Bioprocess Engineering 7 (1992) 343-348
Bioprocess Engineering
9 Springer-Verlag 1992
Enhancement of metabolic rates
of yeast flocculent cells through the use of polymeric additives
N. Lima, J. A. Teixeira a n d M . M o t a *, P o r t o
Abstract. The influence of several polymeric additives on specific glucose uptake rate of flocs of a S. cerevisiae strain - S. cerevisiae NRRL Y 265 was studied. A special continuous membrane micro- reactor was used to measure glucose uptake on the presence of cal- cium and of the tested additives - two cationic polymers - bis(poly- oxyethylene-bis(amine)) 20,000 and BPA 1,000 and one anionic polymer -- Magna Floc LT25.
An increase on glucose uptake rate was always observed when comparing with calcium bound flocs. For bis(polyoxyethylene- bis(amine)) 20,000 the increase was only 19% but for BPA 1,000 a value of more than 50% was observed. For Magna Floc LT25 a two fold increase was measured.
The determination of floc size and porosity in the presence of the additives indicated that, on the basis of these parameters, it was not possible to explain the observed glucose uptake rates. The floc porosites in additive bound flocs were similar and 10% larger than for calcium bound flocs and glucose uptake rate was larger for the largest flocs - Magna Floc LT25 bound flocs were the largest fol- lowed by BPA t,000, bis(polyoxyethylene-bis(amine)) 20,000 and calcium bound flocs. These values disagree with what should be expected in diffusion controlled processes.
The calculation of intercellular floc distance indicated that poly- meric additives act on the reduction of diffusional limitations by increasing the available flux area for glucose inside the flocs. By analysing different kinds of packings, it was also observed that the packing arrangement for yeast cells in flocs is close to the cubic packing. The simulation of this arrangement for the obtained floc sizes confirmed that the 10% increase in floc porosity is sufficient to explain the increase in the available flux area.
1 Introduction
O n e of the m a i n a d v a n t a g e s of the systems t h a t use flocculat- ing m i c r o o r g a n i s m s - bacteria o r yeast - is the possibility of o b t a i n i n g high cell density concentrations, thereby increas- ing the volumetric conversion rate of the r e a c t o r [ 1 - 8 ] .
However, m i c r o b i a l aggregates are characterized b y rela- tively low specific reaction rates. N u t r i e n t s have to reach the cells inside the flocs by diffusing into the floc particles [ 9 - 1 3 ] .
The diffusion of a solute into the floc particles, expressed as effective diffusivity is a function of:
- solute diffusivity in the m e d i u m - floc size
- floc p o r o s i t y - floc t o r t u o s i t y
I n most cases, the substrate diffusion rate t o w a r d s the in- trafloc cells is lower t h a n the cellular m e t a b o l i c rate a n d therefore substrate c a n n o t be m e t a b o l i z e d at the highest possible rate. In such cases, it is said that the overall reaction is mass transfer limited r a t h e r t h a n biochemically limited. I n o r d e r to o b t a i n m a x i m u m productivity, m a s s transfer limita- tions s h o u l d be minimized.
A d j a c e n t cells of flocculent strains are linked to each other t h r o u g h bridges where calcium ions p l a y an i m p o r t a n t role [14-18]. One possible w a y to circumvent mass transfer limitations could be the use of additives t h a t m a y act as bridge extenders, thereby enlarging the accessible space be- tween cells [19].
In this work, the m e a s u r e m e n t of specific glucose u p t a k e rate in a c o n t i n u o u s m i c r o r e a c t o r allowed for the analysis of the influence of several additives in the r e d u c t i o n of diffu- sional limitations in a flocculating strain of S. cerevisiae - S. cerevisiae N R R L Y 265.
Simultaneous floc size analysis a n d intrafloc void volume d e t e r m i n a t i o n s were m a d e to try to correlate the change in glucose u p t a k e rate with the physical characteristics of the flocs t h a t c o n t r o l effective glucose diffusivity.
2 Materials and methods 2.1 Materials
Two strains of Saccharomyces cerevisiae were used:
- flocculating one - S. cerevisiae N R R L Y 265 - a non flocculating one - S. cerevisiae sake
The medium, per cubic meter of t a p water, was c o m p o s e d of K H 2 P O 4 5 kg
(NH4)2SO 4 2 kg
M g S O 4 " 7 H 2 0 0 . 4 k g Yeast extract 1 kg
344
The concentration of calcium chloride, when used, was
1 " 1 0 - 3 M .
The carbon source was glucose, whose initial concentra- tion was 50 kg m - 3 for yeast growth and 5 kg m - 3 on the experiments for the determination of specific glucose uptake rate. The pH was, in all experiments, adjusted to 4.0 + 0.1 by the addition of H3PO 4. The temperature was controlled at 30~ For yeast growth, medium was autoclaved at 121 ~ during 30 minutes.
2.2 Cell preparation
Cells of both strains were grown for 24 hours in 1 liter Erlenmeyer flasks containing the above described medium.
Then they were harvested by centrifugation. The flocculent cells were deflocculated by washing three times with a NaC1 15 kg m - 3 solution at pH 2, after that they were washed three times with ultrapure water. Flocculent cells were then ready to be incubated in the 5 kg m - 3 glucose medium with the desired additive.
Such treated cells were used for measuring floc size, floc porosity and glucose specific uptake rate.
S. cerevisiae sake cells were only washed three times with ultrapure water before being added to the desired medium.
2.3 Analytical
Glucose concentration was measured by the DNS method [20].
Biomass concentration was determined by measuring biomass dry weight [21].
2.4 Additives
The charged polymers added to the medium were:
- Bis (polyoxyethylene-bis (amine)) 20,000 - (P 20,000) - BPA 1,000
- Magna Floc LT25
Bis(polyoxyethylene-bis(amine)) 20,000 is a cationic te- travalent polyethylene glycol derivative with a molecular weight of 20,000 (obtained from S I G M A Co.). BPA 1,000 is a cationic polymer (quaternary amine) of styrene-divinyl- benzene (obtained from R O H M & HASS Co.). Magna Floc LT25 is a high molecular weight anionic polyacrylamide (obtained from A L L I E D C O L L O I D S Ltd.).
In all experiments the additives concentration was 0.01%
(w/v).
2.5 Determination of sedimentation capacity
The flocculation capacity of S. cerevisiae flocculating strain was assayed using a modification of the Helm sedimentation test [15].
After preparing the cells as previously described the sed- imentation capacity was measured as follows:
Bioprocess Engineering 7 (1992) The cells were suspended in a 25 9 1 0 - 6 m 3 beaker con- taining a 1 9 10-3 M CaC12 ultrapure water solution and 0.01% of the desired additive. At defined intervals, samples were taken from a fixed position in the beaker (level corre- sponding to 15 - 10 -6 m3).
The normalized cell concentration, defined as the ratio between actual and initial cell concentration was plotted against sedimentation time. A sedimentation profile was obtained.
Initial cell dry weight concentration in the beaker was 4 k g m -3.
2.6 Measurement of specific glucose uptake rate
The determination of this parameter was made in a specially designed continuous microreactor (Fig. 1). It consisted of a 0.05 m thick plexiglass system formed by two chambers sep- arated by a 0.45 9 10 -6 m membrane filter. The lower cham- ber had a volume of 40.45.10 -6 m 3 and was stirred with a magnetic bar. The volume of the upper chamber was 4.81 9 1 0 - 6 m 3.
The cells were resuspended with the medium containing the desired additive and placed in the lower chamber of the microreactor. After completely tilling this chamber with this medium, the system was sealed and feeding started. Fresh medium with the additive was fed to the microreactor by a peristaltic pump at a dilution rate of 2 h - 1. Samples were collected from the outflow at defined intervals to determi- nate glucose consumption. The cell dry weight was deter- mined after each experiment.
2.7 Floe size analysis
Floc analysis was done by wet sieving the flocs through sieves of standard size.
Flocs treated as described in a previous section were placed in contact with the medium that contained the de- sired additive inside a 250 - 1 0 - 6 m 3 flask and stirred for 15 minutes at the same speed that was used for the determina- tion of the kinetic parameters.
I- lo3 -4
~ A
I . 6 0 ',,I
F i g . 1. E x p l o d e d v i e w o f t h e c o n t i n u o u s m i c r o r e a c t o r . A - F e e d i n l e t ; B - O u t l e t ; C - O - r i n g ; D - B u t t e r f l y s c r e w v a l v e s (all d i m e n - s i o n s a r e e x p r e s s e d i n 1 0 - 6 m )
N. Lima et al.: Enhancement of metabolic rates of yeast flocculent cells through the use of polymeric additives 345 The content o f the flask was then p o u r e d in the sieving
device: a g r o u p o f six s t a n d a r d size sieves - range 37 9 10 - 6 m - 1 8 0 9 10 - 6 m. This system was v i b r a t e d for 5 minutes at a low speed in o r d e r to minimize floc destruction.
2.8 F l o c p o r o s i t y a n a l y s i s
The p o r o s i t y , defined as the ratio between intrafloc water mass a n d total floc mass, was d e t e r m i n e d with a t h e r m o - gravimetric balance (0.01 m g precision) using the p r o c e d u r e described b y U r i b e l a r r e a [22].
2.9 F l o c s p e c i f i c g r a v i t y a n a l y s i s
F l o c specific gravity d e t e r m i n a t i o n was d o n e using a 2 5 . 1 0 - 6 m 3 picnometer. In this technique [9], the water mass (M0) displaced b y a k n o w n flocs mass ( M I ) was m e a - sured. F l o c s specific gravity is the r a t i o M 1 / M 0 . The flocs used in this p r o c e d u r e were treated as for floc p o r o s i t y anal- ysis.
3 R e s u l t s a n d d i s c u s s i o n
W h e n testing the influence of flocculation additives on glu- cose c o n s u m p t i o n rate, o u r initial p u r p o s e was to check whether these additives w o u l d change the s e d i m e n t a t i o n ca- p a c i t y of the flocs.
The results o b t a i n e d with the s e d i m e n t a t i o n tests (Fig. 2) clearly showed that the flocculation ability of S. cerevisiae N R R L Y 265 flocs was not changed by the presence of the tested additives. The o b t a i n e d s e d i m e n t a t i o n profiles were the same in every case.
After confirming the m a i n t e n a n c e of the flocculation ca- p a c i t y of the yeast flocs, the m e a s u r e m e n t of specific glucose u p t a k e rate was made.
These m e a s u r e m e n t s were m a d e at a dilution rate of 2 h - 1 a n d d u r i n g a s h o r t p e r i o d ( 3 0 - 4 0 minutes). W i t h these experimental c o n d i t i o n s it was assumed that no cell g r o w t h w o u l d occur d u r i n g the experiments. Even assuming a m a x - i m u m specific g r o w t h rate as high as 0.4 h - 1, the cells w o u l d have a m i n i m u m d o u b l i n g time of 1.7 h which validates the former assumption.
In all experiments, the a g i t a t i o n speed of the magnetic b a r was 200 r.p.m., assessed b y a s t r o b o s c o p i c tachometer. This speed, as previously shown [13], allowed for the elimination of external diffusional limitations in the flocs.
C o n s i d e r i n g b i o m a s s c o n c e n t r a t i o n X ( k g m - 3 ) , ex- pressed as cell d r y weight, a n d S (kg m - 3) the glucose con- c e n t r a t i o n in the m e d i u m at time t, the specific glucose con- s u m p t i o n rate is defined as:
+ qs = -- 1 I X . d S / d t (1)
a n d so
1 / 2 ( . d S = - q s " d t (2)
100
% 80- 60- ,40- 20.
0 i I "1" 4 min 5
1 2 3
Fig. 2. Floc sedimentation profile for the tested additives. 9 - Ca 2 +;
P- - bis(polyoxyethylene-bis(amine)) 20,000; 9 - BPA t,000; o - Magna Floc LT25
Table 1. Operating conditions and results for the diffusion experi-
ments for the flocculating strain
Additive Initial glu- Dry - qs - qs Correla- cose conc. weight (kg k g - 1 (av) tion co- (kg m - 3) (kg) 9 s - 1) efficient
Ca 2+ 5.81 0.342 6.00.10 -5 0.999
5.04 0.450 P20,000 4.98 0.383
5.62 0.174
BPA 1,000 5.67 0.152
4.58 O.286
Magna 5.48 0.146
Floc LT 25
5.54 0.142
5.82 10 -5 6.80 10 -5 7.28 10 -5 8.50- 10 -5 9.23 10 -s 11.38 10 -5 11.87 10 -5
5.92 - 10 -5 __ 1.3 9 10 -6
7.05 9 I 0 - 5 __3.3.10 -6
8.90 - i0 -5 +5.3.10 -6
11.67.10 -5 ___3.3.10 -6
0.999 0.999 0.998 0.999 0.999 0.997 0.999 Thus, qs can be calculated by the slope of the straight line o b t a i n e d by p l o t t i n g S / X against time. Table 1 displays the conditions of each experiment as well as the values o b t a i n e d for the specific substrate c o n s u m p t i o n rate a n d the correla- tion coefficients for each straight hne.
It is clear, from these results, t h a t the presence of the additive changed the glucose assimilation ability of the test- ed strain S. cerevisiae N R R L Y 265. The additive bis(poly- oxyethylene-bis(amine)) 20,000 h a d the smallest effect a n d only a 19% increase on specific glucose u p t a k e rate was observed. O n the other hand, there was a clear difference between the slope of the calcium straight line a n d the slopes of the BPA 1,000 a n d M a g n a F l o c LT25 straight lines. F o r BPA 1,000 the increase on specific glucose u p t a k e rate was 50% a n d for M a g n a F l o c LT25 there was a 2 fold increase in this parameter.
346 Bioprocess Engineering 7 (1992) Table 2. Operating conditions and results for the diffusion experi-
ments for the non flocculating strain
Additive Initial glu- Dry - q s - q s Correla- cose conc. weight (kg kg- ~ (av) tion co-
(kg m- ~) (kg) 9 s- 1) efficient
Ca 2+ 5.78 0.533 14.37 9 10 -5 0.998
14.57.10- 5 _+ 1.3 9 1 0 - 6
5.78 0.386 14.77 9 10- 5 0.999
Magna 5.22 0.528 15.63 9 10- 5 0.999
Floc LT 25 15.03 - 10- 5
+3.3 9 1 0 - 6
4.09 0.427 14.53 9 10- 5 0.999
Table 3. Measured floc parameters in the presence of the tested additives
Additives Ca z + P 20,000 BPA 1,000 Magna Floc
Parameter LT 25
Floc size 45_+5 59+__5 69+__6 127+7
(10-6 m)
Floc porosity 50.2+1.1 55.0+2.2 55.6___2.3 57.8+3.3 (%)
- q s ' l O - 5 5.92+0.13 7.05-+0.33 8.90+0.53 11.67___0.33
(kg kg- 1 s- 1)
Since the value obtained with M a g n a Floc LT25, was surprisingly high, we wondered if there was no influence of this polymer in the intrinsic metabolic activity of the yeast.
In order to clarify any possible doubt, the influence of the additive in the glucose uptake rate of a n o n flocculating strain of S. eerevisiae was tested. Experiments performed in the described microreactor with S. eerevisiae sake in the presence of C a 2 + and of the additive M a g n a Floc gave the results presented in Table 2. The obtained straight lines were parallel and there was no difference in the specific glucose uptake rate of cells in the presence of C a 2 + or M a g n a Floc LT25.
Also, as expected, the specific glucose uptake rate for S. cerevisiae sake was larger than for the flocculating strain in the presence of M a g n a Floe LT25.
This confirms that the two fold increase in substrate con- sumption observed for the flocculating strain is due only to the physical effects caused by the additive, which most prob- ably causes a reduction in floc internal diffusional limita- tions.
The next step was the determination of floe size and floc porosity (Table 3), since diffusional limitations are controlled by these two parameters. First, the evaluation of floc poros- ity was made, using a thermogravimetric method. It is clear from these determinations that, when compared to calcium, a statistically significant increase (t-test and 95 % confidence interval) was observed in floc porosity in the presence of the tested polymeric additives.
80
% 40
0 80
% 40
i i
0
t,O 120 10"6m2()0
dp
40 120 10-6m200dp
Fig. 3. Floc size distribution for the tested additives. 9 - Ca 2+, I~ - bis(polyoxyethylene-bis(amine)) 20,000; 9 - BPA 1,000; o - Magna Floc LT25
These values agree with reported data. F o r the same cells, Netto [23] reported a floc porosity of 50% in the presence of calcium and Teixeira and M o t a [13] reported an identical value for floes of K. marxianus, also in the presence of cal- cium. These authors also mentioned a 10% increase in floe porosity of K. marxianus in the presence of one of the tested additives - BPA 1,000.
This increase in porosity is not sufficient to explain such an increase in glucose consumption. Furthermore, the porosities for P20,000, BPA 1,000 and M a g n a Floe LT25 being similar, there are significant differences in glucose con- sumption rates.
The floc size for S. cerevisiae floes is the weighted average of the size distribution in the presence of the additives (Fig. 3). These values are a mean of six experimental determi- nations. It m a y be seen that the specific glucose uptake rate increases with the mean floe size, which disagrees with what should be expected in diffusion controlled processes.
The size distribution data, together with floc porosity determinations, indicated that some other factor must be responsible for the significant increase in glucose uptake rate. It must be reminded that a 2-fold increase in glucose uptake rate was observed in the presence of M a g n a Floc when comparing with calcium and that M a g n a Floe b o u n d floes are three times larger than Ca 2 + b o u n d floes.
Therefore it is of fundamental importance to find a pa- rameter that m a y constitute an explanation for the observed values of glucose consumption. F o r each additive, the dis- tance 6 between each individual cell in the floc was estimat- ed. As a matter of fact, this parameter is a measure of the available flux area for glucose. To estimate this parameter, it was necessary to measure flocs specific gravity - d. The obtained calcium b o u n d flocs specific gravity was 1.11. With this value, the specific gravity of the flocs b o u n d by the tested
N. Lima et al.: Enhancement of metabolic rates of yeast flocculent cells through the use of polymeric additives 347
•16_
6 ~ - q~ 10-Fig. 4. Proposed model for yeasts floc packing 5 -~
Table 4. Floc specific gravity and floc void volume for the tested additives
/ /
0 I I
0 011 0.2 0.3 10-6m 0.4.
Additives Ca 2 + P20,000 BPA 1,000 Magna Floc
Parameter LT25
Floc specific gravity l . l t 1.10 1.10 1.09 Floc void volume (%) 55.7 60.5 61.2 63.0
Fig. 5. Variation of specific glucose uptake rate q~ with floc intercel- lular distance 6 for the tested additives. 9 - Ca 2 +; 9 - bis(poly- oxyethylene-bis(amine)) 20,000; 9 - BPA 1,000; o - Magna Floc LT25
additives was calculated. These values and the c o r r e s p o n d - ing floc void volumes are presented in Table 4.
The a d d i t i o n a l following a s s u m p t i o n s were m a d e :
- Yeast cells are a p p r o x i m a t e l y spherical with a d i a m e t e r of 6 . 1 0 - 6 m.
- The distance between yeast cells in C a 2 + flocs is zero. In o t h e r words, this means that with S. cerevisiae cells the m i n i m u m floc void v o l u m e is 55.7%.
- The additive molecules are rigid a n d define the m i n i m u m distance 6 between each individual cell in the flocs, accord- ing to the scheme (Fig. 4).
- The distance 5 is the additive average length.
The system formed by individual cells b o u n d by an addi- tive behaves, for p a c k i n g considerations, like a set of spheres with a (6 + 5) 9 10 - 6 m diameter, p a c k e d as if they were yeast cells b o u n d b y Ca 2+ molecules with a d i a m e t e r of ( 6 + 5 ) 9 10 . 6 m.
The calculated distance between individual cells 5 (10- 6 m) in the flocs a n d specific glucose u p t a k e rate qs (kg kg - 1 s - 1) are correlated b y the e q u a t i o n :
- q s = 5 . 3 3 . 1 0 - 5 + 1 . 4 2 . 10 . 4 . 5 (3)
with a c.c. of 0.90 a n d a percent m e a n d e v i a t i o n of 10.5%.
This relation is p l o t t e d in Fig. 5.
C o n s i d e r i n g that the v a r i a t i o n of floc size a n d floc void v o l u m e c a n n o t explain the observed values of glucose up- t a k e rate it m a y be a d v a n c e d t h a t the tested additives act on the r e d u c t i o n of glucose diffusional limitations m a i n l y by increasing the intercellular distances in the flocs. P r o b a b l y , a small increase in the void volume, m a y increase significant- ly the intercellular distances, thereby changing the available flux a r e a of glucose or any o t h e r nutrient o r product.
A l t h o u g h this a p p r o a c h seemed to be a g o o d analysis of the available data, the relatively low coefficient of correla-
e
7 0
% 60
50
~ i ~ i ~ . ~ 0
\ ~ , . . ... ~.---. ~ . . . . . . . e
Q ~ ' . ~ . ~ ~ ~ ~ ~ ~ - - C
t0
40 80 120 10 -6 rn t60 dp
Fig. 6. Simulated floc void volume e for different floc sizes dp and different interparticle distances 5 for cubic packing, a: 6 = 0; b:
5 = 0.1; c: 5 = 0.2; d: 5 = 0.3; e: 5 = 0.4 (10 - 6 m). 9 -experimental values for Ca 2+ bound flocs; 9 - experimental values for P 20,000 bound flocs; 9 experimental values for BPA 1,000 bound flocs;
o - experimental values for Magna Floc LT25 bound flocs
tion led us to check, for different kinds of packing, how interparticle distance affects floc void volume.
There are four possible a r r a n g e m e n t s for m o n o s i z e d spherical particles [24]: the cubic, the o r t h o r h o m b i c , the te- t r a g o n a l sphenoidal a n d the r h o m b o h e d r a l . Theoretically, when the interparticle distance is zero, the available void volumes are, respectively, 47.64%, 39.54%, 30.19% a n d 25.95%. Therefore we only considered the simulation of the cubic packing, since the theoretical values for the void vol- umes of the other packings are m u c h lower t h a n the experi- m e n t a l results.
F o r the cubic packing, the simulated floc void volumes for different floc sizes a n d different interparticle distances a n d the experimental values for floc size a n d floc void vol- ume for the tested additives are presented in Fig. 6.
348 Bioprocess Engineering 7 (1992) F r o m this analysis, several conclusions can be d r a w n :
- The p o r o s i t y increases as the interparticle distance in- creases.
- The p a c k i n g a r r a n g e m e n t for yeast cells flocs seems well characterized by the cubic packing. As a m a t t e r of fact, the calculated floc void volumes assuming this p a c k i n g ar- r a n g e m e n t and the calculated interparticle distances are close to the experimental values.
- The presence of the additive m a y explain the observed p o r o s i t y values, for yeast flocs.
This analysis reinforces the hypothesis t h a t the available flux area on yeast cell flocs is controlled by the a d d i t i o n of charged polymers.
It m a y be concluded that flocculation additives can be used to reduce diffusional limitations in the flocs a n d so increase the observed reaction rate. Such an i m p r o v e m e n t is accomplished by increasing the available flux area for so- lutes, inside the flocs. P o l y m e r i c additives - anionic and cationic - act by controlling the distance between individual cells in yeast flocs. This hypothesis is confirmed b y calculat- ing the floc void volume for cubic packing. Anyway, this analysis should be enlarged to a wider range of additives with well k n o w n physical and chemical properties, namely, dimensions, structure a n d charge. Also, it is i m p o r t a n t to m e n t i o n t h a t b o t h anionic a n d cationic additives m a y be used to reduce flocs diffusional limitations. Cationic addi- tives m a y act by b i n d i n g negatively charged yeast cell walls.
Anionic additives, m o s t likely, act by bridging calcium ions that are b o u n d to the yeast cell walls.
The utilization of p o l y m e r i c additives with the conse- quent increase in reaction rate m a y be of f u n d a m e n t a l im- p o r t a n c e to o b t a i n high p r o d u c t i v i t y flocculating systems.
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Received July 9, 1991
Manuel Mota (corresponding author) Jos6 A. Teixeira
Nelson Lima
Centro de Engenharia Quimica da Universidade do Porto Rua dos Bragas
4099 Porto Codex Portugal
