Efficiency of Microbial Growth in Biomass Production INTRODUCTION Since Bauchop and Elsden' introduced the ATP yield constant, many papers dealing with different yield constants have However, these papers are mainly on the determination of the yield constant for different substrates and different microorganisms. Only a few papers relate the yield constants to the energy content of the substrates used. In this paper a theory for determining the efficiency of the microbial growth will be presented.
THEORY Forrest and Walker6 deduced that the amount of energy necessary to produce 1 g cell dry weight was 37 mmol ATP when all the cell constituents were synthesized
from glucose or other closely related compounds. This means that for the production of Yx,, g cells an amount of energy corresponding to Yx,.0.037 mol ATP are necessary. For heterotrophic microorganisms the energy production is closely connected to the catabolism of the carbon source. Part of the carbon source is used for synthesis of the carbon skeletons of the C-containing compounds in the cell, another part is used for production of different extracellular metabolites, and the rest is used for energy production. If the content of carbon in the carbon source is b,, the C content in the cells is b x and the C content in the products is b p , then by synthesis of YXlsg cells and Y,,, g products, (b,. Y,,, + 6,. YpIs) g carbon is used. The carbon source used for energy production will therefore be
In most aerobic fermentations the value Ypis is extremely low compared to Yxls and especially in biomass productions the aim is to keep Y p r s= 0. Equation ( 1 ) can then be simplified to
Dependent on catabolism an amount of as mol ATP will be produced for catabolism of 1 mol of the carbon source having the molecular weight M s and the energy will then be 1 - ( b x / b s ). YXiS. (3) a.9 MS
This energy production must be equal to the energy need of the biomass production, Yx,,.0.037:
However, the efficiency of the energy synthesis is considered only to be eq. (4)is then changed to
r)
and
Biotechnology and Bioengineering, Vol. XX, Pp. 1691- 1694 (1978) 0006-3592/78/OO20-1691$01.OO
@ 1978 John Wiley & Sons, Inc.
1692
BIOTECHNOLOGY AND BIOENGINEERING VOL. XX (1978)
Consequently, the yield constant can be found as
or the efficiency can be found as
From eq. (6) it is seen that the yield constant Yx,s is a hyperbolic function of the efficiency, q, as shown in Figure 1. RESULTS AND DISCUSSION
In order to determine the value of q some of the numerous values of yield constants in the literature have been used. In Table I some of the highest reported values of Y,,, are listed and the values of q are given assuming that 6 , is 0.45. From Table I it is seen that the efficiency of microbial growth is 0.235 for growth on carbohydrates and almost the same for growth with amino acids as the only carbon and energy source. The reason for the low efficiency may be found in the energy production in the cells (i.e., low efficiency in oxidative phosphorylation) or it may be found in a destruction of energy-containing compounds (i.e., existence of ATPases). The efficiency of the energy production can be determined by measuring the PI0 ratio. This value can be estimated if the yield constant with respect to oxygen Yxlo, is known since the PI0 ratio is then PI0 = 0.5 Y,,,,
Y,,,
where YATP is the amount of cells producedimol ATP used, i.e., (U0.037) g/mol as indicated above. Kjaergaard' has measured the Y,,,, value for chemostat- as well as batch-grown Bacillus licheniformis with glucose as the sole carbon and energy source. These values are found in Table 11, in which the efficiency of the cell
0
0.2
0.4
0.6
0.8
1.0
R Fig. 1. Theoretic relation between the yield constant Y with respect to the energy and carbon source and the growth efficiency, q.
COMMUNICATIONS T O T H E EDITOR
1693
TABLE I Yield Constants, Yx,s, for Different Organisms Grown on Different Carbon Sources with the Molecular Weights M s, the Carbon Content bs giving a mol ATP by Complete Oxidation, and the Growth Efficiency, 77 a (mol ATP/mol (g/g) (g/mol) substrate)
Ms
Yx,s
Substrate
Microorganism
Glucose
Klebsiella aerogene? Bacillus licheniformis Klebsiella aerogenes3 Klebsiella aerogenes3
Glucose Sucrose Maltose
bs
0.54
180
38
0.40 0.23
0.53
180
38
0.40 0.24
0.58
342
76
0.42 0.25
0.56
342
76
0.42 0.22
-
Average for carbohydrates Proline Ornithine
Bacillus licheniformis ' Bacillus licheniformis
1)
0.235 0.69
114
30
0.53 0.23
0.60
132
33
0.45 0.22
production is also given. It is seen that the P/O ratio, as well as the efficiency, is much lower for batch growth than for continuous growth. Normally it is assumed that the P/O value can be as high as 3.0. Therefore, the low values obtained can be taken as an indication of the fact that some of the low growth efficiency is due to a low energy production efficiency. For batch growth it is seen that the P i 0 ratio is about 1/10 of the maximum-the same as the 7 value. For continuous growth, however, the P i 0 ratio is almost half the maximum value, whereas the 7 value is only about 1/4 of the theoretical maximum. This indicates that the low growth efficiency during a batch process is mostly due to a low energy production efficiency and that the higher efficiency during a continuous process is due to higher growth efficiency and especially a higher energy production efficiency. This is in agreement with the results obtained by Hempfling,8 who found that the presence of glucose lowered the P/O ratio. In continuous cultures the limiting component is only present in very low concentrations indicating that it
TABLE I1 Yield Constants with Respect to Oxygen for B . licheniformis Grown in Chemostat and Batch and Corresponding P/O Ratio and Growth Efficiencies, 1) Type of culture Chemostat Batch
Yxm,
Pi0
1)
71 14
1.3 0.3
0.24 0.12
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BIOTECHNOLOGY AND BIOENGINEERING VOL. XX (1978)
will be much more efficient to perform biomass production by continuous methoda result that is well known from the technique used in the production of bakers’ yeast.g References 1. J. Bauchop and S. R. Elsden, J. Gen. Microbiol., 23, 457 (1960). 2. L. P. Hadjipetrou, J . P. Gerrits, F. A. G. Tenlings, and A. H. Stouthamer, J . Gen. Microbiol., 36, 139 (1964). 3. G. H. Bell, Proc. Biochem., 7(4), 21 (1972). 4. T. C. Bolton, P. J. Rodgers, and A. C. R. Dean, J. Appl. Chem. Biotechnol., 22, 941 (1972). 5. T. P. Coultate and T. K. Sundaram, J . Bacteriul., 121, 55 (1975). 6. W. W. Forrest and D. J. Walker, in Advances on Microbiological Physiology, A. H. Rose and J. F. Wilkinson, Eds., Academic, London, 1971, Vol. 5 , p. 213. 7. L. Kjaergaard, Ph.D. thesis, Technical University of Denmark, Lyngby, Denmark, 1976. 8. W. P. Hempfling, Biochem. Biophys. Res. Commun., 41, 9 (1970). 9. H. Y. Wang, C. L. Cooney, and D. I. C. Wang, Biotechnol. Biueng., 19, 69 (1977).
LEIFKJAERGAARD Department of Applied Biochemistry The Technical University of Denmark Block 223, DK-2800 Lyngby, Denmark Accepted for Publication April 20, 1978
THEORY Forrest and Walker6 deduced that the amount of energy necessary to produce 1 g cell dry weight was 37 mmol ATP when all the cell constituents were synthesized
from glucose or other closely related compounds. This means that for the production of Yx,, g cells an amount of energy corresponding to Yx,.0.037 mol ATP are necessary. For heterotrophic microorganisms the energy production is closely connected to the catabolism of the carbon source. Part of the carbon source is used for synthesis of the carbon skeletons of the C-containing compounds in the cell, another part is used for production of different extracellular metabolites, and the rest is used for energy production. If the content of carbon in the carbon source is b,, the C content in the cells is b x and the C content in the products is b p , then by synthesis of YXlsg cells and Y,,, g products, (b,. Y,,, + 6,. YpIs) g carbon is used. The carbon source used for energy production will therefore be
In most aerobic fermentations the value Ypis is extremely low compared to Yxls and especially in biomass productions the aim is to keep Y p r s= 0. Equation ( 1 ) can then be simplified to
Dependent on catabolism an amount of as mol ATP will be produced for catabolism of 1 mol of the carbon source having the molecular weight M s and the energy will then be 1 - ( b x / b s ). YXiS. (3) a.9 MS
This energy production must be equal to the energy need of the biomass production, Yx,,.0.037:
However, the efficiency of the energy synthesis is considered only to be eq. (4)is then changed to
r)
and
Biotechnology and Bioengineering, Vol. XX, Pp. 1691- 1694 (1978) 0006-3592/78/OO20-1691$01.OO
@ 1978 John Wiley & Sons, Inc.
1692
BIOTECHNOLOGY AND BIOENGINEERING VOL. XX (1978)
Consequently, the yield constant can be found as
or the efficiency can be found as
From eq. (6) it is seen that the yield constant Yx,s is a hyperbolic function of the efficiency, q, as shown in Figure 1. RESULTS AND DISCUSSION
In order to determine the value of q some of the numerous values of yield constants in the literature have been used. In Table I some of the highest reported values of Y,,, are listed and the values of q are given assuming that 6 , is 0.45. From Table I it is seen that the efficiency of microbial growth is 0.235 for growth on carbohydrates and almost the same for growth with amino acids as the only carbon and energy source. The reason for the low efficiency may be found in the energy production in the cells (i.e., low efficiency in oxidative phosphorylation) or it may be found in a destruction of energy-containing compounds (i.e., existence of ATPases). The efficiency of the energy production can be determined by measuring the PI0 ratio. This value can be estimated if the yield constant with respect to oxygen Yxlo, is known since the PI0 ratio is then PI0 = 0.5 Y,,,,
Y,,,
where YATP is the amount of cells producedimol ATP used, i.e., (U0.037) g/mol as indicated above. Kjaergaard' has measured the Y,,,, value for chemostat- as well as batch-grown Bacillus licheniformis with glucose as the sole carbon and energy source. These values are found in Table 11, in which the efficiency of the cell
0
0.2
0.4
0.6
0.8
1.0
R Fig. 1. Theoretic relation between the yield constant Y with respect to the energy and carbon source and the growth efficiency, q.
COMMUNICATIONS T O T H E EDITOR
1693
TABLE I Yield Constants, Yx,s, for Different Organisms Grown on Different Carbon Sources with the Molecular Weights M s, the Carbon Content bs giving a mol ATP by Complete Oxidation, and the Growth Efficiency, 77 a (mol ATP/mol (g/g) (g/mol) substrate)
Ms
Yx,s
Substrate
Microorganism
Glucose
Klebsiella aerogene? Bacillus licheniformis Klebsiella aerogenes3 Klebsiella aerogenes3
Glucose Sucrose Maltose
bs
0.54
180
38
0.40 0.23
0.53
180
38
0.40 0.24
0.58
342
76
0.42 0.25
0.56
342
76
0.42 0.22
-
Average for carbohydrates Proline Ornithine
Bacillus licheniformis ' Bacillus licheniformis
1)
0.235 0.69
114
30
0.53 0.23
0.60
132
33
0.45 0.22
production is also given. It is seen that the P/O ratio, as well as the efficiency, is much lower for batch growth than for continuous growth. Normally it is assumed that the P/O value can be as high as 3.0. Therefore, the low values obtained can be taken as an indication of the fact that some of the low growth efficiency is due to a low energy production efficiency. For batch growth it is seen that the P i 0 ratio is about 1/10 of the maximum-the same as the 7 value. For continuous growth, however, the P i 0 ratio is almost half the maximum value, whereas the 7 value is only about 1/4 of the theoretical maximum. This indicates that the low growth efficiency during a batch process is mostly due to a low energy production efficiency and that the higher efficiency during a continuous process is due to higher growth efficiency and especially a higher energy production efficiency. This is in agreement with the results obtained by Hempfling,8 who found that the presence of glucose lowered the P/O ratio. In continuous cultures the limiting component is only present in very low concentrations indicating that it
TABLE I1 Yield Constants with Respect to Oxygen for B . licheniformis Grown in Chemostat and Batch and Corresponding P/O Ratio and Growth Efficiencies, 1) Type of culture Chemostat Batch
Yxm,
Pi0
1)
71 14
1.3 0.3
0.24 0.12
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BIOTECHNOLOGY AND BIOENGINEERING VOL. XX (1978)
will be much more efficient to perform biomass production by continuous methoda result that is well known from the technique used in the production of bakers’ yeast.g References 1. J. Bauchop and S. R. Elsden, J. Gen. Microbiol., 23, 457 (1960). 2. L. P. Hadjipetrou, J . P. Gerrits, F. A. G. Tenlings, and A. H. Stouthamer, J . Gen. Microbiol., 36, 139 (1964). 3. G. H. Bell, Proc. Biochem., 7(4), 21 (1972). 4. T. C. Bolton, P. J. Rodgers, and A. C. R. Dean, J. Appl. Chem. Biotechnol., 22, 941 (1972). 5. T. P. Coultate and T. K. Sundaram, J . Bacteriul., 121, 55 (1975). 6. W. W. Forrest and D. J. Walker, in Advances on Microbiological Physiology, A. H. Rose and J. F. Wilkinson, Eds., Academic, London, 1971, Vol. 5 , p. 213. 7. L. Kjaergaard, Ph.D. thesis, Technical University of Denmark, Lyngby, Denmark, 1976. 8. W. P. Hempfling, Biochem. Biophys. Res. Commun., 41, 9 (1970). 9. H. Y. Wang, C. L. Cooney, and D. I. C. Wang, Biotechnol. Biueng., 19, 69 (1977).
LEIFKJAERGAARD Department of Applied Biochemistry The Technical University of Denmark Block 223, DK-2800 Lyngby, Denmark Accepted for Publication April 20, 1978
