity of 123.52 h ' without bleeding was obtained in their simulation. it was not shown quantitatively how high productivity can be attained by a multiple stage system. In such a reactor system. Fresh medium supplied to the first stage (or tank) is considered to be sterile. 41 In recent years. Lee et al. For ethanol fermentation. Although these two methods for high cell-density culture are popular in researches aimed at efficient ethanol fermentation. (2) demonstrated that ethanol productivity could be further improved by using cell recycling together with one or two tanks. however. various attempts to improve the productivity of acetic acid by continuous fermentation have been made (3-6). Also. the effect of product inhibition. Most recently. Although Park and Toda (4) also recommend to reuse the bleed which is rich in viable cells from the first cell-recycle fermentor in the following fermentor for further acetic acid production. In the fermentor. One way of obtaining high production rates is to maintain more viable microbial cells in a fermentor compared to conventional batch or continuous production of acetic acid. a fermentor in this system is composed of either N completely mixed tanks connected in series with equal working volumes or a column partitioned into N stages with equal spacings. The uses of immobilized cells and cell recycle with membrane separation are favourable alternatives for this purpose. With recent notable developments in membrane performance. However.1 kg m-3 h. The purpose of this work was to elucidate numerically the effects of various operating parameters on the characteristics of tanks-in-series and multistage column fermentors with cell separation and recycle for acetic acid production. especially on specific product formation rate.462 NISHIWAKI and BOURNE VOL.I at a dilution rate of 3. As shown in Fig. Therefore. substrate .

The culture stream from the Nth stage is introduced to a filtering module to separate cells in the culture through a membrane and the concentrated cells are then recycled to the first stage. (4) The liquid residence time in the recycle loop is negligible. the culture broth in each stage is perfectly mixed and backmixing between adjacent stages is negligible. Hence. To maintain a stable operation. (2) For the column-type fermentor. the flow behaviour of the culture in the whole column as well as that in multiple tanks is expressed by the tanks-in-series model. the specific growth rate can be expressed by the following equation with product inhibition and without the substrate saturation constant in the Monod expression: [ O. 1 Scheme of a multistage fermentor.26h '. = (1) . According to Park and Toda (4). a steady-state in total cells cannot be achieved because of an unlimited accumulation of non-viable cells. Pm= 63. the following assumptions were made: (1) Kinetic expressions for acetic acid fermentation and their parameter values are available in the literature (4) and are stated below.1995 Multistage Fermentor for Acetic Acid 463 (a) (b) fermentor (b) with cell filtering column and recycling. (a) N tanks-in-series Multistage {1. Fig.(P~Pm)} n where . The recycle flow rate is denoted by rF. part of the culture broth is withdrawn from the Nth stage. (3) Membrane separation is perfect and thus the filtrate stream contains no cells. The bleed flow rate is denoted by BE Without such bleeding. consumption and product formation proceed. (5) No resistance to mass transfer exists and the rate of fermentation is determined by its kinetics. substrate and product.61.5 kg m 3 and n = 3. In deriving the mass balance equations for viable and total cells.

. volumetric flow rate of medium feed (m3h-'). min. parameter in Eq. r$XrN. opt. parameter in Eq. b = 386. specific production rate of acetic acid (kg kg-' h-'). v. ith stage or tank. bleed ratio. viable cell concentration (kg m-3). . parameter in Eq. c. . viable cells. v. r.yield of acetic acid based on ethanol consumed (kg kg-'). cell viability. X~._1l+r X~+ 1X1=0 (5) (6) (7) (8 ) (9 ) 10 (11) ND (u1-y)X1=0 1 1 ND gSlX1 0 ND L1X1=0 1 ND ~c. substrate and product in the first and the ith stages can be described as follows: i1. Pr. P. specific death rate (h-'). [Subscripts] av. Based on the above assumptions and kinetics.4 kg m-3 and Pf= l0 kg m-3 as feed concentrations in their experiments and determined the above kinetic parameter values. parameter in Eq. (2) (h). (l+r)Xr1-1-(l+r)Xt~+ l+r X. ~qs. acetic acid production and ethanol consumption are represented by Park and Toda (4) as follows. c. X. v=a+b. Z..8 and c =1.325h. m. parameter in Eq.=O (1+r)S1-1-(l+r)Si- ND gs. (3). Yp~s. i.18 kg kg 1.u-cp2 where a =1. N. substrate (ethanol) concentration (kg m-3). D. Park and Toda (4) used Sf=47. qs=v/Yps (4) (3) (2) where Yp~s =1. 41 Also. the specific rates of cell death.N.. r. respectively: y=klexp(k2D) where k1-0. dimensionless distance. (3) (h). product (acetic acid) concentration (kg m-3).059h -1 and k2-0. i. maximum. average. minimum. b. feed.X1=O l Nomenclature: a. total cell concentration (kg m-3). B. specific consumption rate of ethanol (kg kg-' h-' ). recycle ratio.(l +r)X~1+ ND D r$XN-(l+r)X1+ S f+rSN. optimum. factor by which cells in the recycle stream are concentrated by membrane filter. (1). dilution rate (h-').92 h-1. specific growth rate (h-'). implying that this yield coefficient was constant. total number of stages or tanks. n. k1. productivity of acetic acid (kg m-3h'). [Greek letters] j3.347 h.(1 +r)S1Pf+rPN-(l+r)P1+ i2.ul-r X. the steady-state mass balances for total cells. F. f.464 NISHIWAKI BOURNE and VOL. parameter in Eq.Xi=O 1 l . S.3. (3) (h-'). k2. (2) (h-'). critical.

v. (14) which is derived from Eqs. t from Eqs. Lt. (5)-(8) and using Eqs. (4). (5)-(12) combined with Eqs. by means of the constant mass yield of product from substrate Yp~s: Y P. (3) and (4) with P =P~. The concentrations of total and viable cells.=O 1 (12 ) In these balance equations. the following relation is obtained: This expression shows that the cell separator factor $ is fixed by selecting both the bleed ratio B and the recycle ratio r as operating conditions and increases with the decreases of B and r. B.1m)} l~n X =D(P-Pf)/v X~_ (BD + y)X/BD S=Sf-q5X/D (16) (17) (18) (19) The concentrations of the four components for N=1 are calculated from Eqs. r. the dilution rate D refers to the volumetric feed rate F divided by the total working volume of either all tanks in series or the whole column. When N ==1.+ ND v~X. Also. (16)-(19) in turn after obtaining the values of y and . respectively. and qs~in the ith stage are denoted in Eqs. The acetic acid productivity Pr is defined as follows: Pr-D(PN-Pf) (20) . respectively. (2) and (15). (15) and (17)-(19). (7). These concentrations are independent of the recycle ratio r. From the mass balance for either viable or total cells around the filter module. (5)-(12) by setting r = DO and using Eq. (1)-(4) and (13). (11) and (12).DO a special case of the multistage system. As a numerical technique. (1) and (13) : =BD+r (15) P=Pm {1-(IJ/. When r . substrate and product in each stage for a given set of N. D.1995 Multistage Fermentor for Acetic Acid 465 (l+r)P. the Newton-Raphson iterative method is applicable. the resultant equations as derived from Eqs. (1). -Pf 5= S f-S.the following equations (4) are obtained by setting N= 1 in Eqs. (8). The ith-stage substrate concentration S~is also calculated from the corresponding ith-stage product concentration P. Sf and Pf are obtained by solving numerically Eqs. (13) are identical to Eqs._1-(1+r)P.

the ith tank is equivalent to the ith stage of the column.. X. and Pf=0. The abscissa Z refers to the Figure 2 shows viable cells. as a parameter. Operating parameters are the total number of stages N. with as a parameter. The following fermentor analysis is carried out under the conditions of equal tank volumes and r >_ 1. Broken lines indicate N=1. As can be seen from the values of Fig. the corresponding axial profiles of S. and P~ are greater for a larger N.2 2. Xr~ and X~ in each stage are larger than those for N=1 and increase with an increase of N. Si and F. their stepwise concentration profiles for each N along the whole fermentor are almost flat.466 This definition means that NIsHIwMU and BOURNE acetic acid in the bleed stream VOL. respectively. On the other hand. lb. of total and viable cells. Also. 41 is recoverable as product. Xt. respectively. substrate and product in each total h-`. stage with D=1.1. of stages r=2. N of the calculated concentrations of total cells. substrate the total number of stages dimensionless distance from the top of a column fermentor as shown in Fig. Concentrations number B=0. RESULTS AND DISCUSSION Concentrations in stages an example and product. Sf=47. For a tanks-in-series fermentor.4 and 0-15 kg m-3 are used for Sf and Pf. the ranges 25-47. the recycle ratio r and the feed concentrations Sf and Pf. Also. in each stage.. the bleed ratio B.4kgm_3 .

and then decrease promptly. approaching their respective maximum concentrations. The relation between such a D~ and other parameters will be stated Fig. the washout of cells occurs because of insufficient residence time for cell growth. Figure 3 shows an example of the calculated effect of the dilution rate D on the final-stage concentrations of total cells. because product inhibition in the first stage is reduced.1995 Multistage Fermentor for Acetic Acid 467 S. the outlet concentrations SN and PN decrease and increase with increasing N. product Sf=47. and P. Examining the concentrations of the four components in each stage for B less than that in Fig. 3. the substrate multistage fermentor consumption are reduced and product formation compared to N=1. both XtN and XN increase. For each N. as D becomes large. for each N in this case increases slightly. However. substrate and product. concentrations as a parameter. Effect and r=2. in the first stage for the Thus. respectively. As a result.4 number of stages Broken kg m_3 and . of dilution with rate total on final-stage Pf=O.. SN and PN. lower considerably the increase larger because of further accumuof X. in this case brings higher and concentrations of the corresponding FN and SN. lines indicate of total N=1N and viable substrate B =0. while X<.05. XN. 2. it is found that the corresponding X. respectively. becomes lation of non-viable cells. the cell growth proceeds further and viable cells increase with increasing N compared to N=1. viable cells. cells. At a certain critical dilution rate D. Such increases lead to substrate consumption and acetic acid production to a high level in the following stages. with the total number of stages N as a parameter. respectively. XtN.

although vavincreases slightly with the increase of N. differences between these two values for each N and also between vavvalues for different N stages are withir. (5). except D very close to D~.l+ r B vavB IN (21) ND(1+r) 1. Solid indicate viability in each stage and average viability. both X1N and X~ are higher when more stages are used. Figure 4 shows a calculation example of the viabilities v~and vas. moreover. The equation for giving vas. Sf=47. The effects of D and N on SN are the inverse of those on PN. r=1. For each N. as in the above case of cell concentrations. cell viability in the whole D = l. the stepwise profile of v.1. Eq. 4. and dotted lines The broken line . (6). (9) and (10) and combining them to eliminate Lt and then Xr~by substitution. PN decreases with increases in D and alsc becomes higher for larger N values. along the entire fermentor tends to a convex one with increasing N and thus vN becomes less than the corresponding vas. the bleed ratio B and the recycle ratio r on vas. it is found that the value of vavincreases with increasing D and B and is almost independent of r.l+ r +y When N-1 or r= oo. in each stage refers to X1/Xr~ the average and cell viability vavin the whole fermentor to the arithmetic mean of v.468 NIsHIWAKI and BOURNE VOL. Using Eq. which is defined here as a constant v~ (-X/X1) can be obtained by setting X. =Xrwav in Eqs.1. Examining the effects of the dilution rate D. However.2 h-'. 10o. (21) is reduced to: V= BD+ BD y (22) Fig. respectively. B =0. The cell viability v. 41 later.. thf resultant equation for vavis represented as follows: 11N ND ( l+r) 1. (13). At any constant D less than D~. Cell viability The viability of cells is the fraction of viable cell concentration relative to total cell concentration. Cell viability in each stage and average fermentor with total number of stages as a parameter.1. indicates N=1.4 kg m-3 and Pf=0.

Critical dilution rate When the value of D becomes larger than a certain critical dilution rate D~. Eq. =Pf fora. When N =1 or r = c. Pr for each N and Pf increases and has its maximum L). The equation for D~ can be derived by setting P. (23) agree exactly with those in Fig. while the effect of B on D~ is great. using Eq. r=2. in Fig. cell washout occurs. (1) with P =Pf and y to Eq. the resultant expression for D. the value of D~ is determined by a trial-and-error method. at a certain of the dilution rate of product Pf as As D increases. Sf=47. Broken lines indicate N=1. 3. (21) agree well with the arithmetic means of v. Dotted lines refer to the relation between maximum productivity and optimum dilution rate. (23) also contains D~. (18) for N=1. 4 and also under other conditions. is written as: D~= N(1+r) 1'tT 1lB+ r . (23) is also applicable for other conditions. As shown by the dotted lines.4kgm-3 and Pf=10 (A) and 0 (B) kgm-3. Thus. (2) with D =Dr. it can be seen that the effects of N and r on D~ are not significant. Then Pr decreases steeply. the Fig.~N (23) where u refers to Eq. It is found that Eq. From calculation results for various conditions. B=0. as mentioned above in Fig. The values of D~ calculated from Eq. . since the right-hand side of Eq. Furthermore. 3. Productivity Figure 5 shows the acetic acid productivity Pr as a function D with the total number of stages N and the feed concentration parameters. The values of vav calculated from Eq.05. (23) results in Eq. (6) and (10) and combining them to delete X~ by substitution.1995 Multistage Fermentor for Acetic Acid 469 This equation is identical to that obtained from Eq. Effect of dilution rate on acetic acid productivity with total number of stages and feed concentration of product as parameters. (13). in Eqs. 5. (15) with D = D~.

Ac R h. multistage fermentors are superior to single-stage fermentors. 47.3 and Pf= 0. Fig. PN and SN.ccm~ll Y . which is the minimum acetic acid concentration used for vinegar (4). at the same Doptas those in Fig. The relation between SN and Doptseems nearly symmetric with respect to a horizontal line of about 27 kg m-3 against the case of PN. The effects of N and r on Dopt are comparatively small or negligible. i. 6 for Prm. total 6. Hence. Relation of stages. Both Prm and D0 at r = for each N are identical to those for N=1 at the same B.. B. 8 shows the corresponding final-stage concentrations of total cells and viable cells. and between bleed P maximum ratio 0. Such values of Prm and Doptwere determined for various sets of N. B and r as parameters in the case of Sf= 47. 6 for Prm. Although Prm becomes high with increasing N. PN at constant N and r becomes slightly higher for a smaller B. ra~cac firct ararlrlallu and than rc r rlly rlrla tc crrnwlnn 0opt (hFig. Also. and productivity recycle ratio line and optimum dilution rate with number Sf as parameters. to N=1. Figure 7 shows the corresponding final-stage concentrations of product and substrate. PN in most cases is over 40 kg m-3. at the same Dopt as those in Fig.rr mc. r. respectively. Prm for multiple stages is higher at any B and r compared to that for N=1 and increases with decreasing both B and r. only the lines of r =1 are shown in the figure. For N = 3 and 5.4 kg m. Also. the increase of Prm per stage for multiple stages is largest when N = 2.470 NISHIWAKI and BOURNE VOL. the optimum dilution rate Doptgiving Prm is almost independent of N and is larger for a smaller Pf. Similarly.4kgm_3 The broken refers .2. 41 attainable maximum productivity Prm becomes higher with increasing N and with decreasing Pf. except for the range of B larger than about 0.e.. equal to the concentrations in the filtrate and bleed streams. Sf and Pf as operating conditions. PN at a constant B increases with increasing N and with decreasing r. Figure 6 presents the relation between Prm and Doptwith N. XtNand XN.. respectively.

both Prm and D0Pt are found to decrease with increasing Pf. same 7. Also. same 8. there is no effect of the feed concentration of substrate Sf on Prm and D0 because Eqs. of product and substrate at the optimum Fig. 6 and the relation between XN and D0 for constant N and r values is almost linear. the influences of B and r on them for a constant Pf are similar to those in Fig. 6. Corresponding dilution rates final-stage as those concentrations in Fig. (6). (9) and (l0) are independent of Sf and S1. 6.1995 Multistage Fermentor for Acetic Acid 471 Fig. B and r on XN are similar with those on Prm in Fig. In the case that the feed concentration of product Pf is nonzero. (5). On the other hand. of total and viable cells at the optimum accumulation of non-viable cells. 6. . The effects of N. Corresponding dilution rates final-stage as those concentrations in Fig. AtN also increases with increasing IV and with decreasing r.

B and r on the substrate conversion for a constant Sf are the same as those either on PN at a constant Pf or on Pr at a constant D. The effects of operating parameters on various fermentor characteristics were examined and the following results were obtained. Hence. 6 for Prm and Dopt. The difference between the cell viability in each stage and the average cell viability in the whole fermentor is not significant and the average viability is given by Eq. the substrate conversion becomes high with increasing N and with decreasing B and r. (14) with i =N and Eq. it is found that the conversion increases with decreasing Sf and as a result. (21). (20). As presented in Figs. its steady-state performance was investigated numerically for continuous acetic acid production. 3 for P -O). Sf for a feasible operation must be greater than Sfmin. examining the effect of Pf on Sfmin.472 NISHIWAKI and BOURNE VOL. as those in Fig. Examining the effect of Sf on the substrate conversion. the substrate conversion is found to increase with decreasing Pf. The critical dilution rate for the washout of cells is expressed by Eq. Determining such values of Sfminfor various given sets of N. from Fig. D. For example. This raises the overall substrate consumption and acetic acid production. The viable cell concentration in each stage for multistage fermentors is higher compared to a single fermentor and increases with increasing the total number of stages and with decreasing the bleed ratio. CONCLUSIONS Applying the tanks-in-series model and the kinetic expressions and parameter values from the literature (4) to a multistage fermentor with cell separation and recycle. 5 and 6. each left end of the solid and broken lines corresponds to complete substrate consumption (See Fig. these values agree well with those from the following equation: S _ fminI Pr Y p5D (25) which is obtained by setting SN = 0 in Eq. Also. 7 for SN and PN at D0 giving Prm. 5. as can be seen from Fig. except Sf. For the same conditions. B. (24). r and Pf by changing Sf. 41 Substrate conversion Using Eq. Also. the maximum acetic acid productivity increases with increasing the total number of . Sf minincreases with increasing N and with decreasing B and r. overall substrate conversion at the fermentor outlet is written as: Sf-SN _ PN-Pf _ Pr Sf YpsSf YpsSfD I I ( 24 ) This equation means that the effects of N. by comparing Pr values at a constant D. (23). there exists a minimum feed concentration of substrate Sfmin for complete substrate consumption in the final stage. In this figure. it is found that Sfmin increases with decreasing Pf.

App!.. Simulation study on bleed effect in cell-recycle culture of Acetobacter aceti. 25.. and Wandrey.. Biotechnol. 69. 34. Production of vinegar by immobilized cells. A. Ethanol fermentation with cell recycling: Computer simulation. 344-349 (1990). (25) holds. Product and substrate inhibition and optimization of fermentor design. Gen. Rapid ethanol fermentation of cellulose hydrolysate.. M. 21. 20. 102-110 (1990). the total cell concentration then becomes very high. the optimum dilution rate giving the maximum productivity increases with decreasing the bleed ratio and the feed concentration of product. Continuous production of acetic acid using immobilized Acetobacter aceti in a three-phase fluidized bed bioreactor... Von Eysmondt. J. 221-233 (1990). REFERENCES 1) 2) 3) 4) 5) 6) Ghose.. Lee. II. as shown in Fig.. There exists a minimum feed concentration of substrate for its complete consumption and Eq. R. J. S. G.. Biotechno!. Mori. Microbiol. C.. T. K. Park. and Coulman. 1 is based on the kinetic model of Park and Toda (4) and is sufficiently promising for the continuous production of acetic acid that it should now be checked experimentally. Process Biochem. K.. S. D. 67-74 (1985).. Although the maximum productivity can be enhanced by reducing the bleed ratio. 497-511 (1983). The substrate conversion increases with decreasing the feed concentration of substrate. J.... F. App!.. and Furusaki. 1401-1420 (1979). J. Y.1995 Multistage Fermentor for Acetic Acid 473 stages and with decreasing the bleed ratio. Acetic acid production by Acetogenium kivui in continuous culture-Kinetic studies and computer simulations. Also. Biotechnol. and Toda. and Tyagi. . Pollard. Bioeng. Microbiol. Sun. Bioeng. the recycle ratio and the feed concentration of product. D. A. Y. 36. Bioeng. J. Ferment. The computed performance of the fermentor configuration proposed in Fig. Vasic-Racki. 8.