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A new method for pH stabilization of the lactoacidic fermentation
ELSEVIER
A new method for pH stabilization of the lactoacidic fermentation Ludmila
Peeva* and Georgy Peev?
*Institute of Chemical Engineering, Bulgarian Academy of Sci., Sofia, Bulgaria ?Department of Chemical Engineering, institute of Chemical Technology, Soj?a, Bulgaria
It has been established that the neutralization of lactic acid obtained during the lactose fermentation with the strain Lactobacillus casei can be accomplished by compatibility of the process with the enzyme hydrolysis of urea using immobilized m-ease. The kinetics offermentation ajier the exponential phase of cell growth and the kinetics of urea degradation have been investigated. Mathematical models for both bioconversions have been derived. On the basis of these models, an algorithm for determination of experimental conditions which allow regulation of pH within narrow limits has been developed. It has been shown that the compatibility under conditions determined in this way results in a considerable enhancement of the lactose bioconversion as compared to the process with the addition of a neutralizing agent such as a 12% (v/v) water solution of ammonia. The multiple 0 I997 Elsevier Science Inc. use of immobilized enzyme has been found to be limited.
Keywords: bioprocess
Lactic acid, lactose; modelling compatibility
of fermentation
kinetics; pH regulation
Introduction Lactoacidic fermentation is a process with a well-expressed inhibition by the reaction product. The inhibiting action of lactic acid is mainly due to the decrease in the pH of the fermentation medium in the course of the process.’ This results in an incomplete substrate conversion and relatively low final concentrations of the product. Several ways have been suggested to overcome this disadvantage: -
-
Selection of new strains with an increased adaptation at high acid concentrations in the nutrition medium;’ Inhibitor withdrawal from the fermentation medium in the course of the process. Various methods have been suggested to this aim such as extraction, adsorption, ion-exchange, electrodialysis, etc.;3-s Neutralization of the lactic acid obtained by periodic addition of sodium,4*9 calcium, or ammonium hydroxide7*“*” during the process.
This leads to a considerable dilution of the fermentation medium. An intensive mixing and a sensitive, quick responding regulation device are also necessary to maintain the pH within narrow limits in the whole reaction volume.
Address reprint requests to Dr. Georgy Peev, Dept. of Chemical Engineering, Inst. of Chemical Technology, 1756Sofia, Darvenitza, Bulgaria Received 2 November 1995; revised 14 November 1996; accepted 27 November 1996
Enzyme and Microbial Technology 21:176-181, 1997 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
by urea hydrolysis;
For these reasons, the neutralization complicates the process and increases its costs. These disadvantages could be avoided if the neutralizing agent could be obtained directly in the fermentation medium. Such a possibility is the enzyme hydrolysis of urea by urease which produces ammonium hydroxide. It permits the development of a new method based on the simultaneous running of two biological processes at equivalent rates in one reactor which rest&s in the direct production of ammonium lactate in the fermentation medium: Lrrctobacillus G&Q,
cosei
*
CH,CHOHCOOH
(1)
+ CO2 t
(2)
urease
CO(NH,),
-+ NH,OH
CHJHOHCOOH COONH4 + HZ0
+ NH,OH
+ CH,CHOH (3)
It could be expected that due to the precise maintenance of pH within optimum limits in the whole reaction volume, this method would result in a considerable enhancement of the lactoacidic fermentation. The aim of the present work is to prove the compatibility of the enzyme hydrolysis of urea with the lactoacidic fermentation, develop an algorithm for calcuIating the conditions for running these reactions at equivalent rates, and verify its applicability.
0141.0229/97/$17.00 PII SOl41-0229(96)00260-8
New method
Materials and methods
for pH regulation:
L. Peeva and G. Peev
(v/v) water solution of ammonia through a dosimeter by means of a peristaltic
pump.
Cells, nutrition media, and enzyme The L. cusei -NBIMCC-1013 strain used in all our experiments was supplied by the National Bank of Industrial Microorganisms and Cell Cultures (Sofia, Bulgaria). It was maintained on semisynthetic medium A consisting (in g 1-l) lactose, 11; yeast extract, 10; peptone, 10; CH,COONa, 5; MgSO, - 7H,O, 0.1; MnSO, * 4H,O, 0.05; agar, 20; distilled water, 1 1. The medium pH was adjusted to 7 by addition of H,SO, and NaOH. In order to obtain
an inoculum for experiments, the bacterial cells were transferred from the medium A onto a semisynthetic medium B (pH 6.8) which contained (in g) lactose, 11; yeast extract, 5.5; peptone, 12.5; KH,PO,, 0.25; CH,COONa, 10; MgSO, * 7H,O, 0.1; MnSO, - 4H,O, 0.05; and FeSO, * 7H,O, 0.05 in 1 1 distilled water. All media ingredients were supplied by Fluka (Buchs, Switzerland). The media A and B were sterilized at 121°C under an overpressure of 101.4 KPa for 20 min. The culture was incubated for 24 h at 38°C on a thermostated rotatory shaker (New Brunswick Scientific, Edison, NJ) at 100-120 rpm. The immobilized urease was supplied by the Department of Biotechnology in the Institute of Chemical Technology (Sofia, Bulgaria). The enzyme immobilization is realized on a cellulose carrier obtained by the method of Chen and Tsao from an 8.33% solution of cellulose in a mixture of acetone and dimethylsulfoxide (1: 1 v/v). The cellulose was obtained from acetylcellulose (waste films) by hydrolysis in a 0.5 N solution of NaOH in a mixture of water and ethyl alcohol (95:5 v/v). The method of immobilization and the properties of the product are described in details elsewhere. I2
Methods for analysis The concentration of lactose and lactic acid were determined by means of high resolution liquid chromatography following the method of Lazarus and Seymour.13 For this purpose, a PerkinElmer liquid chromatograph Series 10 with a Bio-Rad Aminex ion-exclusion column HPX-87H (300 X 7.8 mm) was used. Detection was accomplished by a refractive index detector LC-25 (Perkin-Elmer). Sulfuric acid 0.005 mol 1-l was used as a mobile phase with a flow rate of 0.5 ml min-‘. The cell concentration was found turbidimetrically from the suspension adsorption at 550 nm using a spectrophotometer for which a calibration curve had been obtained previously. The concentration of urea was determined spectrophotometrtally by its reaction with p-dimethylaminobenzaldehyde which produces a yellow-green complex compound.‘4 To a given volume of urea containing sample, 5 cm3 of p-dimethylaminobenzaldehyde (Merck Darmstadt, Germany) solution (5 mass %) in 2 N HCI (Merck) was added and after a dilution up to 50 cm3 with distilled water, the sample was kept for 15 min before measurement on a Specol 11 (Carl Zeiss Jena, Germany) at 420 nm using a 10 mm cuvette. A calibrated line was obtained experimentally in advance and its mathematical description used to calculate the urea concentration.
Experimental
program,
results, and discussion
The basis for searching the conditions for maintenance of a constant pH value by a compatibility of the two processes could be the assumption that during the simultaneous running they do not affect each other. From this prerequisite, the investigation of the kinetics of each process and its mathematical modelling is a preliminary condition for evaluation of the method feasibility.
Kinetics of lactoacetic fermentation In this case, the reactor was filled with 1 1 of fermentation medium B and 100 cm3 inoculum. Five different initial
concentrations of lactose were used: 13.7; 21.5; 30; 37.6, and 50 g 1-l. The maintenance of a constant pH value was realized by periodic addition of 12% (v/v) ammonium hydroxide. It has been previously establishedI that for this strain of L. casei, lactic acid production is mainly nongrowth associated. Similar results have been obtained with other strains;‘0 thus, a model for the second part of the bioprocess was inducted. This part begins approximately 12 h after the inoculation when the exponential phase of cell growth is ended and the lactic acid obtained in appreciable quantity starts its inhibiting action. An analysis of the mechanisms accounted for in various models’,‘0~‘6-‘9 show that the kinetics of lactic acid and biomass production can be described by the differential equations: dP -dt. = pX(l
- kiP”)
dX dt = l.~,,,X(l
- k,P”) - k,X
The coefficients in Eqs. (4) and (5) were found by an optimization procedure based on the simplex method using a TUTSIMB (Meerman Automation) dynamic simulator. The values obtained are given in Table I. It shows that the exponent (Y strongly depends on the lactose initial concentration and for the calculation of (Y, the following relationship was derived. OL= 6.13P,,,
- 0.056
(6)
P max is the theoretical
lactic acid concentration [mol l-‘1 which should be obtained after a complete fermentation of the substrate and is determined as P max= 4SL”
Experimentat
set-up
The experiments were performed in a bioreactor (Quickfit, England) with a volume of l-l stirred magnetically at 400-450 ‘pm. The temperature of the solutions was maintained at 37 2 1°C by means of a thermostat and a heating coil situated in the reactor. The pH of the reaction medium was measured using a combined sterilized electrode (Ingold, Switzerland) connected to a pH meter (Radelkis OP-21 l/l, + 0.02 pH units) and recorded by computer. A constant pH value of the lactose fermentation was maintained either by enzyme hydrolysis of the urea or by the addition of a 12%~
(7)
The values of the other coefficients deviate (Table I) in limits allowable for a biological process which depends on many factors and with further application of the model they were taken as p = 0.625 X 10e3 and k, = 0.2.
Kinetics of enzyme degradation
of urea
Water solutions of urea with concentrations of 0.5, 1.0, 2.0 and 5.5 g 1-l were subjected to enzyme hydrolysis. The maintenance of pH within the limits 5.5-6 was realized by
Enzyme Microb. Technol.,
1997, vol. 21, August
15
177
Papers Table 1 Coefficients in Eqs. (4) and (5) obtained with different initial concentrations of lactose and calculated compatibility of the lactose fermentation and urea bioconversion at T = 38°C and pH = 5.5-6 Coefficients Initial lactose concentration s, (g I-‘) 13.7 21.5 30 37.6 50
for
Conditions for compatibility in maintaining constant pH
in Eqs. (4) and (5)
Initial urea concentration suO (g 1-V
p x 103
e
k,
ki
l&B,
0.6 0.725 0.9 0.4 0.5
1.04 1.49 2.2 2.5 3.5
0.16 0.18 0.19 0.24 0.24
4 4 4 4 4
0.27 0.27 0.27 0.27 0.27
periodic addition of a 0.1 N water solution of The amount of the enzyme preparation was 2 experimental data confirmed that the process scribed by Michaelis-Menten kinetics. Applied acid neutralizing agent it has the form
conditions
lactic acid. g 1-l. The can be defor a lactic
5 8 11 12 15
Preparation amount M (g I-‘) 12.5 11 18.5 9 19
(4)-(6) allows calculation of the initial concentration of urea and the amount of enzyme preparation which are necessary to obtain an equivalent production of ammonia during the fermentation run. These conditions were found by numerically solving the set of Eqs. (4)-(6) and (8) at the requirement i=N
(8)
C
(Pi - P,,)* = min
(11)
i=l
Eq. (8) is obtained reaction. H*N-CO---NH2
accounting
for the stoichiometry
+ HZ0 = 2NI-&OH + CO2 T
of the
(12)
If the two biological processes do not influence each other, KS in Eq. (8) and k, in Eq. (12) can be taken from the investigation of urea hydrolysis. The enzyme quantity E is proportional to the preparation amount M and V talc max- Map M talc = (13) V exP max
dS, dP, -2-=-~-jl::dSII-l~~dP,~S, dt dt
The values of V,,, and KS were found to be 6.59 X lop4 mol 1-l h-’ and 0.0098 mol l-l, respectively.
Discussion and conditions for compatibility Figures la-lc represent a part of experimental data for the changes in lactic acid and biomass concentrations with time when the fermentation process is performed separately and the respective theoretical curves calculated by Eqs. (4)-(6). It can be seen that the coincidence for the lactic acid production is very good while the prediction of biomass production deviates considerably from the experiment with the increase in fermentation time. The last is probably due to the fact that the turbidimetric method determines the total amount of cells in the system. It gives satisfactory results in the initial stage of the process when the degree of cells dying is negligible. With the increase in this degree, the difference between the producing cells and those found by turbidimetry increases. Although the method has such a disadvantage, it is the most frequently used. The determinations of the living cells are too prolonged and inconvenient for a current control in the process. The exact prediction of the lactic acid production by Eqs. Enzyme Microb.
V max= k>E
(9)
from which there follows
178
Actually, the solution gives SuO and V,,, in Eq. (8) but the quantity of enzyme preparation follows from the relationship
Technol.,
1997, vol. 21, August
The results obtained in this way are given in Table 1. Figures la-lc show the outputs of ammonia with the time calculated by Eq. (8) at the respective urea initial concentrations and enzyme quantities. It can be seen that these outputs follow the lactic acid production with satisfying accuracy.
Simultaneous per$ormance of the processes In this case, the reactor was charged with 1 1 fermentation medium B and 100 cm3 inoculum. After 12 h, the necessary amounts of urea and enzyme preparation were added. Lactose initial concentrations of 11, 24.8, 31.4, and 43.4 g 1-l were used. Preliminary experiments show that 12 h after the inoculation, the concentrations of lactic acid and biomass were not completely reproducible despite the maintenance of constant process parameters (temperature, pH, etc.) and therefore could not be predicted in advance. These concentrations, however, are initial conditions (X = X,, P = P, at t = 0) of the set of Eqs. (4)-(6) and (8) and affect appreciably its solution for SU, and V,,, at the requirement.” This imposed the determination of X0 and P, in a sample of the fermentation medium 12 h after the inoculation and to calculate So,, and M on the base of the results 15
New method
for pH regulation:
L. Peeva and G. Peev
Table 2 Initial conditions
for simultaneous performance of lactose fermentation and urea hydrolysis at equivalent rates, T = 38°C. and pH = 5.5-6
24
Initial conditions for compatibility
8
Run No.
j 0 B
1 2 3 4
4
0
0.0 0
10
20 time
30
c
4
,’
20 time
1.0
30
z
40
[h]
,
I 0
J- 60
l
=
11.50 24.80 31.40 43.40
0.57 5.11 6.25 6.70
18.00 21 .oo 19.81 16.9
Urea, Enzyme S preparation, (g I”-“rr M (4 1-‘1 2.2’7 9.48 12.24 16.26
32.58 30.00 32.42 33.33
obtained. This procedure took approximately 10 min. Table 2 shows the initial conditions and calculated amounts of urea and preparation for representative experiments performed in the way described. Part of the data are illustrated in Figures 2a-2c as a dependence of the degree of lactose fermentation on time. The values of the medium pH during the process are also given. These demonstrate a small and smooth increase within the allowable limits. For a comparison, the conversions obtained under the same conditions (initial lactose concentration, temperature, quantity of inoculum, etc.) but with pH maintenance in the range 5.5-6 by adding a 12% (v/v) water solution of ammonia are shown. In all experiments, a considerable (2-3 times) enhancement of the lactose fermentation performed simultaneously with the urea enzyme hydrolysis can be observed. This acceleration could result from the maintenance of a constant pH at the microlevel in the whole reaction volume while, with the addition of ammonium hydroxide, some local pH fluctuations are probable due to nonideal mixing and the relatively slow reaction time of the pH probe. The time for homogenization in the reactor after an addition of neutralizing agent can be described by the equation
0
1
10
Biomass, x, (g I_‘)
[h]
20
0
Lactic acid, PO (9 I_‘)
40
1.0
0.0
Initial lactose concentration S,, (9 1-l)
Amount of urea and enzyme preparation added (calculated in advance)
0.8
rn = k,(D/d)’
0.0
/ 0
0 10
20
30
40
50
time [ h] Figure 1 Comparison of the experimental concentrations of lactic acid (0) and biomass (0) with those calculated by Eqs. (4)-(6) (solid lines) and expected production of ammonia at the designated conditions for compatibility (dotted lines) with initial lactose concentrations: 13.7 g I-’ (a), 30 g I-’ (b); and 50 g I-’ (c)
(14)
where k, depends on the mixer type, reactor geometry, and Reynolds number, Re. ” For a two-blade stirrer, nonbaffled vessel, and Re = 1.2 X 104, k, = 20.*’ This value for the Reynolds number corresponds to the conditions of urea bioconversion; therefore, the homogenization time for this process according to Eq. (14) should be 17 s (D = 10 cm, d = 4 cm). For the lactose fermentation, 7 should be much more because of the higher viscosity of lactose water solutions compared to urea ones. It is important to emphasize that the reported value of k, is for a degree of homogenization equal to 0.67. The local pH fluctuations could result in a (stress) decrease of the cell productivity. It is interesting to mention that despite the essential enhancement of the lactose fermentation in all experiments, the pH was held within the optimum limit of 5-6 (Figures 2a-2c). This implies that the enzyme hydrolysis of urea has also been accelerated due to the same reason. The biosystem used has a well-established
Enzyme Microb. Technol.,
1997, vol. 21, August
15
179
Papers ability for autocontrol owing to the mutual enhancement of the two processes mainly because of the smooth pH regulation within narrow limits. It can be calculated from Figures 2a-2c that the average productivity of the reactor varies from OS-l.19 g 1-l hh’. These values cannot be precisely compared with other studies due to the differences in the process conditions. In the lactic acid fermentation of sucrose by Lactobacillus delbreuckii at T = 39°C and pH = 6 (controlled by the addition of 2.5 N solution NaOH) a productivity of 0.313 g I-’ hh’ is reported.4 Lactose conversion by L. cusei at T = 37°C and pH = 6.5 (maintained by concentrated NH,OH) has produced 0.98 g 1-l hh’ lactate.” The last cells immobilized in Ca-alginate gel have converted glucose at a rate of 0.328 g 1-l h-l.’ For glucose, lactose and whey permeate fermentation by Luctobacillus helveticus at T = 42°C and pH = 5.9 (controlled by 3 N NH,OH), the reported productivity is 0.72-1.68 g I-’ hh’.” The extraction of lactic acid by ion-exchange resins has resulted in productivities of 0.4825 and 1.66 g 1-l hK’.4 These data show that the productivity obtained in our experiments is among the better ones for a batch fermentation process carried out by Luctobacillus. After each experiment, the immobilized urease was separated from the solution and was used with the following one. This multiple application of the preparation is of essential importance for the process economy. After a sequence of four cycles, however, a continuous loss of immobilized enzyme activity was observed. This phenomenon can be attributed to a mechanical destruction of the carrier and enzyme washing away or (as well as) to a prothease activity of L. cusei.
8
6%
4 dv/dt=0.131 2
0 20
30
40
50
time [h] 1.0
12
10
0.8
F ii i ‘0 g ._ $!
8 0.6 6% 0.4
z S 0.2
dr$dt=0.068
Conclusions 20
30
time [h]
F
0.8
8
b
$
0.6 -6
=Q
-4
The simultaneous performance of a lactose lactoacidic fermentation and a urea enzyme hydrolysis offers the possibility for smooth pH regulation of the system within narrow limits and considerable enhancement of the reactions. The calculation of experimental conditions necessary for maintenance of constant pH on the basis of models describing the kinetics of the processes performed separately results in their successful compatibility although there is the presence of a mutual influence. The multiple use of immobilized urease is limited. For the effective practical application of the method, a precise economic analysis is necessary.
List of symbols d
dr//dt=O.O18 q 50
D E
I 0
k,
-PH
0.0 0
-2
100
150
time [h] Figure 2 Lactose conversion (0) and pH dium (0) versus time with simultaneous mentation and urea hydrolysis compared pH = 5.5-6 with an addition of neutralizing initial concentrations: 11 g I-’ (a); 32 g I-’
180
Enzyme Microb. Technol.,
of the reaction meperformance of ferto the conversion at agent (0) at lactose (b); and 44 g I-’ (c)
1997, vol. 21, August
Stirrer diameter [ml Reactor diameter [ml Enzyme quantity [mol l-‘1 Coefficient accounting for the cells dying
W’l ki k, KS M
15
Coefficient of inhibition by the reaction product [1 mol-‘1 Rate constant of enzyme-substrate complex decomposition [hh’] Michaelis-Menten constant [mol l- ‘1 Amount of the immobilized urease preparation [g l- ‘1
New method
; p, Re = nd21v sl_ &I t T Vmax X a P rl CL max V
T
Stirrer revolutions [s- ‘1 Lactic acid concentration [mol l-‘1 Concentration of ammonia [mol l-‘1 Reynolds number Lactose concentration [mol l-l] Concentration of urea [mol l-‘1 Time of the process [h] Temperature Maximum rate of urea degradation [mol l-- ’ h-‘1 Concentration of biomass [g l-‘1 Exponent Biomass productivity coefficient [mol g-- ’ h-‘1 Conversion degree Maximum cells specific growth rate [h- ‘1 Reaction media kinematic viscosity [ml s-l] Time for homogenization in reactor [s]
6.
7.
8. 9.
10.
Il.
12.
13.
Subscripts 14.
0
exp talc
Initial Experimental Calculated
15.
16.
References 1. 2.
3.
4.
5.
Yeh, P. L.-H., Bajpai, R. K., and Iannotti, E. L. An improved kinetic model for lactic acid fermentation. J. Ferm. Bioeng. 1991,71(l), 75-77 Demirci, A. and Pometto, A. L. Enhanced production of D(-)-lactic acid by mutants of Lactobacillus delbrueckii ATCC-9649. J. Ind. Microbial. 1992, 11(l). 23-28 Davison, B. H. and Scott, D. C. A proposed biparticle fluidized-bed reactor for lactic acid fermentation and simultaneous adsorption. Biotechnol. Bioeng. 1992,39, 365-368 Srivastava, A., Roychoudhury, P. K., and Sahai, V. Extractive lactic acid fermentation using ion-exchange resin. Biotechnol. Bioeng. 1992. 39, 607-613 Jianlong, W.. Ping, L., and Ding, Z. Extractive fermentation of lactic
17.
18.
19.
20.
for pH regulation:
L. Peeva and G. Peev
acid by immobilized Luctobucillus casei using ion-exchange resin. Biotechnol. Tech. 1994, S( 12), 905-908 Boyaval, P., Corre, C.. and Terre, S. Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis. Biotechnol. l&t. 1987. 9(3), 207-212 Nomura, Y., Iwahara, M., and Hongo, M. Lactic acid production by electrodialysis fermentation using immobilized growing cells. Biotechnol. Bioeng. 1987,3,788 -793 Yabannavar, V. M. and Wang. D. 1. C. Extrwtive fermentation for lactic acid production. Biotechnol. Bioeng. 1991. 37, 1095-I 100 Cachon. R. and Divies, C. Kinetics of lactate fermentation and citrate bioconversion by Lactocnccus luctis ssp. factis in batch culture. J. Appl. Bacterial. 1993. 75, 387-392 Roy, D.. Leduy, A., and Goulet, J. Kinetics of growth and lactic acid production from whey permeate by Luctobacillus hulveticus. Con. J. Chem. Eng. 1987. 65, 597-603 Taniguchi, M., Kotani. N., and Kobayashi, T. High-concentration cultivation of lactic acid bacteria in fermentor with cross-flow filtration. J. Ferment. Technof. 1987. 65(2), 179 -184 Krysteva. M.. Peev. G., and Sokolov, T. Continuous conversion of urea in wastewaters with the aid of urease covalently immobilized to granulated microcrystallized cellulose. Biotechnol. Chem. 1989. 1, 24-27 Lazarus, R. A. and Seymour. J. L. Determination of 2-keto-tgulonic acid and other ketoaldonic and aldonic acids produced by ketogenic bacterial fermentation. Anal. B&hem. 1986. 157. 360-366 Watt, G. W. and Chrisp. J. D. Spectrophotometric method for determination of urea. Anal. Chem. 1954, 26(3), 452-453 Kosseva, M., Beschkov. V.. and Pilafova, E. Modelling of lactic acid production from lactose by immobilized Lactobacillus rusei cells. Proc. Bioproc. Eng.. Sofia, Bulgaria, 1994, 75-82 Samuel, W. A., Lee. Y. Y.. and Anthony, W. B. Lactic acid fermentation of crude sorghum extract. Biotechnol. Bioeng. 1980, 22, 757-777 Luedeking, R. and Piret. E. L. A kinetic study of the lactic acid fermentation. Batch process at controlled pH. J. B&hem. Microbiol. Technol. Eng. 1959, 1, 393-412 Jorgensen. M. H. and Nikolajsen, K. Mathematic model for lactic acid formation with Streptococcus cremoris from glucose. Appl. Microbial. Biotechnol. 1987, 25, 3 13-3 16 Leh. M. B. and Charles, M. Lactic acid production by batch fermentation of whey permeate: A mathematical model. J. fnd. Microbial. 1989, 4, 65-70 Braginski, L. N.. Begachev. V. I., and Barabash. V. M. Mixing in homogeneous media. In: Miring in Liquid Media. Leningrad, “Chimia”. 1984, 110-l 13
Enzyme Microb. Technol.,
1997, vol. 21, August
15
181
A new method for pH stabilization of the lactoacidic fermentation Ludmila
Peeva* and Georgy Peev?
*Institute of Chemical Engineering, Bulgarian Academy of Sci., Sofia, Bulgaria ?Department of Chemical Engineering, institute of Chemical Technology, Soj?a, Bulgaria
It has been established that the neutralization of lactic acid obtained during the lactose fermentation with the strain Lactobacillus casei can be accomplished by compatibility of the process with the enzyme hydrolysis of urea using immobilized m-ease. The kinetics offermentation ajier the exponential phase of cell growth and the kinetics of urea degradation have been investigated. Mathematical models for both bioconversions have been derived. On the basis of these models, an algorithm for determination of experimental conditions which allow regulation of pH within narrow limits has been developed. It has been shown that the compatibility under conditions determined in this way results in a considerable enhancement of the lactose bioconversion as compared to the process with the addition of a neutralizing agent such as a 12% (v/v) water solution of ammonia. The multiple 0 I997 Elsevier Science Inc. use of immobilized enzyme has been found to be limited.
Keywords: bioprocess
Lactic acid, lactose; modelling compatibility
of fermentation
kinetics; pH regulation
Introduction Lactoacidic fermentation is a process with a well-expressed inhibition by the reaction product. The inhibiting action of lactic acid is mainly due to the decrease in the pH of the fermentation medium in the course of the process.’ This results in an incomplete substrate conversion and relatively low final concentrations of the product. Several ways have been suggested to overcome this disadvantage: -
-
Selection of new strains with an increased adaptation at high acid concentrations in the nutrition medium;’ Inhibitor withdrawal from the fermentation medium in the course of the process. Various methods have been suggested to this aim such as extraction, adsorption, ion-exchange, electrodialysis, etc.;3-s Neutralization of the lactic acid obtained by periodic addition of sodium,4*9 calcium, or ammonium hydroxide7*“*” during the process.
This leads to a considerable dilution of the fermentation medium. An intensive mixing and a sensitive, quick responding regulation device are also necessary to maintain the pH within narrow limits in the whole reaction volume.
Address reprint requests to Dr. Georgy Peev, Dept. of Chemical Engineering, Inst. of Chemical Technology, 1756Sofia, Darvenitza, Bulgaria Received 2 November 1995; revised 14 November 1996; accepted 27 November 1996
Enzyme and Microbial Technology 21:176-181, 1997 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
by urea hydrolysis;
For these reasons, the neutralization complicates the process and increases its costs. These disadvantages could be avoided if the neutralizing agent could be obtained directly in the fermentation medium. Such a possibility is the enzyme hydrolysis of urea by urease which produces ammonium hydroxide. It permits the development of a new method based on the simultaneous running of two biological processes at equivalent rates in one reactor which rest&s in the direct production of ammonium lactate in the fermentation medium: Lrrctobacillus G&Q,
cosei
*
CH,CHOHCOOH
(1)
+ CO2 t
(2)
urease
CO(NH,),
-+ NH,OH
CHJHOHCOOH COONH4 + HZ0
+ NH,OH
+ CH,CHOH (3)
It could be expected that due to the precise maintenance of pH within optimum limits in the whole reaction volume, this method would result in a considerable enhancement of the lactoacidic fermentation. The aim of the present work is to prove the compatibility of the enzyme hydrolysis of urea with the lactoacidic fermentation, develop an algorithm for calcuIating the conditions for running these reactions at equivalent rates, and verify its applicability.
0141.0229/97/$17.00 PII SOl41-0229(96)00260-8
New method
Materials and methods
for pH regulation:
L. Peeva and G. Peev
(v/v) water solution of ammonia through a dosimeter by means of a peristaltic
pump.
Cells, nutrition media, and enzyme The L. cusei -NBIMCC-1013 strain used in all our experiments was supplied by the National Bank of Industrial Microorganisms and Cell Cultures (Sofia, Bulgaria). It was maintained on semisynthetic medium A consisting (in g 1-l) lactose, 11; yeast extract, 10; peptone, 10; CH,COONa, 5; MgSO, - 7H,O, 0.1; MnSO, * 4H,O, 0.05; agar, 20; distilled water, 1 1. The medium pH was adjusted to 7 by addition of H,SO, and NaOH. In order to obtain
an inoculum for experiments, the bacterial cells were transferred from the medium A onto a semisynthetic medium B (pH 6.8) which contained (in g) lactose, 11; yeast extract, 5.5; peptone, 12.5; KH,PO,, 0.25; CH,COONa, 10; MgSO, * 7H,O, 0.1; MnSO, - 4H,O, 0.05; and FeSO, * 7H,O, 0.05 in 1 1 distilled water. All media ingredients were supplied by Fluka (Buchs, Switzerland). The media A and B were sterilized at 121°C under an overpressure of 101.4 KPa for 20 min. The culture was incubated for 24 h at 38°C on a thermostated rotatory shaker (New Brunswick Scientific, Edison, NJ) at 100-120 rpm. The immobilized urease was supplied by the Department of Biotechnology in the Institute of Chemical Technology (Sofia, Bulgaria). The enzyme immobilization is realized on a cellulose carrier obtained by the method of Chen and Tsao from an 8.33% solution of cellulose in a mixture of acetone and dimethylsulfoxide (1: 1 v/v). The cellulose was obtained from acetylcellulose (waste films) by hydrolysis in a 0.5 N solution of NaOH in a mixture of water and ethyl alcohol (95:5 v/v). The method of immobilization and the properties of the product are described in details elsewhere. I2
Methods for analysis The concentration of lactose and lactic acid were determined by means of high resolution liquid chromatography following the method of Lazarus and Seymour.13 For this purpose, a PerkinElmer liquid chromatograph Series 10 with a Bio-Rad Aminex ion-exclusion column HPX-87H (300 X 7.8 mm) was used. Detection was accomplished by a refractive index detector LC-25 (Perkin-Elmer). Sulfuric acid 0.005 mol 1-l was used as a mobile phase with a flow rate of 0.5 ml min-‘. The cell concentration was found turbidimetrically from the suspension adsorption at 550 nm using a spectrophotometer for which a calibration curve had been obtained previously. The concentration of urea was determined spectrophotometrtally by its reaction with p-dimethylaminobenzaldehyde which produces a yellow-green complex compound.‘4 To a given volume of urea containing sample, 5 cm3 of p-dimethylaminobenzaldehyde (Merck Darmstadt, Germany) solution (5 mass %) in 2 N HCI (Merck) was added and after a dilution up to 50 cm3 with distilled water, the sample was kept for 15 min before measurement on a Specol 11 (Carl Zeiss Jena, Germany) at 420 nm using a 10 mm cuvette. A calibrated line was obtained experimentally in advance and its mathematical description used to calculate the urea concentration.
Experimental
program,
results, and discussion
The basis for searching the conditions for maintenance of a constant pH value by a compatibility of the two processes could be the assumption that during the simultaneous running they do not affect each other. From this prerequisite, the investigation of the kinetics of each process and its mathematical modelling is a preliminary condition for evaluation of the method feasibility.
Kinetics of lactoacetic fermentation In this case, the reactor was filled with 1 1 of fermentation medium B and 100 cm3 inoculum. Five different initial
concentrations of lactose were used: 13.7; 21.5; 30; 37.6, and 50 g 1-l. The maintenance of a constant pH value was realized by periodic addition of 12% (v/v) ammonium hydroxide. It has been previously establishedI that for this strain of L. casei, lactic acid production is mainly nongrowth associated. Similar results have been obtained with other strains;‘0 thus, a model for the second part of the bioprocess was inducted. This part begins approximately 12 h after the inoculation when the exponential phase of cell growth is ended and the lactic acid obtained in appreciable quantity starts its inhibiting action. An analysis of the mechanisms accounted for in various models’,‘0~‘6-‘9 show that the kinetics of lactic acid and biomass production can be described by the differential equations: dP -dt. = pX(l
- kiP”)
dX dt = l.~,,,X(l
- k,P”) - k,X
The coefficients in Eqs. (4) and (5) were found by an optimization procedure based on the simplex method using a TUTSIMB (Meerman Automation) dynamic simulator. The values obtained are given in Table I. It shows that the exponent (Y strongly depends on the lactose initial concentration and for the calculation of (Y, the following relationship was derived. OL= 6.13P,,,
- 0.056
(6)
P max is the theoretical
lactic acid concentration [mol l-‘1 which should be obtained after a complete fermentation of the substrate and is determined as P max= 4SL”
Experimentat
set-up
The experiments were performed in a bioreactor (Quickfit, England) with a volume of l-l stirred magnetically at 400-450 ‘pm. The temperature of the solutions was maintained at 37 2 1°C by means of a thermostat and a heating coil situated in the reactor. The pH of the reaction medium was measured using a combined sterilized electrode (Ingold, Switzerland) connected to a pH meter (Radelkis OP-21 l/l, + 0.02 pH units) and recorded by computer. A constant pH value of the lactose fermentation was maintained either by enzyme hydrolysis of the urea or by the addition of a 12%~
(7)
The values of the other coefficients deviate (Table I) in limits allowable for a biological process which depends on many factors and with further application of the model they were taken as p = 0.625 X 10e3 and k, = 0.2.
Kinetics of enzyme degradation
of urea
Water solutions of urea with concentrations of 0.5, 1.0, 2.0 and 5.5 g 1-l were subjected to enzyme hydrolysis. The maintenance of pH within the limits 5.5-6 was realized by
Enzyme Microb. Technol.,
1997, vol. 21, August
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177
Papers Table 1 Coefficients in Eqs. (4) and (5) obtained with different initial concentrations of lactose and calculated compatibility of the lactose fermentation and urea bioconversion at T = 38°C and pH = 5.5-6 Coefficients Initial lactose concentration s, (g I-‘) 13.7 21.5 30 37.6 50
for
Conditions for compatibility in maintaining constant pH
in Eqs. (4) and (5)
Initial urea concentration suO (g 1-V
p x 103
e
k,
ki
l&B,
0.6 0.725 0.9 0.4 0.5
1.04 1.49 2.2 2.5 3.5
0.16 0.18 0.19 0.24 0.24
4 4 4 4 4
0.27 0.27 0.27 0.27 0.27
periodic addition of a 0.1 N water solution of The amount of the enzyme preparation was 2 experimental data confirmed that the process scribed by Michaelis-Menten kinetics. Applied acid neutralizing agent it has the form
conditions
lactic acid. g 1-l. The can be defor a lactic
5 8 11 12 15
Preparation amount M (g I-‘) 12.5 11 18.5 9 19
(4)-(6) allows calculation of the initial concentration of urea and the amount of enzyme preparation which are necessary to obtain an equivalent production of ammonia during the fermentation run. These conditions were found by numerically solving the set of Eqs. (4)-(6) and (8) at the requirement i=N
(8)
C
(Pi - P,,)* = min
(11)
i=l
Eq. (8) is obtained reaction. H*N-CO---NH2
accounting
for the stoichiometry
+ HZ0 = 2NI-&OH + CO2 T
of the
(12)
If the two biological processes do not influence each other, KS in Eq. (8) and k, in Eq. (12) can be taken from the investigation of urea hydrolysis. The enzyme quantity E is proportional to the preparation amount M and V talc max- Map M talc = (13) V exP max
dS, dP, -2-=-~-jl::dSII-l~~dP,~S, dt dt
The values of V,,, and KS were found to be 6.59 X lop4 mol 1-l h-’ and 0.0098 mol l-l, respectively.
Discussion and conditions for compatibility Figures la-lc represent a part of experimental data for the changes in lactic acid and biomass concentrations with time when the fermentation process is performed separately and the respective theoretical curves calculated by Eqs. (4)-(6). It can be seen that the coincidence for the lactic acid production is very good while the prediction of biomass production deviates considerably from the experiment with the increase in fermentation time. The last is probably due to the fact that the turbidimetric method determines the total amount of cells in the system. It gives satisfactory results in the initial stage of the process when the degree of cells dying is negligible. With the increase in this degree, the difference between the producing cells and those found by turbidimetry increases. Although the method has such a disadvantage, it is the most frequently used. The determinations of the living cells are too prolonged and inconvenient for a current control in the process. The exact prediction of the lactic acid production by Eqs. Enzyme Microb.
V max= k>E
(9)
from which there follows
178
Actually, the solution gives SuO and V,,, in Eq. (8) but the quantity of enzyme preparation follows from the relationship
Technol.,
1997, vol. 21, August
The results obtained in this way are given in Table 1. Figures la-lc show the outputs of ammonia with the time calculated by Eq. (8) at the respective urea initial concentrations and enzyme quantities. It can be seen that these outputs follow the lactic acid production with satisfying accuracy.
Simultaneous per$ormance of the processes In this case, the reactor was charged with 1 1 fermentation medium B and 100 cm3 inoculum. After 12 h, the necessary amounts of urea and enzyme preparation were added. Lactose initial concentrations of 11, 24.8, 31.4, and 43.4 g 1-l were used. Preliminary experiments show that 12 h after the inoculation, the concentrations of lactic acid and biomass were not completely reproducible despite the maintenance of constant process parameters (temperature, pH, etc.) and therefore could not be predicted in advance. These concentrations, however, are initial conditions (X = X,, P = P, at t = 0) of the set of Eqs. (4)-(6) and (8) and affect appreciably its solution for SU, and V,,, at the requirement.” This imposed the determination of X0 and P, in a sample of the fermentation medium 12 h after the inoculation and to calculate So,, and M on the base of the results 15
New method
for pH regulation:
L. Peeva and G. Peev
Table 2 Initial conditions
for simultaneous performance of lactose fermentation and urea hydrolysis at equivalent rates, T = 38°C. and pH = 5.5-6
24
Initial conditions for compatibility
8
Run No.
j 0 B
1 2 3 4
4
0
0.0 0
10
20 time
30
c
4
,’
20 time
1.0
30
z
40
[h]
,
I 0
J- 60
l
=
11.50 24.80 31.40 43.40
0.57 5.11 6.25 6.70
18.00 21 .oo 19.81 16.9
Urea, Enzyme S preparation, (g I”-“rr M (4 1-‘1 2.2’7 9.48 12.24 16.26
32.58 30.00 32.42 33.33
obtained. This procedure took approximately 10 min. Table 2 shows the initial conditions and calculated amounts of urea and preparation for representative experiments performed in the way described. Part of the data are illustrated in Figures 2a-2c as a dependence of the degree of lactose fermentation on time. The values of the medium pH during the process are also given. These demonstrate a small and smooth increase within the allowable limits. For a comparison, the conversions obtained under the same conditions (initial lactose concentration, temperature, quantity of inoculum, etc.) but with pH maintenance in the range 5.5-6 by adding a 12% (v/v) water solution of ammonia are shown. In all experiments, a considerable (2-3 times) enhancement of the lactose fermentation performed simultaneously with the urea enzyme hydrolysis can be observed. This acceleration could result from the maintenance of a constant pH at the microlevel in the whole reaction volume while, with the addition of ammonium hydroxide, some local pH fluctuations are probable due to nonideal mixing and the relatively slow reaction time of the pH probe. The time for homogenization in the reactor after an addition of neutralizing agent can be described by the equation
0
1
10
Biomass, x, (g I_‘)
[h]
20
0
Lactic acid, PO (9 I_‘)
40
1.0
0.0
Initial lactose concentration S,, (9 1-l)
Amount of urea and enzyme preparation added (calculated in advance)
0.8
rn = k,(D/d)’
0.0
/ 0
0 10
20
30
40
50
time [ h] Figure 1 Comparison of the experimental concentrations of lactic acid (0) and biomass (0) with those calculated by Eqs. (4)-(6) (solid lines) and expected production of ammonia at the designated conditions for compatibility (dotted lines) with initial lactose concentrations: 13.7 g I-’ (a), 30 g I-’ (b); and 50 g I-’ (c)
(14)
where k, depends on the mixer type, reactor geometry, and Reynolds number, Re. ” For a two-blade stirrer, nonbaffled vessel, and Re = 1.2 X 104, k, = 20.*’ This value for the Reynolds number corresponds to the conditions of urea bioconversion; therefore, the homogenization time for this process according to Eq. (14) should be 17 s (D = 10 cm, d = 4 cm). For the lactose fermentation, 7 should be much more because of the higher viscosity of lactose water solutions compared to urea ones. It is important to emphasize that the reported value of k, is for a degree of homogenization equal to 0.67. The local pH fluctuations could result in a (stress) decrease of the cell productivity. It is interesting to mention that despite the essential enhancement of the lactose fermentation in all experiments, the pH was held within the optimum limit of 5-6 (Figures 2a-2c). This implies that the enzyme hydrolysis of urea has also been accelerated due to the same reason. The biosystem used has a well-established
Enzyme Microb. Technol.,
1997, vol. 21, August
15
179
Papers ability for autocontrol owing to the mutual enhancement of the two processes mainly because of the smooth pH regulation within narrow limits. It can be calculated from Figures 2a-2c that the average productivity of the reactor varies from OS-l.19 g 1-l hh’. These values cannot be precisely compared with other studies due to the differences in the process conditions. In the lactic acid fermentation of sucrose by Lactobacillus delbreuckii at T = 39°C and pH = 6 (controlled by the addition of 2.5 N solution NaOH) a productivity of 0.313 g I-’ hh’ is reported.4 Lactose conversion by L. cusei at T = 37°C and pH = 6.5 (maintained by concentrated NH,OH) has produced 0.98 g 1-l hh’ lactate.” The last cells immobilized in Ca-alginate gel have converted glucose at a rate of 0.328 g 1-l h-l.’ For glucose, lactose and whey permeate fermentation by Luctobacillus helveticus at T = 42°C and pH = 5.9 (controlled by 3 N NH,OH), the reported productivity is 0.72-1.68 g I-’ hh’.” The extraction of lactic acid by ion-exchange resins has resulted in productivities of 0.4825 and 1.66 g 1-l hK’.4 These data show that the productivity obtained in our experiments is among the better ones for a batch fermentation process carried out by Luctobacillus. After each experiment, the immobilized urease was separated from the solution and was used with the following one. This multiple application of the preparation is of essential importance for the process economy. After a sequence of four cycles, however, a continuous loss of immobilized enzyme activity was observed. This phenomenon can be attributed to a mechanical destruction of the carrier and enzyme washing away or (as well as) to a prothease activity of L. cusei.
8
6%
4 dv/dt=0.131 2
0 20
30
40
50
time [h] 1.0
12
10
0.8
F ii i ‘0 g ._ $!
8 0.6 6% 0.4
z S 0.2
dr$dt=0.068
Conclusions 20
30
time [h]
F
0.8
8
b
$
0.6 -6
=Q
-4
The simultaneous performance of a lactose lactoacidic fermentation and a urea enzyme hydrolysis offers the possibility for smooth pH regulation of the system within narrow limits and considerable enhancement of the reactions. The calculation of experimental conditions necessary for maintenance of constant pH on the basis of models describing the kinetics of the processes performed separately results in their successful compatibility although there is the presence of a mutual influence. The multiple use of immobilized urease is limited. For the effective practical application of the method, a precise economic analysis is necessary.
List of symbols d
dr//dt=O.O18 q 50
D E
I 0
k,
-PH
0.0 0
-2
100
150
time [h] Figure 2 Lactose conversion (0) and pH dium (0) versus time with simultaneous mentation and urea hydrolysis compared pH = 5.5-6 with an addition of neutralizing initial concentrations: 11 g I-’ (a); 32 g I-’
180
Enzyme Microb. Technol.,
of the reaction meperformance of ferto the conversion at agent (0) at lactose (b); and 44 g I-’ (c)
1997, vol. 21, August
Stirrer diameter [ml Reactor diameter [ml Enzyme quantity [mol l-‘1 Coefficient accounting for the cells dying
W’l ki k, KS M
15
Coefficient of inhibition by the reaction product [1 mol-‘1 Rate constant of enzyme-substrate complex decomposition [hh’] Michaelis-Menten constant [mol l- ‘1 Amount of the immobilized urease preparation [g l- ‘1
New method
; p, Re = nd21v sl_ &I t T Vmax X a P rl CL max V
T
Stirrer revolutions [s- ‘1 Lactic acid concentration [mol l-‘1 Concentration of ammonia [mol l-‘1 Reynolds number Lactose concentration [mol l-l] Concentration of urea [mol l-‘1 Time of the process [h] Temperature Maximum rate of urea degradation [mol l-- ’ h-‘1 Concentration of biomass [g l-‘1 Exponent Biomass productivity coefficient [mol g-- ’ h-‘1 Conversion degree Maximum cells specific growth rate [h- ‘1 Reaction media kinematic viscosity [ml s-l] Time for homogenization in reactor [s]
6.
7.
8. 9.
10.
Il.
12.
13.
Subscripts 14.
0
exp talc
Initial Experimental Calculated
15.
16.
References 1. 2.
3.
4.
5.
Yeh, P. L.-H., Bajpai, R. K., and Iannotti, E. L. An improved kinetic model for lactic acid fermentation. J. Ferm. Bioeng. 1991,71(l), 75-77 Demirci, A. and Pometto, A. L. Enhanced production of D(-)-lactic acid by mutants of Lactobacillus delbrueckii ATCC-9649. J. Ind. Microbial. 1992, 11(l). 23-28 Davison, B. H. and Scott, D. C. A proposed biparticle fluidized-bed reactor for lactic acid fermentation and simultaneous adsorption. Biotechnol. Bioeng. 1992,39, 365-368 Srivastava, A., Roychoudhury, P. K., and Sahai, V. Extractive lactic acid fermentation using ion-exchange resin. Biotechnol. Bioeng. 1992. 39, 607-613 Jianlong, W.. Ping, L., and Ding, Z. Extractive fermentation of lactic
17.
18.
19.
20.
for pH regulation:
L. Peeva and G. Peev
acid by immobilized Luctobucillus casei using ion-exchange resin. Biotechnol. Tech. 1994, S( 12), 905-908 Boyaval, P., Corre, C.. and Terre, S. Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis. Biotechnol. l&t. 1987. 9(3), 207-212 Nomura, Y., Iwahara, M., and Hongo, M. Lactic acid production by electrodialysis fermentation using immobilized growing cells. Biotechnol. Bioeng. 1987,3,788 -793 Yabannavar, V. M. and Wang. D. 1. C. Extrwtive fermentation for lactic acid production. Biotechnol. Bioeng. 1991. 37, 1095-I 100 Cachon. R. and Divies, C. Kinetics of lactate fermentation and citrate bioconversion by Lactocnccus luctis ssp. factis in batch culture. J. Appl. Bacterial. 1993. 75, 387-392 Roy, D.. Leduy, A., and Goulet, J. Kinetics of growth and lactic acid production from whey permeate by Luctobacillus hulveticus. Con. J. Chem. Eng. 1987. 65, 597-603 Taniguchi, M., Kotani. N., and Kobayashi, T. High-concentration cultivation of lactic acid bacteria in fermentor with cross-flow filtration. J. Ferment. Technof. 1987. 65(2), 179 -184 Krysteva. M.. Peev. G., and Sokolov, T. Continuous conversion of urea in wastewaters with the aid of urease covalently immobilized to granulated microcrystallized cellulose. Biotechnol. Chem. 1989. 1, 24-27 Lazarus, R. A. and Seymour. J. L. Determination of 2-keto-tgulonic acid and other ketoaldonic and aldonic acids produced by ketogenic bacterial fermentation. Anal. B&hem. 1986. 157. 360-366 Watt, G. W. and Chrisp. J. D. Spectrophotometric method for determination of urea. Anal. Chem. 1954, 26(3), 452-453 Kosseva, M., Beschkov. V.. and Pilafova, E. Modelling of lactic acid production from lactose by immobilized Lactobacillus rusei cells. Proc. Bioproc. Eng.. Sofia, Bulgaria, 1994, 75-82 Samuel, W. A., Lee. Y. Y.. and Anthony, W. B. Lactic acid fermentation of crude sorghum extract. Biotechnol. Bioeng. 1980, 22, 757-777 Luedeking, R. and Piret. E. L. A kinetic study of the lactic acid fermentation. Batch process at controlled pH. J. B&hem. Microbiol. Technol. Eng. 1959, 1, 393-412 Jorgensen. M. H. and Nikolajsen, K. Mathematic model for lactic acid formation with Streptococcus cremoris from glucose. Appl. Microbial. Biotechnol. 1987, 25, 3 13-3 16 Leh. M. B. and Charles, M. Lactic acid production by batch fermentation of whey permeate: A mathematical model. J. fnd. Microbial. 1989, 4, 65-70 Braginski, L. N.. Begachev. V. I., and Barabash. V. M. Mixing in homogeneous media. In: Miring in Liquid Media. Leningrad, “Chimia”. 1984, 110-l 13
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