A series of experiments demonstrating that on-line measurements of intracellular NADH by culture fluorescence can be used to monitor and control the fermentation process are described. A distinct advantage of intercellular NADH measurements over other monitoring techniques such as pH and dissolved oxygen is that it directly measures real time events occurring within the cell rather than changes in the environment. When coupled with other measurement parameters, it can provide a finer degree of sophistication in process control

Armiger, W. B.

Forro, J. R.

Lee, J. F.

Montalvo, L. M.

English

NASA Technical Reports Server

FED-BATCH CONTROL BASED UPON THE MEASUREMENT 
OF INTRACELLULAR NADH 
W. B. Armiger, J. F. Lee, L. M. Montalvo 
and J. R. Forro 
BioChem Technology, Inc. 
Malvern, PA 
I NTROD U CT I ON 
This paper describes a series of experiments demonstrating that on-line meas- 
urements of intracellular NADH by culture fluorescence can be used to monitor 
and control fermentation processes. A distinct advantage of intracellular 
NADH measurements over other monitoring techniques such as pH and dissolved 
oxygen is that it directly measures real-time events occurring within the 
cell rather than changes in the environment and when coupled with other meas- 
urement parameters can provide a finer degree of sophistication in process 
control. 
Culture fluorescence measurement techniques were first introduced by Duysens 
and Ames2.l They noted that the fluorescence spectrum for suspensions of 
aerobic baker's yeast was similar to the one for NADH and that the fluores- 
cence signal could be significantly enhanced by additions of ethanol or glu- 
cose. Damped oscillations in fluorescence were observed after the establish- 
ment of anaerobic conditions. These oscillations were similar to those re- 
ported earlier by Chance' in yeast using absorption spectrophotometry. 
Chance and Thorel13 found that most cellular fluorescence in eucaryotic cells 
was localized within mitochondria where NADH is concentrated because of its 
role in respiration. Chance et. a1 .4 investigated the initiation of fluores- 
cence oscillations in suspensions o f  starved yeast. The oscillations are 
caused by the sudden rates of glycolysis (Pasteur Effect). This work 
established a laboratory technique to follow intracellular metabolism. 
Harrison and Chance5 later built an instrument capable of measuring culture 
fluorescence within a fermentor. With this system, they were able to monitor 
aerobic/anaerobic transitions in continuous cultures of Klebsiella aeroqenes. 
Using a similar device, Zabriskie and Humphrey6 established a linear rela- 
tionship between the log of culture fluorescence and the log of cell con- 
centration. Ristroph et. at.' studied the relationship between culture 
fluorescence and the growth of Candida utilis in a fed-batch fermentation 
during which ethanol was added in discrete pulses. In this process, exces- 
sive ethanol feeding rates cause acetate to accumulate as a by-product. In 
the absence of ethanol, the yeast can assimilate the accumulated acetate. 
After each ethanol pulse, culture fluorescence peaks and declines gradually 
Y j  "EECEDING PAGE BLANK NOT F K M S  
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as the ethanol is consumed. At the point where ethanol is exhausted and 
acetate consumption begins, the fluorescence declines more rapidly. Corre- 
sponding changes in the respiratory quotient indicate the metabolic changes 
accompanying the diphastic growth. The reproducible response of culture 
fluorescence to ethanol and acetate metabolism suggests that the instrument 
could be used to control the ethanol feeding schedule to improve process 
yield and productivity. 
NADH IN METABOLISM 
A highly simplified summary of basic cellular metabolism, illustrated in 
figure 7-1, shows how NAD+ is constantly cycled between the oxidized form 
(NAD+) and the reduced form (NADH). As the organism utilizes substrate and 
converts it into carbon dioxide, metabolic intermediates and other catabo- 
lites, NAD+ is reduced to NADH. In aerobic cells, oxygen is used to reoxi- 
dize the NADH to NAD+ during oxidative phosphorylation and energy in the 
form of ATP is produced for use in other metabolic reactions (e.g., growth). 
Anaerobic organisms operate in a similar manner, but use electron acceptors 
other than oxygen. 
Figure 7-1 may also be used to illustrate the effect of various parameters 
on the reducing level of the cell - that is, the ratio of NADH concentration 
to the total NADH + NAD+ concentration in the cell. If in an aerobic system 
the oxygen supply is limited while there is an adequate supply of substrate 
present, catabolism will continue to function, but reoxidation of NADH by 
respiration stops. 
unity. 
levels fall and the reducing level approaches zero. 
Under these conditions the reducing level approaches 
If on the other hand, substrate becomes limiting or depleted, NADH 
By measurement and control of the reducing state of the cell (i.e., the 
ratio of NADH to total NADH + NAD+), it is possible to maintain the culture 
in the desired condition for selected growth or metabolic activity. 
THE FLUORESCENT SIGNAL 
The fluorescent signal received from a fermentation culture is a composite 
signal which is a function of the number of cells, the reducing state of the 
cell, and environmental effects. This is illustrated in figure 7-2 for a 
fed-batch process which may be viewed as a high frequency signal superim- 
posed on a low frequency signal. If the signal is properly decomposed it 
can reveal information regarding the process. The cell concentration and 
environmental effects will vary slowly while the metabolic effects (reducing 
state) due to varying nutrient or oxygen can change rapidly. Decomposition 
94 
of the fluorescent signal into its major components can then yield data for 
process control. 
FERMENTOR STUD I ES 
Figure 7-3 is data obtained during growth of a mammalian cell culture showing 
fluorescence (NFU values), total cell count (-), percent viability (+), and 
temperature. Figure 7-4 depicts similar results obtained with a Pediococcus 
and shows NFU, pH, and viable cell counts (El). In both cases there i s  a 
good correlation between cell growth and measured culture fluorescence, but 
it is also evident that the fluorescent signal has other components - lack 
of correlation between cell count and fluorescence in early stages of Pedio- 
coccus growth was due, for example, to pH effects on cells and background 
(environmental). 
Actual data illustrating how the signal can be decomposed into its specific 
elements is shown in the next series of figures. Figure 7-5 shows the re- 
sponse o f  a culture of the yeast Saccharom.yces to changes in dissolved oxygen 
levels. Yeast cells were placed in a nongrowth environment and allowed to 
metabolize their internal glycogen reserve - yielding a relatively constant 
fluorescent signal. 
At 1.5 hours the air to the fermentor was turned off. As the DO level began 
to fall and approach zero, there was a step increase in the culture fluores- 
cence signal due to an increase in intracellular NADH. This corresponds to 
the organism undergoing a metabolic transition from an aerobic to an anaero- 
bic state. It should be noted that the step increase in fluorescence act- 
ually occurs prior to a DO reading o f  zero. This  i s  a consequence of the 
more rapid response of the F1uroMeasureTM Detector relative to a DO probe. 
By the F1uroMeasureTM monitoring intracellular conditions, it indicates that 
the cell is internally anaerobic before the broth i s  depleted o f  oxygen, The 
air flow was turned back on at 2.0 hours and there was a rapid decrease in 
the NFU reading back to the level corresponding to aerobic metabolism. After 
2.0 hours the culture continued to be in a resting condition. After 2.5 
hours there was a rapid decrease in the fluorescence (NFU) signal as the gly- 
cogen stores of the yeast became depleted. At approximately 3.0 hours the DO 
returned to 100% and the level o f  culture fluorescence (NFU) reached its min- 
imum energy value as metabolism ceased. 
In the absence of glucose and glycogen, yeast will aerobically metabolize 
ethanol via the pathway illustrated in figure 7-6. 
is metabolized through acetaldehyde to acetate and in both reactions NADH is 
produced. Acetate is further metabolized through acetyl-coA into the TCA 
cycle. 
In this pathway ethanol 
95 
Using high pressure liquid chromatography to monitor the change in extra- 
cellular ethanol and acetate concentrations, an ethanol addition cycle was 
studied and the results are shown in figure 7-7. At 2.4 hours ethanol was 
added at a concentration of 250 PPM. The ethanol was rapidly absorbed by 
the cells and its concentration dropped to less than 100 PPM within the 
first minute after its addition. This was followed by a more gradual de- 
cline in the ethanol concentration between 2.5 and 3.0 hours until ethanol 
was entirely depleted. 
ethanol concentration is greater than 50 PPM, there is a steady state condi- 
tion in the NFU signal. As the ethanol concentration drops below this level 
and approaches the Ks value, it becomes rate limiting and there is a rapid 
decrease in the level of intracellular NADH. This indicates that the cells 
are in a transition from one steady state reducing level to another lower 
reducing level. At 3 hours there is an inflection point corresponding to the 
exhaustion of ethanol. During this same time period there is an accumulation 
of acetate in the media. The acetate concentration, which was initially 
zero, increases to greater than 100 PPM while the ethanol is being utilized. 
When the ethanol is depleted, acetate re-enters the cell and is then metab- 
olized. Depletion of  the acetate corresponds to the next inflection point 
in the curve a t  3.15 hours. 
It should be noted that when the extracellular 
Extending this data to various cell concentrations it is found that a series 
o f  curves can be developed relating the NFU value to the active catabolic 
pathways. The bottom curve, representing the 
lowest reducing state for the culture, is the NFU values for cells which are 
in the resting state devoid of metabolizable substrate. The top curve, 
representing the highest steady state cellular reducing level, i s  derived 
from the NFU value for cells converting glucose to ethanol under anaerobic 
conditions. Similar curves have been generated for aerobic conversion of 
ethanol to acetate and for acetate metabolism. For any cell concentration, 
the on-line NFU value can be used to determine the active metabolic pathways. 
Thus, the reducing state of the cell is a basis for process control. 
This is shown in figure 7-8. 
Fed-batch fermentations with Saccharomyces were conducted using a glucose 
feed rate based on a known starting biomass concentration and an estimated 
biomass doubling time. Figure 7-9 shows the NFU and cell mass data for one 
of these runs. For the first 6 hours after inoculation NFU levels remained 
constant and then began to rise. Cell mass increased only slightly during 
this time. The sharp drop off in the NFU value at about 7.5 hours was due to 
stoppage of the glucose feed. The glucose feed was restored at about 9 hours 
and both cell mass and NFU readings continued to increase. 
For each cell mass value measured in this experiment a corresponding minimum 
(resting cell) and maximum (glucose to ethanol pathway) reducing value was 
determined by reference to figure 7-8. 
utilization were also determined (+-). 
Reducing levels for aerobic ethanol 
These values were then plotted along 
96 
with the NFU readings from the fed-batch fermentation (fig. 7-9) and are 
shown in figure 7-10. 
glucose theoretically can be converted to cell mass. Production of ethanol 
results in reduced yields and lower productivity. 
mass production, therefore, the reducing state should be control led between 
the two extreme conditions. 
During production of yeast, 50% of the available 
For optimal yeast cell 
As can be seen from figure 7-10 initial NFU values were high, indicating 
overfeeding with resulting ethanol production. This was confirmed by off- 
line HPLC analysis for ethanol. As cell mass began to increase, the NFU 
reading remained constant and approached a midpoint range between the two 
extreme values, At about 7.5 hours, stoppage of the feed resulted in an 
immediate drop in NFU values to the resting cell level, When the feed was 
restarted at about 9 hours, NFU values increased immediately and then dropped 
again. 
proper balance between substrate consumption and increase in cell mass. 
ginning at about 12 hours; however, an imbalance between these two parameters 
occurred again resulting in the production of ethanol as evidenced by the 
increase in NFU values and confirmed by HPLC analysis. A series of oscilla- 
tions was then noted in which additional ethanol was produced indicative of 
the Crabtree effect. 
From about 11 to 12 hours into the run the NFU values indicated a 
Be- 
From about 15 to 20 hours into the run the glucose feed rate was constant and 
the cell mass increased to a level where it was no longer being overfed. At 
this stage the glucose feed was stopped and NFU values dropped and remained 
at a level corresponding to aerobic ethanol metabolism. 
CONCLUSION 
These data demonstrate that the measurement of intracellular NADH can be 
utilized to monitor the internal reducing state o f  cells. The internal re- 
ducing state reflects the major metabolic events which are occurring in the 
culture. Since these data describe pathways which are common to those found 
in many procaryotic and eucaryotic organisms, similar information may be used 
to control a wide variety of fermentations and cell cultures. 
It should be possible to develop control algorithms for product induction or 
feeding strategies to maintain a culture at a selected reducing level corre- 
sponding to the desired product output. 
97 
REFERENCES 
1. Duysens, L, N. M., and Amesa, J., IIFluorescence Spectrophotometry of 
Reduced Phosphopyridine Nucleotide in Intact Cells in the New-Ultra- 
violet and Visible Region," Biochem. et Biophys. Acta., 24:19, 1957. 
2. Chance, B., "Spectrophotometry of Intracellular Respiratory Pigments," 
Science, 120:767, 1954, 
3. Chance, B., and Thorell, B., IIFluorescence Measurements o f  Mitochondrial 
Pyridine Nucleotide in Aerobiosis and Anaerobiosis," Nature, 184:931, 
1959. 
4. Chance, B., Estabrook, R. W.,  and Ghosh, A,, llDamped Sinusoidal Oscilla- 
tions o f  Cytoplasmic Reduced Pyridine Nucleotide in Yeast Cells," PNAS, 
51:1244, 1964. 
5. Harrison, D. E. F., and Chance, B., IIFluorimetric Technique for Monitor- 
ing Changes in the Level o f  Reduced Nicotinamide Nucleotides in Contin- 
uous Culture o f  Microorganisms," Appl. Microbiol., 19:446, 1970. 
6. Zabriskie, D. W., and Humphrey, A. E., "Estimation of Fermentation Bio- 
mass Concentration by Measuring Culture Fluorescence," Appl. Environ. 
Microbiol., 35:337, 1978. 
7. Ristroph, D, L., Watteeuw, C. M., Armiger, W. B., and Humphrey, A. E., 
"Experience in the Use o f  Culture Fluorescence for Monitoring Fermenta- 
tions," J. Ferment. Techno1 ., 55:599, 1977. 
Figure 7-1.- Oiagram of basic cellular metabolism showing involvement of 
oxidized and reduced pyridine nucleotides. 
98 
Figure 7-2.- Representation of culture fluorescence as a function of the 
number of cells, the reducing state of cells, and environmental effects. 
Bi cChem Technology, Inc. 
Flurot leasure S y s t e m  
R u n  Number  CW3-10 
L a b o r t c r y  Data c u b - ? O A  
Startin5 Dace 1 L - X -  1984 
S t a r t i n p  T i m e  18:07:00 
Figure 7-3.- Fluorescent changes during growth of Mouse L29 cells 
in suspension culture. 
99 
B i o C h e m  T e c h n o l o g y .  Inc. 
FluroMeasure System 
R u n  Number ~ L T - Q ~ F I  
Laboratory  Data MLT-O7AL 
Starting D a t e  06-26-1985 
Starting T i m e  07:40:00 
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The Range of Variables i n  HLT-07AL 
V a r  i ab 1 e Name Range Llni t 
cell c o n  0 . 00--- 200.00 c / m l  l O L 7  
Figure 7-4.- Fluorescent changes during growth o f  a Pediococcus sp. 
Figure 7-5.- Fluorescent response o f  Saccharomyces to changes 
in dissolved oxygen levels. 
100 
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Figure 7-7.- Correlation of fluorescence with ethanol and acetate 
concentration during ethanol metabolism in starved Saccharomyces. 
Run Number EioChrm Technology, Inc. 
Laadrtory ~ a t a  REDRESS FluroMeasure S y s t e m  
S t a r t i  nq Date 
Start:ng Time 
1 B 1 B 8 8 . 8 8  F1 S E 
The Range of V + r i & l e s  i n  REGRESE 
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+ ETQH 
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0.00---1000.00 NFU 
0 . 00--- 1000.00 NFU 
0.00---1000.00 NFU 
Figure 7-8.- Fluorescence values at various cellular reducing states 
as a function of dry cell mass. 
102 
R u n  Number 
Lacoratory Data mf-06al 
Starting Date 
Starting Time 
Ei oChem Techno1 c q y ,  I n c .  
FluroMeasure S y s t e m  
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Figure 7-9.- Fluorescence and cell mass changes during fed-batch growth 
of Saccharomyces on glucose. 
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Figure 7-10.- Measured fluorescent values versus theoretical minimum and 
maximum values for a Saccharomyces fed-batch glucose fermentation. 
103 


Fed-batch control based upon the measurement of intracellular NADH

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