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Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells
Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells M o t o h i k o H i k u m a , Haruo Obana and T a k e o Yasuda Central Research Laboratories, A f i n o m o t o Co. Inc., 1 Suzuki-cho, Kawasaki-ku, Kawasaki, 210, Japan
and Isao Karube and Shuich S u z u k i Research Laboratory o f Resources Utilization, Tokyo Institute o f Technology, Nagatsuta-cho , Midori-ku, Yokohama, 22 7, Japan
(Received 3 December 1979; revised 12 February 1980) A microbial sensor consisting o f immobilized living whole cells o f Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination o f total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption o f oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane o f the oxygen electrode and the current o f the electrode decreased markedly with time until steady state was reached. The response time was ~ 10 min by the steady state method and 1 rain by the pulse method. A linear relationship was fo u n d between the decrease in current and the concentration o f glucose (<1 mm), fructose (~1 mM) and sucrose (~0. 8 mM). The ratio o f the sensitivity o f the microbial sensor to glucose, fructose and sucrose was 1.00: 0.80: O.92. The decrease in current was reproducible to within 2% o f the relative standard deviation when a sample solution containing glucose (0.8 raM) was employed Jor experiments. The selectivity o f the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity o f the response o f the microbial sensor for glucose, Jructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acM production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.
Introduction On-line measurements o f substrate concentrations in culture broths are required in the fermentation industry. In the cultivation of microorganisms in cane molasses, which contains various sugars, determination of the total assimilable sugars in a broth is important for the control of the fermentation process. Reduced sugars and sucrose in culture broth are determined by the ferricyanide method. 1 However, the m e t h o d is not completely reliable because unassimilable substances are also determined. On the other hand, various enzyme electrodes have been developed for the determination of sugars. 2,3 In order to determine the total amount o f assimilable sugars, many kinds of enzyme electrode can be employed. But, as enzymes are generally expensive and unstable, enzyme electrodes are not suitable for the determination of assimilable sugars in fermentation broths. Recently, microbial sensors have consisted of immobilized microorganisms, and electrochemical devices have been developed by the authors and applied to the estimation of
234
Enzyme Microb. Technol., 1980, Vol. 2, July
biochemical oxygen demand (BOD) 4 and the determination of alcohols, s acetic acid 6 and vitamins. 7 As previously reported, assimilation of organic compounds by microorganisms can be determined from the respiratory activity of the microorganisms, which can be directly measured by an oxygen electrode. 4-6 It is therefore possible to devise a microbial sensor for total assimilable sugars using immobilized microorganisms. The microorganism employed for the sensor was, for preference, the same species as that being cultivated in the fermentation. The method was applied to the determination o f total assimilable sugars in a fermentation broth for glutamic acid production.
Materials and m e t h o d s Materials
Glucose, fructose and sucrose were purchased from Junsei Chemical Co. Ltd. Other reagents were commercially 0141 --0229/80/030234--05 $02.00 ~ 1980 IPC Business Press
Amperometric determination o f sugars in fermentation broths: Motohiko Hikuma et al.
available analytical reagents or laboratory grade materials, Deionized water was used in all procedures.
ioo
Microorganisms Brevibacterium lactofermentum AJ 1511 was grown under aerobic conditions at 30°C for 24 h in a medium, pH 6.0, containing 4% glucose, 0.1% KH2 PO4, 0.1% MgSO4.7H20, 2 p.p.m FeSO4.7HzO, 2 p.p.m MnSO4, 24 p.p.m soybean extract, 0.1 p.p.m thiamin-HC1 and 0.6% urea. The cells were harvested by centrifugation and washed three times with sterilized water.
8(3
g
A
Assembly o f the microbial electrode The scheme of the microbial electrode is illustrated in
Figure 1. An oxygen electrode (Model 777, Beckman Inst. Co.) consisted of a Teflon membrane, a platinum.cathode, a silver anode and electrolyte. A strip of nylon net (1 cm x 1 cm, 20 mesh) was coated with the cells (0.015 g). The nylon net retaining microorganisms was placed on the surface of the Teflon membrane of the electrode and covered with a cellophane membrane(no. 105-1058, Technicon) so that the microorganisms were entrapped between the two membranes.
4C
(3 Et
10
20 Time (min)
30
40
Figure 3 Response curves of the microbial sensor. A sample solution containing 1 mM glucose was injected into the system f o r : A , 0.5; B, 2; C, 16 min. The experiment was carried out under the standard conditions described in the Experimental section
Apparatus Figure 2 shows a schematic diagram of the system. The system consisted of a flow cell (diameter 13 ram, height 4 mm; volume 0.5 ml) containing the microbial electrode, a water bath, a peristaltic pump (model I, Technicon), a transmitter of the oxygen electrode (type 777 Beckman) and a recorder (model LER-12A, Yokogawa Electric Works).
Procedure
H Figure 1 Scheme of the microbial electrode for total assimilable sugars. A, Silver anode; B, platinum cathode; C,D, rubber rings; E, electrolyte gel; F, Teflon membrane; G, microorganisms retained on nylon net; H, cellophane membrane
,A Somple ~ ~ Top woter Air
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.,.,j TL Figure 2 Schematic diagram of the sensor system A, peristalticpump; B, water bath; C, flow cell;D, microbial electrode; E, transmitter; F, recorder
The temperature of the flow cell was maintained at 30 +0.2°C by a water bath. Tap water saturated with oxygen was transferred to the flow cell at a flow rate of 3.9 ml min-1 together with 500 ml min-a of air. When the output current of the microbial electrode reached a constant value, a sample solution was injected into the system at a flow rate of 0.42 ml min-a at 0.5, 2 and 16 min intervals. In this paper, the sample concentration means that in the flow cell.
Results and discussion
Response o f the electrode Figure 3 shows typical response curves of the microbial sensor for glucose. The current at zero time was obtained with tap water saturated with dissolved oxygen. This current reflects the endogenous respiration level of the immobilized microorganisms. When the sample solution containing glucose w.as injected into the system for 16 min, glucose permeated through the cellophane membrane and was assimilated by the immobilized microorganisms. Oxygen
Enzyme Microb. Technol., 1980, Vol. 2, July 235
Papers consumption by the microorganisms caused a decrease in dissolved oxygen around the membrane, and the current of the electrode decreased markedly with time and reached steady state within 10 min (the response tinae). The steady state indicates that the consumption of oxygen by the microorganism and that diffused from a sample solution to the membrane were in equilibrium. When the tap water was again transferred to the flow cell, the microbial sensor current returned to the initial level within 15 rain. The time required for the determination was too long using the steady state method. Therefore, the pulse method was employed, in which a sample solution was transferred to the flow cell for a certain time. When a sample solution was transferred to the flow cell for 0.5 and 2 min, the microbial electrode current decreased, as shown in Figure 3. The maximum fall in current (the difference in current between the initial and the maximum) reached 55 and 85% of that obtained by the steady state method within 1 and 3 min (response time), respectively. The total time required for the assay was 60 min using the steady state method and 10 or 20 min using the pulse method. We therefore used the pulse method, transferring a sample solution for 0.5 min, for further work.
Current-concentration relationship for assimilable sugars Glucose solution (1 mM) was used as the standard. A maximum decrease in current (peak height) for a sample was normalized by the following equation in order to correct for variations in the current-concentration relationship of the microbial sensor: Normalized peak height = =
observed peak height peak height of the standard solution
(1)
Figures 4 and 5 show calibration curves of the microbial sensor for assimilable sugars such as glucose, fructose and sucrose. Peak heights were normalized against the standard solution (1 mM glucose solution) by equation (1). The
z
I 02
I 04
I 06
Concentration
(rnM)
I 0.8
1.0
Figure 4 C u r r e n t - - c o n c e n t r a t i o n r e l a t i o n s h i p o f glucose (o) and f r u c t o s e (o). A sample s o l u t i o n (0.21 ml) c o n t a i n i n g various a m o u n t s o f glucose and f r u c t o s e was injected into the system f o r 0.5 min
236 Enzyme Microb. Technol., 1980, Vol. 2, July
o
/:
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( )
i
v J=:
o.61I
N :=
04-
Q2
v0
I
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i
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Concentrotion (mM) Figure 5 C u r r e n t - - c o n c e n t r a t i o n r e l a t i o n s h i p o f sucrose (o) and glucose (©). The e x p e r i m e n t a l c o n d i t i o n s were the same as those described in Figure 4
calibration curves for glucose, fructose and sucrose respectively, can be represented by the following equations derived by the method of least squares: Ii = 0.07 + 0.95 C1 (2) Iz = 0.05 + 0.77 Cz (3) I3 = 0.03 + 0.91 C3 (4) where C is the concentration of sugar (mM) in the flow cell, I is the normalized peak height and subscripts 1,2 and 3 represent glucose, fructose and sucrose, respectively. The ratio of the sensitivity to glucose (180 mg 1-1 ), fructose (180 mg1-1 ) and sucrose (360 mgl -~ ) was 1.00:0.80:0.92. The sensitivity of the microbial sensor for sugars corresponds to the amount of oxygen consumed by the immobilized microorganisms assimilating them. BOD values for glucose, fructose and sucrose are, for example, 115, 128 and 225 g 02 / mol, respectively ~ and the ratio of each is 1.00 : 1.11 : 1.96. tlowever, the microbial sensor showed a particularly low relative sensitivity for sucrose in view of the BOD value for sucrose. This may be caused by the slow rate of decomposition of sucrose by the immobilized microorganisms. Since the respiratory activity of microorganisms depends on the pH and temperature, their influence on the current output of the microbial sensor was examined. The response (peak height) of the sensor for 0.8 mM glucose solution was almost constant between pH 6 and 8. The maximum response (peak height) of the sensor to the same standard solution was observed from 30 to 37°C. However, the response was smaller at temperatures below 30°C and above 37°C because the immobilized microorganisms were inactivated under these conditions. The effect of salts on the activity of microorganisms is well known. We therefore examined the influence of potassium phosphate, ammonium sulphate, potassium chloride and other salts which are generally present in fermentation broths. When the 0.8 mM glucose solution containing 1 mM potassium phosphate was transferred to the sensor, a greater response (3%) was observed. However, no influence was observed when a sample solution con tained 0.002 mM polasslum phosphate in the flow cell, which corresponds to the
Amperometric determination of sugars in fermentation broths: Motohiko Hikuma et aL [0,
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Concentration (mM) Figure 6 Current--concentration relationship of galactose (e), maltose (A), lactose (m) and glucose (o). The experimental conditions were the same as those described in Figure 4
02
......
J
~ _ _
I
04 0.6 Concentration (mM)
Q8
1.0
Figure 7 Current--concentration relationship for glutamic acid (e) and glucose (o). The experimental conditions were the same as those described in Figure 4
level in fermentation broths. The influence of other salts on the current output was less than that of potassium phosphate. The reproducibility of the microbial electrode was examined. Introduction of 0.8 m e glucose solution into the system was repeated 20 times, and the relative standard deviation (coefficient of variation) was 2%.
Additivity o f the response by the microbial electrode sensor
Selectivity o f the microbial sensor Figures 6 and 7 show the relationship between the con-
When a mixture of assimilable sugars was applied to the system, the observed normalized peak height was compared with that calculated by the following equation:
centrations of glucose, other sugars and glutamic acid and normalized peak height. The sensor scarcely responded to the sugars, which might be slowly utilized by the microorganisms; 9 on the other hand, the sensor gave little response
to glutamic acid. Therefore, the selectivity of the microbial electrode sensor for assimilable sugars was satisfactory for fermentation control.
I = 0 . 1 5 +0.95 Ca +0.77 Cz +0.91 C3 (5) where I is the normalized peak height and C1, C2 and C3 are the concentrations of the assimilable sugars, as defined
Table 1 A d d i t i v i t y of response for assimilable sugars Sugar concentration in sample solution
No.
Glucose (mM) 1-1 1 --2 1 --3 1-4 1 --5 1 --6 2-1 2-2 2--3 2--4 2--5 3-1 3--2 3--3 3--4 3--5 3-6 4--1 4--2 4--3 4-4 4--5 4--6 5--1 5--2 5--3 5--4
0.50 0.50 0.50 0.50 0.50 0.5O 0.50 0.50 0.50 0.50 0.50
Fructose (mM)
Sucrose (mM)
0.075 0.15 0.25 0.375 O.50 0.075 0.15 0.25 0.275
0.075 0.15 0.25 0.375 0.50
0.50 0.50 0.50 0.50 0.50 0.50
0.10 0.167 0.25 0.33
0.075 0.15 0.25 0.375 0.50 0.10 0.167 0.25 0.33
O.5O 0.50 0.50 0.50 0.50 0.50 0.10 0.167 0.25 0.33
Observed (mM)
Calculated (raM)
0.55 0.64 0.68 0.78 0.88 0.95 0.55 0.66 0.75 0.82 0.90 0.48 0.59 0.66 0.76 0.87 0.96 0.50 0.62 0.68 0.75 0.81 0.90 0.40 0.59 0.81 0.95
0.54 0.65 0.71 0.79 0.88 0.98 0.54 0.64 0.71 0.80 0.91 0.44 0.58 0.65 0.74 0.86 0.98 0.49 0.60 0.66 0.73 0.83 0.92 0.42 0.59 0.81 1.02
Difference (raM)
(%)
-0.01 0.01 0.03 0.01 0.00 0.03 -0.01 -0.02 -0.04 -0.02 0.01 -0.04 --0.01 --0.01 --0.02 -0.01 0.02 --0.01 0.02 --0.02 --0.02 0.02 0.02 0.02 0.00 0.00 0.07
(-2) ( 2) ( 4) ( 1) ( O) ( 3) (-2) (-3) (-5) (-2) ( 1) (-8) (--2) (--2) (--3) ( 1) ( 2) (--2) ( 3) (--3) (--3) ( 2) ( 2) ( 5) ( 0) ( 0) ( 7)
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 0 , V o l . 2, J u l y
237
Papers above. Equation (5) means that the response of the microbial sensor to a mixture of the assimilable sugars is equal to the algebraic sum of the responses to the individual assimilable sugars. The difference between the observed and the calculated values was within 8%, as shown in Table 1. These results suggest that total assimilable sugars can be measured by the microbial sensor. Application and reusability of the microbial sensor
The microbial sensor for total assimilable sugars was applied to a fermentation broth (cane molasses) for glutamic acid production. The broth was directly applied to the system after diluting 30 times with tap water. In this method, the concentration of assimilable sugars in the broth was determined as glucose, because the sensor was calibrated with a standard solution containing glucose. On the other hand, the concentration of reducing sugars in the broth was determined by the ferricyanide method. 1° The results are shown in Table 2. Results obtained by the conventional method were higher than those obtained by the microbial sensor because the conventional method was influenced by reducing substances other than the assimilable sugars. After cultivation for 30 h, the assimilable sugars and glutamic acid in the broth were determined by paper chromatography and Warburg's method using glutamate decarboxylase, 11 respectively. The assimilable sugars were hardly detectable (glutamic acid concentration 7.5 g 1 ~. At that time, the microbial sensor gave 5 g 1-1 which was smaller than the value of 16 g 1 =obtained by the conventional method. The reusability of the electrode was examined by using a standard solution containing glucose and the broths. When the microbial sensor was used continuously for a long time, the nraximum current decrease of the electrode for a standard solution gradually drifted (within 15% during 10 days use) thus, occasional calibration of the electrode was required. For example, two standard solutions (0.4 and 1.0 mM glucose) were employed after every 10 samples, giving two points of calibration. The values obtained were then reproducible to within 4% of the relative error, and the concentration of total assimilable sugars was accurately determined by the microbial sensor. This sensor could be used for more than 10 days and 960 assays, indicating good survival of the bacteria in the electrode,
238
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 0 , V o l . 2, J u l y
Table 2 Comparison of total assimilable sugars determined by the microbial electrode and the conventional method Cultivation a time (h) 0 8
15 24 30
Total sugars b (g I -~ )
Glutamic acid (g I - j )
Microbial electrode
Ferricyanide method
39
67
-
13 8 6 5
41 23 18 16
75
aFeeding method was used; bcalculated as glucose
and its suitability for the determination of assimilable sugars in broths. Further developmental study is intended to improve the selectivity of the microbial sensor.
Acknowledgements The authors are grateful to Mr T. Kiya, Dr Y. Sakata and Dr K. Mitsugi, Central Research Laboratories, Ajinomoto Co., for their helpful advice and encouragement during this study and also to Mr T. Shirakawa for his assistance.
References I 2 3 4 5 6 7 8
9 I0 11
Reducing sugar and sucrose in food products' Industrial method no. 142 71 A,Technicon Industrial Systems, 1972 Guilbault, G. G. Handbook of Enzymatic Methods ofA nalysis M. Dekker, New York, 1976 Satoh, S., Karube, 1. and Suzuki, S. BiotechnoL Bioeng. 1976, 18, 269 Hikuma, M., Suzuki, It., Yasuda, Y., Karube, I. and Suzuki, S. Eur. J. NppL Microbiol. Bioteehnol. 1979, 8,289 Hikuma, M., Kubo, T., Yasuda, T., Karubc, 1. and Suzuki, S. Biotectmol. Bioeng. 1979, 21, 1845 Hikuma, M.,Kubo, T.,Yasuda, T., Karube, l., and Suzuki, S. Anal. Chim. Acta 1979, 109, 33 Matsunaga,T., Karube, 1. and Suzuki, S. Anal. Chim Acta 1978, 99, 233 Handbook ofEnvironnzental 6bnlrol, (Bond, R. G. and Straub, C. P., eds) CRC Press, Cleveland, Ohio, 1973, vol 3, pp. 671 686 Yamada,K. and Komagata, K. J. Gen. ,4ppl. Microhiol. 1972, 18, 399 Paper671romatography (ltais, 1. M. and Macck, K., eds) Academic Press, New York, 1963, pp. 289 330 'Monosodium glutamate in fermenter solutions' Industrial method no. 210 72 A, TechniconlndustrialSystems, i973
and Isao Karube and Shuich S u z u k i Research Laboratory o f Resources Utilization, Tokyo Institute o f Technology, Nagatsuta-cho , Midori-ku, Yokohama, 22 7, Japan
(Received 3 December 1979; revised 12 February 1980) A microbial sensor consisting o f immobilized living whole cells o f Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination o f total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption o f oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane o f the oxygen electrode and the current o f the electrode decreased markedly with time until steady state was reached. The response time was ~ 10 min by the steady state method and 1 rain by the pulse method. A linear relationship was fo u n d between the decrease in current and the concentration o f glucose (<1 mm), fructose (~1 mM) and sucrose (~0. 8 mM). The ratio o f the sensitivity o f the microbial sensor to glucose, fructose and sucrose was 1.00: 0.80: O.92. The decrease in current was reproducible to within 2% o f the relative standard deviation when a sample solution containing glucose (0.8 raM) was employed Jor experiments. The selectivity o f the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity o f the response o f the microbial sensor for glucose, Jructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acM production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.
Introduction On-line measurements o f substrate concentrations in culture broths are required in the fermentation industry. In the cultivation of microorganisms in cane molasses, which contains various sugars, determination of the total assimilable sugars in a broth is important for the control of the fermentation process. Reduced sugars and sucrose in culture broth are determined by the ferricyanide method. 1 However, the m e t h o d is not completely reliable because unassimilable substances are also determined. On the other hand, various enzyme electrodes have been developed for the determination of sugars. 2,3 In order to determine the total amount o f assimilable sugars, many kinds of enzyme electrode can be employed. But, as enzymes are generally expensive and unstable, enzyme electrodes are not suitable for the determination of assimilable sugars in fermentation broths. Recently, microbial sensors have consisted of immobilized microorganisms, and electrochemical devices have been developed by the authors and applied to the estimation of
234
Enzyme Microb. Technol., 1980, Vol. 2, July
biochemical oxygen demand (BOD) 4 and the determination of alcohols, s acetic acid 6 and vitamins. 7 As previously reported, assimilation of organic compounds by microorganisms can be determined from the respiratory activity of the microorganisms, which can be directly measured by an oxygen electrode. 4-6 It is therefore possible to devise a microbial sensor for total assimilable sugars using immobilized microorganisms. The microorganism employed for the sensor was, for preference, the same species as that being cultivated in the fermentation. The method was applied to the determination o f total assimilable sugars in a fermentation broth for glutamic acid production.
Materials and m e t h o d s Materials
Glucose, fructose and sucrose were purchased from Junsei Chemical Co. Ltd. Other reagents were commercially 0141 --0229/80/030234--05 $02.00 ~ 1980 IPC Business Press
Amperometric determination o f sugars in fermentation broths: Motohiko Hikuma et al.
available analytical reagents or laboratory grade materials, Deionized water was used in all procedures.
ioo
Microorganisms Brevibacterium lactofermentum AJ 1511 was grown under aerobic conditions at 30°C for 24 h in a medium, pH 6.0, containing 4% glucose, 0.1% KH2 PO4, 0.1% MgSO4.7H20, 2 p.p.m FeSO4.7HzO, 2 p.p.m MnSO4, 24 p.p.m soybean extract, 0.1 p.p.m thiamin-HC1 and 0.6% urea. The cells were harvested by centrifugation and washed three times with sterilized water.
8(3
g
A
Assembly o f the microbial electrode The scheme of the microbial electrode is illustrated in
Figure 1. An oxygen electrode (Model 777, Beckman Inst. Co.) consisted of a Teflon membrane, a platinum.cathode, a silver anode and electrolyte. A strip of nylon net (1 cm x 1 cm, 20 mesh) was coated with the cells (0.015 g). The nylon net retaining microorganisms was placed on the surface of the Teflon membrane of the electrode and covered with a cellophane membrane(no. 105-1058, Technicon) so that the microorganisms were entrapped between the two membranes.
4C
(3 Et
10
20 Time (min)
30
40
Figure 3 Response curves of the microbial sensor. A sample solution containing 1 mM glucose was injected into the system f o r : A , 0.5; B, 2; C, 16 min. The experiment was carried out under the standard conditions described in the Experimental section
Apparatus Figure 2 shows a schematic diagram of the system. The system consisted of a flow cell (diameter 13 ram, height 4 mm; volume 0.5 ml) containing the microbial electrode, a water bath, a peristaltic pump (model I, Technicon), a transmitter of the oxygen electrode (type 777 Beckman) and a recorder (model LER-12A, Yokogawa Electric Works).
Procedure
H Figure 1 Scheme of the microbial electrode for total assimilable sugars. A, Silver anode; B, platinum cathode; C,D, rubber rings; E, electrolyte gel; F, Teflon membrane; G, microorganisms retained on nylon net; H, cellophane membrane
,A Somple ~ ~ Top woter Air
El
~
Waste
!
'
/
i
.,.,j TL Figure 2 Schematic diagram of the sensor system A, peristalticpump; B, water bath; C, flow cell;D, microbial electrode; E, transmitter; F, recorder
The temperature of the flow cell was maintained at 30 +0.2°C by a water bath. Tap water saturated with oxygen was transferred to the flow cell at a flow rate of 3.9 ml min-1 together with 500 ml min-a of air. When the output current of the microbial electrode reached a constant value, a sample solution was injected into the system at a flow rate of 0.42 ml min-a at 0.5, 2 and 16 min intervals. In this paper, the sample concentration means that in the flow cell.
Results and discussion
Response o f the electrode Figure 3 shows typical response curves of the microbial sensor for glucose. The current at zero time was obtained with tap water saturated with dissolved oxygen. This current reflects the endogenous respiration level of the immobilized microorganisms. When the sample solution containing glucose w.as injected into the system for 16 min, glucose permeated through the cellophane membrane and was assimilated by the immobilized microorganisms. Oxygen
Enzyme Microb. Technol., 1980, Vol. 2, July 235
Papers consumption by the microorganisms caused a decrease in dissolved oxygen around the membrane, and the current of the electrode decreased markedly with time and reached steady state within 10 min (the response tinae). The steady state indicates that the consumption of oxygen by the microorganism and that diffused from a sample solution to the membrane were in equilibrium. When the tap water was again transferred to the flow cell, the microbial sensor current returned to the initial level within 15 rain. The time required for the determination was too long using the steady state method. Therefore, the pulse method was employed, in which a sample solution was transferred to the flow cell for a certain time. When a sample solution was transferred to the flow cell for 0.5 and 2 min, the microbial electrode current decreased, as shown in Figure 3. The maximum fall in current (the difference in current between the initial and the maximum) reached 55 and 85% of that obtained by the steady state method within 1 and 3 min (response time), respectively. The total time required for the assay was 60 min using the steady state method and 10 or 20 min using the pulse method. We therefore used the pulse method, transferring a sample solution for 0.5 min, for further work.
Current-concentration relationship for assimilable sugars Glucose solution (1 mM) was used as the standard. A maximum decrease in current (peak height) for a sample was normalized by the following equation in order to correct for variations in the current-concentration relationship of the microbial sensor: Normalized peak height = =
observed peak height peak height of the standard solution
(1)
Figures 4 and 5 show calibration curves of the microbial sensor for assimilable sugars such as glucose, fructose and sucrose. Peak heights were normalized against the standard solution (1 mM glucose solution) by equation (1). The
z
I 02
I 04
I 06
Concentration
(rnM)
I 0.8
1.0
Figure 4 C u r r e n t - - c o n c e n t r a t i o n r e l a t i o n s h i p o f glucose (o) and f r u c t o s e (o). A sample s o l u t i o n (0.21 ml) c o n t a i n i n g various a m o u n t s o f glucose and f r u c t o s e was injected into the system f o r 0.5 min
236 Enzyme Microb. Technol., 1980, Vol. 2, July
o
/:
0.8-
( )
i
v J=:
o.61I
N :=
04-
Q2
v0
I
L
I
i
02
Q4
06
08
IO
Concentrotion (mM) Figure 5 C u r r e n t - - c o n c e n t r a t i o n r e l a t i o n s h i p o f sucrose (o) and glucose (©). The e x p e r i m e n t a l c o n d i t i o n s were the same as those described in Figure 4
calibration curves for glucose, fructose and sucrose respectively, can be represented by the following equations derived by the method of least squares: Ii = 0.07 + 0.95 C1 (2) Iz = 0.05 + 0.77 Cz (3) I3 = 0.03 + 0.91 C3 (4) where C is the concentration of sugar (mM) in the flow cell, I is the normalized peak height and subscripts 1,2 and 3 represent glucose, fructose and sucrose, respectively. The ratio of the sensitivity to glucose (180 mg 1-1 ), fructose (180 mg1-1 ) and sucrose (360 mgl -~ ) was 1.00:0.80:0.92. The sensitivity of the microbial sensor for sugars corresponds to the amount of oxygen consumed by the immobilized microorganisms assimilating them. BOD values for glucose, fructose and sucrose are, for example, 115, 128 and 225 g 02 / mol, respectively ~ and the ratio of each is 1.00 : 1.11 : 1.96. tlowever, the microbial sensor showed a particularly low relative sensitivity for sucrose in view of the BOD value for sucrose. This may be caused by the slow rate of decomposition of sucrose by the immobilized microorganisms. Since the respiratory activity of microorganisms depends on the pH and temperature, their influence on the current output of the microbial sensor was examined. The response (peak height) of the sensor for 0.8 mM glucose solution was almost constant between pH 6 and 8. The maximum response (peak height) of the sensor to the same standard solution was observed from 30 to 37°C. However, the response was smaller at temperatures below 30°C and above 37°C because the immobilized microorganisms were inactivated under these conditions. The effect of salts on the activity of microorganisms is well known. We therefore examined the influence of potassium phosphate, ammonium sulphate, potassium chloride and other salts which are generally present in fermentation broths. When the 0.8 mM glucose solution containing 1 mM potassium phosphate was transferred to the sensor, a greater response (3%) was observed. However, no influence was observed when a sample solution con tained 0.002 mM polasslum phosphate in the flow cell, which corresponds to the
Amperometric determination of sugars in fermentation broths: Motohiko Hikuma et aL [0,
el "T
?
!
z=
0.6?
o E
04
z
i, o
"o2
~
c4
--
o6
08
I
o
Concentration (mM) Figure 6 Current--concentration relationship of galactose (e), maltose (A), lactose (m) and glucose (o). The experimental conditions were the same as those described in Figure 4
02
......
J
~ _ _
I
04 0.6 Concentration (mM)
Q8
1.0
Figure 7 Current--concentration relationship for glutamic acid (e) and glucose (o). The experimental conditions were the same as those described in Figure 4
level in fermentation broths. The influence of other salts on the current output was less than that of potassium phosphate. The reproducibility of the microbial electrode was examined. Introduction of 0.8 m e glucose solution into the system was repeated 20 times, and the relative standard deviation (coefficient of variation) was 2%.
Additivity o f the response by the microbial electrode sensor
Selectivity o f the microbial sensor Figures 6 and 7 show the relationship between the con-
When a mixture of assimilable sugars was applied to the system, the observed normalized peak height was compared with that calculated by the following equation:
centrations of glucose, other sugars and glutamic acid and normalized peak height. The sensor scarcely responded to the sugars, which might be slowly utilized by the microorganisms; 9 on the other hand, the sensor gave little response
to glutamic acid. Therefore, the selectivity of the microbial electrode sensor for assimilable sugars was satisfactory for fermentation control.
I = 0 . 1 5 +0.95 Ca +0.77 Cz +0.91 C3 (5) where I is the normalized peak height and C1, C2 and C3 are the concentrations of the assimilable sugars, as defined
Table 1 A d d i t i v i t y of response for assimilable sugars Sugar concentration in sample solution
No.
Glucose (mM) 1-1 1 --2 1 --3 1-4 1 --5 1 --6 2-1 2-2 2--3 2--4 2--5 3-1 3--2 3--3 3--4 3--5 3-6 4--1 4--2 4--3 4-4 4--5 4--6 5--1 5--2 5--3 5--4
0.50 0.50 0.50 0.50 0.50 0.5O 0.50 0.50 0.50 0.50 0.50
Fructose (mM)
Sucrose (mM)
0.075 0.15 0.25 0.375 O.50 0.075 0.15 0.25 0.275
0.075 0.15 0.25 0.375 0.50
0.50 0.50 0.50 0.50 0.50 0.50
0.10 0.167 0.25 0.33
0.075 0.15 0.25 0.375 0.50 0.10 0.167 0.25 0.33
O.5O 0.50 0.50 0.50 0.50 0.50 0.10 0.167 0.25 0.33
Observed (mM)
Calculated (raM)
0.55 0.64 0.68 0.78 0.88 0.95 0.55 0.66 0.75 0.82 0.90 0.48 0.59 0.66 0.76 0.87 0.96 0.50 0.62 0.68 0.75 0.81 0.90 0.40 0.59 0.81 0.95
0.54 0.65 0.71 0.79 0.88 0.98 0.54 0.64 0.71 0.80 0.91 0.44 0.58 0.65 0.74 0.86 0.98 0.49 0.60 0.66 0.73 0.83 0.92 0.42 0.59 0.81 1.02
Difference (raM)
(%)
-0.01 0.01 0.03 0.01 0.00 0.03 -0.01 -0.02 -0.04 -0.02 0.01 -0.04 --0.01 --0.01 --0.02 -0.01 0.02 --0.01 0.02 --0.02 --0.02 0.02 0.02 0.02 0.00 0.00 0.07
(-2) ( 2) ( 4) ( 1) ( O) ( 3) (-2) (-3) (-5) (-2) ( 1) (-8) (--2) (--2) (--3) ( 1) ( 2) (--2) ( 3) (--3) (--3) ( 2) ( 2) ( 5) ( 0) ( 0) ( 7)
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 0 , V o l . 2, J u l y
237
Papers above. Equation (5) means that the response of the microbial sensor to a mixture of the assimilable sugars is equal to the algebraic sum of the responses to the individual assimilable sugars. The difference between the observed and the calculated values was within 8%, as shown in Table 1. These results suggest that total assimilable sugars can be measured by the microbial sensor. Application and reusability of the microbial sensor
The microbial sensor for total assimilable sugars was applied to a fermentation broth (cane molasses) for glutamic acid production. The broth was directly applied to the system after diluting 30 times with tap water. In this method, the concentration of assimilable sugars in the broth was determined as glucose, because the sensor was calibrated with a standard solution containing glucose. On the other hand, the concentration of reducing sugars in the broth was determined by the ferricyanide method. 1° The results are shown in Table 2. Results obtained by the conventional method were higher than those obtained by the microbial sensor because the conventional method was influenced by reducing substances other than the assimilable sugars. After cultivation for 30 h, the assimilable sugars and glutamic acid in the broth were determined by paper chromatography and Warburg's method using glutamate decarboxylase, 11 respectively. The assimilable sugars were hardly detectable (glutamic acid concentration 7.5 g 1 ~. At that time, the microbial sensor gave 5 g 1-1 which was smaller than the value of 16 g 1 =obtained by the conventional method. The reusability of the electrode was examined by using a standard solution containing glucose and the broths. When the microbial sensor was used continuously for a long time, the nraximum current decrease of the electrode for a standard solution gradually drifted (within 15% during 10 days use) thus, occasional calibration of the electrode was required. For example, two standard solutions (0.4 and 1.0 mM glucose) were employed after every 10 samples, giving two points of calibration. The values obtained were then reproducible to within 4% of the relative error, and the concentration of total assimilable sugars was accurately determined by the microbial sensor. This sensor could be used for more than 10 days and 960 assays, indicating good survival of the bacteria in the electrode,
238
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 0 , V o l . 2, J u l y
Table 2 Comparison of total assimilable sugars determined by the microbial electrode and the conventional method Cultivation a time (h) 0 8
15 24 30
Total sugars b (g I -~ )
Glutamic acid (g I - j )
Microbial electrode
Ferricyanide method
39
67
-
13 8 6 5
41 23 18 16
75
aFeeding method was used; bcalculated as glucose
and its suitability for the determination of assimilable sugars in broths. Further developmental study is intended to improve the selectivity of the microbial sensor.
Acknowledgements The authors are grateful to Mr T. Kiya, Dr Y. Sakata and Dr K. Mitsugi, Central Research Laboratories, Ajinomoto Co., for their helpful advice and encouragement during this study and also to Mr T. Shirakawa for his assistance.
References I 2 3 4 5 6 7 8
9 I0 11
Reducing sugar and sucrose in food products' Industrial method no. 142 71 A,Technicon Industrial Systems, 1972 Guilbault, G. G. Handbook of Enzymatic Methods ofA nalysis M. Dekker, New York, 1976 Satoh, S., Karube, 1. and Suzuki, S. BiotechnoL Bioeng. 1976, 18, 269 Hikuma, M., Suzuki, It., Yasuda, Y., Karube, I. and Suzuki, S. Eur. J. NppL Microbiol. Bioteehnol. 1979, 8,289 Hikuma, M., Kubo, T., Yasuda, T., Karubc, 1. and Suzuki, S. Biotectmol. Bioeng. 1979, 21, 1845 Hikuma, M.,Kubo, T.,Yasuda, T., Karube, l., and Suzuki, S. Anal. Chim. Acta 1979, 109, 33 Matsunaga,T., Karube, 1. and Suzuki, S. Anal. Chim Acta 1978, 99, 233 Handbook ofEnvironnzental 6bnlrol, (Bond, R. G. and Straub, C. P., eds) CRC Press, Cleveland, Ohio, 1973, vol 3, pp. 671 686 Yamada,K. and Komagata, K. J. Gen. ,4ppl. Microhiol. 1972, 18, 399 Paper671romatography (ltais, 1. M. and Macck, K., eds) Academic Press, New York, 1963, pp. 289 330 'Monosodium glutamate in fermenter solutions' Industrial method no. 210 72 A, TechniconlndustrialSystems, i973