BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIX, PAGES 413-424 (1977)
Aeration without Air: Oxygen Supply by Hydrogen Peroxide H. G . SCHLEGEL,* Institut fiir Mikrobiologie der Gesellschaft fiir Strahlen und Umweltforschung m bH und der Universitat, Gottingen, Federal Republic of Germany
Summary Oxygen has been supplied to suspensions of microorganisms kept under nitrogen by the addition of hydrogen peroxide. If catalaae was present in the suspension and the flow was adjusted to the rate of oxygen consumption, the cells grew at rates identical to the controls incubated under air. The applicability of oxygen supply by hydrogen peroxide and its limits are discuesed.
INTRODUCTION Compared with the solubility of conventional energy and carbon sources, the solubility of oxygen is very low. Therefore oxygen cannot be stored in a nutrient solution. Only oxygen dissolved in water is taken u p by the cells. Suspensions of aerobic microorganisms depend on the continual supply of oxygen to, and its dissolution in, the nutrient solution. Normally, oxygen is supplied by air. The efficiency of aeration is a function of those parameters which are described in Fick’s Law of Diffusion. The phase boundaries can be increased by several means, however, only in limits. The oxygen partial pressure may be increased as well; however, the rate of transfer of oxygen from the gas into the liquid phase depends on the diffusion constant D ; therefore the oxygen transfer rate can be manipulated only within narrow limits. The question has been raised whether an oxygen concentrate can be added to the nutrient solution in liquid form; this would drastically simplify the means of aeration. I n addition, oxygen could be easily supplied t o those cells which do not tolerate the high shearing forces due t o strong aeration and agitation. *Present address: Institute of Microbiology, Grisebachstrasse 8, 3400 Gottingen, Federal Republic of Germany. 413
@ 1977 by John Wiley & Sons, Inc.
414
SCHLEGEL
Perhydrol (30% HzOz)is such a n oxygen concentrate; about 100 liter oxygen is produced from the hydrogen peroxide contained in 1 liter perhydrol. One liter perhydrol is equivalent t o 500 liter air which is sufficient to produce about 135 g (dry wt) cells when glucose is the substrate. The experiments to be described later aimed a t examining the conditions under which a suspension of growing microorganisms can be supplied with oxygen through continuous addition of diluted perhydrol solutions. A short communication was presented a t the Fifth International Fermentation Symposium.
MATERIALS AND METHODS Organisms The organisms used were Alcaligenes eutrophus H 16 (ATCC 17699, DSM 428) and the mutant PHB-4 (DSM 541) derived from the wild-type strain and unable to accumulate poly(0-hydroxybutyric acid),2 Acinetobacter calcoaceticus (DSM 586) , Pseudomonas putida (DSM 291,) Paracoccus denitrijicans (DSM 413), Candida oleophila (ATCC 20177, DSM 343) , and Saccharom yces cerevisiae.
Growth Experiments Alcaligenes eutrophus, Pseudomonas putida, and Paracoccus denitrijicans were grown in a mineral nutrient solution3 supplemented with 1% fructose; Acinetobacter calcoaceticus was grown with 1% glucose. Candida oleophila and S. cerevisiae were grown in a medium containing 0.3’% each of Difco nutrient broth, Difco yeast extract, and 1% glucose. After overnight growth, the cells were harvested by centrifugation and were resuspended in a warm nutrient solution t o a certain cell density, transferred to 100 ml Erlenmeyer flasks, and shaken in a thermoconstant bath a t 30°C. At 1 hr intervals samples were taken and diluted for turbidity measurements. The optical density was measured in a Zeiss Filterphotometer (PL 4) a t 436 or 546 nm. The stopcocks had facilities for gasing, for the addition of perhydrol, and for sampling. A Vario-Perpex pump from LKB (Bromma, Sweden) served for the supply of perhydrol. I n most experiments, 2.5 ml diluted perhydrol solution per hour were added to 30 ml cell suspension. As the suspension was continually diluted by the perhydrol solution, the optical density values had to be corrected for dilution and the corrected values were considered for preparing the semilogarithmic plot of the growth curve (Fig. 1.)
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
415
Alcaligenes eutrophus 15.01 H16- PHB-4
-1
Fructose.aerobically
1.5
0 I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
Time in hours Fig. 1. Comparison of growth curves of Alcaligenes eutrophus. Small Erlenmeyer vessels each containing 30 ml suspension of growing cells were shaken in a waterbath at 30°C. From vessel I small samples were taken to measure the OD. Nutrient medium was continually pumped into vessel 11, a t a rate of 5.0 ml/hr. Five ml samples were taken at intervals of 1hr and used for turbidity measurements. Black circles represent the actual readings; open circles represent the turbidity values (calculated) which the suspension would have reached without continual dilution. Dotted line I1 is the fictive growth curve resulting from adding the interval stretches I1 to each other.
Chemicals Bovine liver catalase (20 mg/ml) was purchased from Boehringer, Mannheim; perhydrol (30% hydrogen peroxide) and other chemicals from Merck, Darmstadt.
RESULTS Sensitivity of Cells to Hydrogen Peroxide The sensitivity to hydrogen peroxide was tested in growth experiments with Alcaligenes eutrophus as the model organism. Perhydrol was diluted in a 0.5% fructose nutrient solution at a serial dilution ratio of 1:2; the nutrient solution was inoculated by l o 7 cells per ml. Readings were taken after three to five days. When the initial HzOz concentration was l l m M , the cells did not grow; however, growth was not impaired at 8mM HzO2. From the determination of catalase activities of cell suspensions (vide infra) it became obvious that the hydrogen peroxide in the nutrient medium must have been cleaved a t a high rate as soon as the cells were inoculated. There-
416
SCHLEGEL
fore, the cells were exposed to the peroxide only for a few minutes. Cell respiration proved to be even less sensitive. Cellular respiration was measured manometrically; when fructose was used as a substrate significant inhibition occurred at an initial concentration of 22mM H202. Since the cells contained catalase, the concentration of H202 in the suspension continuously decreased. The inhibitory effect estimated was apparently due to irreversible cell damage which had already occurred immediately after addition of the hydrogen peroxide solution to the cell suspension. The manometric experiments only indicated that the contact of the cells with lOOmM H202 solution did not yet cause irreversible damage to the respiratory metabolism. If catalase was added to the cell suspension (usually 1 pl. per 1ml nutrient solution or bacterial suspension) prior to the addition of hydrogen peroxide solution, there was no inhibitory effect on respiration or on growth up to 1M concentration of HzOt. Furthermore, it was examined whether accessory substances which are present in commercial perhydrol solutions, e.g. , hydroquinone, added as a stabilizer, or other contaminating compounds, exert inhibitory effects. From perhydrol solutions containing 30 or 43% hydrogen peroxide the hydrogen peroxide was removed by the addition of catalase. This solution was sterilized and added to the nutrient solution at a 1 : 1 ratio; growth was not a t all impaired or inhibited. Perhydrol solutions apparently do not contain any contaminations inhibitory to Alcaligenes eutrophus. Catalase Content of Cells Microbial growth with the oxygen produced by catalytic cleavage of hydrogen peroxide had to be studied. Preliminary experiments had indicated that the cellular catalase is not sufficient to cleave the hydrogen peroxide added to the cell suspension, and to keep it at a low nontoxic concentration. For comparison the catalase activity of intact cells was of interest, however. Catalase activity was measured manometrically in a Warburg apparatus within a range of 0.74 to 50.8mM H202(Figs. 2a-2c). The rate of oxygen evolution decreased during the course of the reaction. The higher the HzOz concentration and the lower the cell density, the faster was the decrease. The plot of initial velocities versus substrate concentrations indicates that substrate saturation was not reached (Fig. 2c). At low H202concentrations, a linear correlation exists between initial velocity of oxygen evolution and H202 concentration. The specific catalase activity of Alcaligenes eutrophus PHB-4 was
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
417
A
25.4 m~
H202
-concentration (mM)
- b
10
20
30
40 min
10
20
30
4 0 min
Fig. 2. Manometric measurement of catalase activity of intact cells of AL caligenes eutrophus H 16 at different hydrogen peroxide concentrations. Cell suspensions grown on fructose were diluted to an OD (436 nm) of 0.05. Fivetenths ml (a) or 1.0 ml (b) were pipetted in the main compartment. Twotenths ml of diluted hydrogen peroxide solution were tipped in a t zero time. Within the range of concentrations tested (0.79-50.8mM HsOz) the initial velocities of Hz02decomposition were almost proportional to the H202 concentration (c: data from a: A,data from b: 0 ) .
4 18
SCHLEGEL
calculated by interpolation and amounts to 59.5 pmol HzOzper min and mg cell dry wt a t 12.5mM H202and 30°C. The unit of catalase activity is defined as the conversion of 1 pmol HzOz/min a t 25°C and a t 12.5mM HzOzmeasured during the initial 30 sec. The catalase activities of some other microorganisms were determined by the same method; they did not differ very much, they are (in pmol HzOz cleaved/min.mg cell dry wt at 12.5mM HzOz and 30°C): Acinetobacter calcoaceticus, 92; Pseudomonas putida, 50; Paracoccus denitrificans, 15; C . oleophila, 4.1 ; X. cerevisiae, 6.6.
Respiratory Rates The experiments on hydrogen peroxide supply required approximate values of the respiratory rates of the microorganisms to be studied in order to adjust the flow of H 2 0 2to the consumption of oxygen. The rates were measured manometrically with the growth substrates dissolved in phosphate buffer a t the pH of optimal growth; they amounted in Alcaligenes eutrophus to 1.82; Acinetobacter calcoaceticus, 3.32; Pseudomonas putida, 1.18; Paracoccus denitrificans, 1.04; C . oleophila, 2.36; S. cerevisiae, 1.28 pmol O2per hr and ml of suspension with a n oxygen demand (OD) of 1.0. From these data the oxygen or hydrogen peroxide demand rate of the cells was calculated.
Growth with Hydrogen Peroxide Growth experiments in Erlenmeyer flasks were carried out in order to examine whether oxygen liberated by catalytic cleavage of hydrogen peroxide within the cell suspension supports growth. Each flask contained 30 ml of cell suspension with an OD of 1.0. Routinely, 1 pl. catalase/ml and 0.5% fructose were added to the suspension. I n controls under air the cells grew exponentially for 6 hr. When the space above the suspension was filled with OZ-free nitrogen, the cells did not grow. However, if a H202 solution of appropriate concentration was continuously added to the anaerobic suspension, the cells grew a t a rate almost identical to that under air. Usually, 2.5 ml of HzOz solution per hr were pumped into 30 ml of suspension; at the end of a 1 hr interval a n equal volume of suspension was taken for the determination of the optical density. Under these conditions, the growth rate was high (Figs. 3 and 4) and was approximately equal to that under air, provided the rate of the addition of H202was equal to the rate of oxygen consumption by the cells. A lower flow rate as well as a higher flow rate of hydrogen peroxide resulted in minor growth rates.
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
419
Alcaligenes eutrophus 6.0- H16-PHB-4
*
41)-
I
I
I
I
I
I
1
2
3
4
5
6
Time in hours Fig. 3. Growth of cells of Alcaligenes eutrophus H 16-PHB-4 under an oxygenfree nitrogen atmosphere during oxygen supply by continual supply of diluted peroxide solutions. Several vessels each with 30 ml of a cell suspension of an initial OD of 1.0 and an oxygen-free nitrogen atmosphere were shaken a t 30°C. A continuous flow of 35 (a),140 (A), and 700 pmol HzOz (in 2.5 ml solution) was delivered into the suspension per hour; the peroxide was cleaved by catalase (1 pl./ml suspension). Oxygen liberated enabled the cells t o grow at a rate approximately equal to the rate of cells shaken under air (0).
(m)
With the addition of a peroxide solution, the cells even grew in a nonaerated, magnetically stirred submerged culture with an average generation time of 3.2 hr (Fig. 5). The bacterial and yeast species so far studied grew as well under these conditions (Figs. 4 and 5). Growth was followed by turbidimetric measurements. The use of the strain PHB-4, a PHB deficient mutant of Alcaligenes eutrophus, guaranteed that the increase in turbidity was due to growth and not to the accumulation of the storage polymer. Pseudomonas putida does not form storage substances. I n order to avoid misinterpretation protein content was determined in the initial and final samples. The increase in total protein agreed well with the increase of the turbidity of the suspension. Variation of Catalase Concentration
The catalase added to the suspension has the function of cleaving the hydrogen peroxide t o provide oxygen and to keep the concentration of H202minimal. The commercial bovine liver catalase used has a specific activity of 50,000 u/mg protein. The amount of catalase usually added to 30 ml of cell suspension was 30 pl. (= 0.6 mg) and would be able to cleave 1.8 X 106 pmol H202(per hr) a t a 12.5mM H202 concentration. However, only 35 pmol H202 were
420
SCHLEGEL Pseudomonas putida
Fig. 4. Growth of Paracoccus denilrijicans (on fructose), Pseudomonas putida (on fructose), C . oleophila (on glucose), and S . cerm'siae (on glucose) with oxygen supplied by hydrogen peroxide. H2O2 (amount added per hour indicated in pmol) was supplied as described in Figure 3.
added per hour; thus, catalase was present in high excess. The high catalase concentration guaranteed a peroxide concentration in the suspension far below the toxicity threshold. Experiments carried out with 1/10 or 1/100 of the usual concentration of catalase indicated that the catalase concentration chosen on the basis of preliminary experiments was approximately optimal (Fig. 6). Lower concentrations of catalase resulted in decreased growth rates, whereas higher catalase concentrations did not result in increased growth rates. The response to decreased catalase concentrations was not correlated to the catalase activity of the cells. The intracellular catalase does not, apparently, contribute to the cleavage of the peroxide added to the medium.
OXYGEN SUPPLY BY HYDROGEN PEROXIDE L
- Alcaligenes 6.00- H16-PHB-4 m 5.0 4.0E
(D
*m
eutrophus
-
.em
I
-.=
3.0-
5
20-
.-u) U
421
, . '
,d' 500 - 2 000 p o l e s li2O2
1
,
I
,
I
,
2
3
4
5
6
Time in hours
Fig. 5. Growth of Alcaligenes eutrophus PHB-4 in submerged culture with oxygen supplied by hydrogen peroxide. Three-hundred ml cell suspension with an initial OD of 1.11 containing 300 pl. catalase were stirred a t 150 rpm in an Erlenmeyer flask; the air space was flushed with 0%free nitrogen. A 175mM hydrogen peroxide solution was pumped into the suspension and the flow rate Control did not receive H ~ O (0). I was increased from 2.5 to 11.0 ml per hr
(m).
Acinetobacter calcoaceticus
Time in hours Figure 6. Variation of catalase concentration. Suspensions of Acinetobacter calcwceticus and C . oleophila were grown as described in Figure 3, however, with continual addition of 420 pmol HzOzper hour and with varied concentrations of catalase in the suspension (0.001 to 1.0 pl. catalase/ml). Symbols: 0-0 control under air without H10,; 0--0 control under N2without H ~ O Zall; others under Nz in the presence of catalase (numbers indicate pl. catalase/30 ml suepension).
-
422
SCHLEGEL
Since during oxygen supply by HzOz the respiratory carbon dioxide is not removed (swept out) by a stream of inert gas, the influence of high COz tensions on growth had to be checked. Alcaligenes eutrophus PHB-4 and C . oleophila were grown in their respective nutrient media, fructose minimal medium and peptone-YE-NB medium. The gas atmosphere was SO% carbon dioxide and 20% oxygen; 0.42% sodium bicarbonate was added to the medium to keep the same p H as in the control experiment run under air. Minor changes of the p H were observed and corrected. There was a drastic effect caused by COz in the growth rate. The doubling times were significantly higher in the presence of COz: 325 min compared t o 140 min under air for Alcaliyenes eutrophus and 200 min compared to 75 min under air for C . oleophila. These experiments indicate that the respiratory carbon dioxide accumulated in the medium may have drastic effects on the growth as well as other metabolic activities such as accumulation of storage materials and excretion of primary or secondary metabolites.
DISCUSSION The experiments described show that the supply of oxygen t o cell suspensions by hydrogen peroxide is possible. I n the presence of catalase in the medium and at minimal agitation hydrogen peroxide is decomposed fast enough to keep the steady-state concentration of H202 below the threshold of toxicity. At low and medium cell densities the cell suspensions reach the growth rates of the controls aerated conventionally. Furthermore, the cell growth indicates that during catalytic cleavage of hydrogen peroxide in the nutrient medium no toxic byproducts and superoxide radicals, which are formed. The reaction of H202 are produced during aerobic metabolism, and the formation of the most aggressive hydroxyl radicals4 had a t least t o be taken into consideration. During cleavage of HzOz by catalase not even singlet oxygen arises; rather, only triplet oxygen is f ~ r m e d . The ~ growth experiments done with Alcaliyenes eutrophus indicate that commercial hydrogen peroxide neither contains toxic byproducts, which impair growth, nor gives rise to the formation of toxic, reactive species of oxygen during catalytic cleavage. Initially, changing growth responses were observed when oxygen was supplied through hydrogen peroxide. Two reasons have to
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
423
be considered: 1) overdosage of oxygen and 2) the rise of carbon dioxide concentrations. 1). Aeration by oxygen supply through air is a self-regulating system. The steady-state concentration of oxygen in a cell suspension is a function of the oxygen absorption rate and the oxygen consumption rate. If the concentration of dissolved oxygen decreases due to increased cellular respiration, the oxygen absorption rate rises automatically as i t is proportional to the oxygen deficit in the solution. During conventional aeration, the maintenance of medium or low oxygen concentrations in the cell suspension is therefore guaranteed. The oxygen concentration cannot exceed that observed in water saturated with air and cannot reach high values exerting toxic effects. Oxygen supply by hydrogen peroxide is not a self-regulatory system. Continuous measurement of the PO, and controlled addition of HzOz is needed t o keep the oxygen concentration in the suspension approximately a t the optimal growth value. Therefore, H z 0 2 aeration requires that one know the oxygen concentration optimal for growth of the microorganism or tissue cells t o be grown. I n addition, the use of oxygen probes and autotitrators for peroxide supply is needed. 2). Conventional aeration fulfills two functions : oxygen supply and carbon dioxide removal. During HzOzaeration, the respiratory carbon dioxide-as well as possibly other volatile metabolic products-are not swept out,, as only oxygen and no inert gas is added. Carbon dioxide is of great influence on cellular metabolism; as is well known it cannot be dispensed with. However, the effects of high C 0 2 concentrations on cellular metabolism are known only from a few examples, and further studies are needed. Oxygen supply by hydrogen peroxide may have various advantages. Especially that those effects which aim a t the enlargement of phase boundaries and a t the increase of flow velocities can be restricted. Therefore, H202 aeration may prove to be the method of choice when microorganisms or animal or plant tissue cells are cultivated which do not tolerate high shearing forces. I n fermentors, the use of H202, in addition t o conventional aeration, may be a means to reach higher cell densities. I n research experiments, the supply of oxygen by H202 lends itself to all systems which require a homogeneous phase free from air bubbles. The excellent technical assistance of Miss Maria Meyer is gratefully acknowledged. The study followed discussions with Dr. R. Gabellieri at Solvay, Brussels.
424
SCHLEGEL
References 1. H. G. Schlegel, 5th International Fermentation Symposium; Abstracts of Papers, H. Dellweg, Ed. , Verlag Versuchs- und Lehranstalt fiir Spiritusfabrikation und Fermentationstechnologie im Institut fiir Giirungsgewerbe und Biotechnologie, Berlin, 1976. 2. H. G. Schlegel, R. Lafferty, and I. Krauss, Arch. Mikrobiol., 71, 283 (1970). 3. H. G. Schlegel, H. Kaltwasser, and G. Gottschalk, Arch. Mikrobiol., 38, 55 (1961). 4. G. Cohen and R. E. Heikkila, J . BioE. Chem., 249, 2447 (1974). 5. D. J. T. Porter and L. L. Ingraham, Biochim. Bzophys. Acta, 334.97 (1974).
Accepted for Publication October 25, 1976
Aeration without Air: Oxygen Supply by Hydrogen Peroxide H. G . SCHLEGEL,* Institut fiir Mikrobiologie der Gesellschaft fiir Strahlen und Umweltforschung m bH und der Universitat, Gottingen, Federal Republic of Germany
Summary Oxygen has been supplied to suspensions of microorganisms kept under nitrogen by the addition of hydrogen peroxide. If catalaae was present in the suspension and the flow was adjusted to the rate of oxygen consumption, the cells grew at rates identical to the controls incubated under air. The applicability of oxygen supply by hydrogen peroxide and its limits are discuesed.
INTRODUCTION Compared with the solubility of conventional energy and carbon sources, the solubility of oxygen is very low. Therefore oxygen cannot be stored in a nutrient solution. Only oxygen dissolved in water is taken u p by the cells. Suspensions of aerobic microorganisms depend on the continual supply of oxygen to, and its dissolution in, the nutrient solution. Normally, oxygen is supplied by air. The efficiency of aeration is a function of those parameters which are described in Fick’s Law of Diffusion. The phase boundaries can be increased by several means, however, only in limits. The oxygen partial pressure may be increased as well; however, the rate of transfer of oxygen from the gas into the liquid phase depends on the diffusion constant D ; therefore the oxygen transfer rate can be manipulated only within narrow limits. The question has been raised whether an oxygen concentrate can be added to the nutrient solution in liquid form; this would drastically simplify the means of aeration. I n addition, oxygen could be easily supplied t o those cells which do not tolerate the high shearing forces due t o strong aeration and agitation. *Present address: Institute of Microbiology, Grisebachstrasse 8, 3400 Gottingen, Federal Republic of Germany. 413
@ 1977 by John Wiley & Sons, Inc.
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SCHLEGEL
Perhydrol (30% HzOz)is such a n oxygen concentrate; about 100 liter oxygen is produced from the hydrogen peroxide contained in 1 liter perhydrol. One liter perhydrol is equivalent t o 500 liter air which is sufficient to produce about 135 g (dry wt) cells when glucose is the substrate. The experiments to be described later aimed a t examining the conditions under which a suspension of growing microorganisms can be supplied with oxygen through continuous addition of diluted perhydrol solutions. A short communication was presented a t the Fifth International Fermentation Symposium.
MATERIALS AND METHODS Organisms The organisms used were Alcaligenes eutrophus H 16 (ATCC 17699, DSM 428) and the mutant PHB-4 (DSM 541) derived from the wild-type strain and unable to accumulate poly(0-hydroxybutyric acid),2 Acinetobacter calcoaceticus (DSM 586) , Pseudomonas putida (DSM 291,) Paracoccus denitrijicans (DSM 413), Candida oleophila (ATCC 20177, DSM 343) , and Saccharom yces cerevisiae.
Growth Experiments Alcaligenes eutrophus, Pseudomonas putida, and Paracoccus denitrijicans were grown in a mineral nutrient solution3 supplemented with 1% fructose; Acinetobacter calcoaceticus was grown with 1% glucose. Candida oleophila and S. cerevisiae were grown in a medium containing 0.3’% each of Difco nutrient broth, Difco yeast extract, and 1% glucose. After overnight growth, the cells were harvested by centrifugation and were resuspended in a warm nutrient solution t o a certain cell density, transferred to 100 ml Erlenmeyer flasks, and shaken in a thermoconstant bath a t 30°C. At 1 hr intervals samples were taken and diluted for turbidity measurements. The optical density was measured in a Zeiss Filterphotometer (PL 4) a t 436 or 546 nm. The stopcocks had facilities for gasing, for the addition of perhydrol, and for sampling. A Vario-Perpex pump from LKB (Bromma, Sweden) served for the supply of perhydrol. I n most experiments, 2.5 ml diluted perhydrol solution per hour were added to 30 ml cell suspension. As the suspension was continually diluted by the perhydrol solution, the optical density values had to be corrected for dilution and the corrected values were considered for preparing the semilogarithmic plot of the growth curve (Fig. 1.)
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
415
Alcaligenes eutrophus 15.01 H16- PHB-4
-1
Fructose.aerobically
1.5
0 I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
Time in hours Fig. 1. Comparison of growth curves of Alcaligenes eutrophus. Small Erlenmeyer vessels each containing 30 ml suspension of growing cells were shaken in a waterbath at 30°C. From vessel I small samples were taken to measure the OD. Nutrient medium was continually pumped into vessel 11, a t a rate of 5.0 ml/hr. Five ml samples were taken at intervals of 1hr and used for turbidity measurements. Black circles represent the actual readings; open circles represent the turbidity values (calculated) which the suspension would have reached without continual dilution. Dotted line I1 is the fictive growth curve resulting from adding the interval stretches I1 to each other.
Chemicals Bovine liver catalase (20 mg/ml) was purchased from Boehringer, Mannheim; perhydrol (30% hydrogen peroxide) and other chemicals from Merck, Darmstadt.
RESULTS Sensitivity of Cells to Hydrogen Peroxide The sensitivity to hydrogen peroxide was tested in growth experiments with Alcaligenes eutrophus as the model organism. Perhydrol was diluted in a 0.5% fructose nutrient solution at a serial dilution ratio of 1:2; the nutrient solution was inoculated by l o 7 cells per ml. Readings were taken after three to five days. When the initial HzOz concentration was l l m M , the cells did not grow; however, growth was not impaired at 8mM HzO2. From the determination of catalase activities of cell suspensions (vide infra) it became obvious that the hydrogen peroxide in the nutrient medium must have been cleaved a t a high rate as soon as the cells were inoculated. There-
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fore, the cells were exposed to the peroxide only for a few minutes. Cell respiration proved to be even less sensitive. Cellular respiration was measured manometrically; when fructose was used as a substrate significant inhibition occurred at an initial concentration of 22mM H202. Since the cells contained catalase, the concentration of H202 in the suspension continuously decreased. The inhibitory effect estimated was apparently due to irreversible cell damage which had already occurred immediately after addition of the hydrogen peroxide solution to the cell suspension. The manometric experiments only indicated that the contact of the cells with lOOmM H202 solution did not yet cause irreversible damage to the respiratory metabolism. If catalase was added to the cell suspension (usually 1 pl. per 1ml nutrient solution or bacterial suspension) prior to the addition of hydrogen peroxide solution, there was no inhibitory effect on respiration or on growth up to 1M concentration of HzOt. Furthermore, it was examined whether accessory substances which are present in commercial perhydrol solutions, e.g. , hydroquinone, added as a stabilizer, or other contaminating compounds, exert inhibitory effects. From perhydrol solutions containing 30 or 43% hydrogen peroxide the hydrogen peroxide was removed by the addition of catalase. This solution was sterilized and added to the nutrient solution at a 1 : 1 ratio; growth was not a t all impaired or inhibited. Perhydrol solutions apparently do not contain any contaminations inhibitory to Alcaligenes eutrophus. Catalase Content of Cells Microbial growth with the oxygen produced by catalytic cleavage of hydrogen peroxide had to be studied. Preliminary experiments had indicated that the cellular catalase is not sufficient to cleave the hydrogen peroxide added to the cell suspension, and to keep it at a low nontoxic concentration. For comparison the catalase activity of intact cells was of interest, however. Catalase activity was measured manometrically in a Warburg apparatus within a range of 0.74 to 50.8mM H202(Figs. 2a-2c). The rate of oxygen evolution decreased during the course of the reaction. The higher the HzOz concentration and the lower the cell density, the faster was the decrease. The plot of initial velocities versus substrate concentrations indicates that substrate saturation was not reached (Fig. 2c). At low H202concentrations, a linear correlation exists between initial velocity of oxygen evolution and H202 concentration. The specific catalase activity of Alcaligenes eutrophus PHB-4 was
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
417
A
25.4 m~
H202
-concentration (mM)
- b
10
20
30
40 min
10
20
30
4 0 min
Fig. 2. Manometric measurement of catalase activity of intact cells of AL caligenes eutrophus H 16 at different hydrogen peroxide concentrations. Cell suspensions grown on fructose were diluted to an OD (436 nm) of 0.05. Fivetenths ml (a) or 1.0 ml (b) were pipetted in the main compartment. Twotenths ml of diluted hydrogen peroxide solution were tipped in a t zero time. Within the range of concentrations tested (0.79-50.8mM HsOz) the initial velocities of Hz02decomposition were almost proportional to the H202 concentration (c: data from a: A,data from b: 0 ) .
4 18
SCHLEGEL
calculated by interpolation and amounts to 59.5 pmol HzOzper min and mg cell dry wt a t 12.5mM H202and 30°C. The unit of catalase activity is defined as the conversion of 1 pmol HzOz/min a t 25°C and a t 12.5mM HzOzmeasured during the initial 30 sec. The catalase activities of some other microorganisms were determined by the same method; they did not differ very much, they are (in pmol HzOz cleaved/min.mg cell dry wt at 12.5mM HzOz and 30°C): Acinetobacter calcoaceticus, 92; Pseudomonas putida, 50; Paracoccus denitrificans, 15; C . oleophila, 4.1 ; X. cerevisiae, 6.6.
Respiratory Rates The experiments on hydrogen peroxide supply required approximate values of the respiratory rates of the microorganisms to be studied in order to adjust the flow of H 2 0 2to the consumption of oxygen. The rates were measured manometrically with the growth substrates dissolved in phosphate buffer a t the pH of optimal growth; they amounted in Alcaligenes eutrophus to 1.82; Acinetobacter calcoaceticus, 3.32; Pseudomonas putida, 1.18; Paracoccus denitrificans, 1.04; C . oleophila, 2.36; S. cerevisiae, 1.28 pmol O2per hr and ml of suspension with a n oxygen demand (OD) of 1.0. From these data the oxygen or hydrogen peroxide demand rate of the cells was calculated.
Growth with Hydrogen Peroxide Growth experiments in Erlenmeyer flasks were carried out in order to examine whether oxygen liberated by catalytic cleavage of hydrogen peroxide within the cell suspension supports growth. Each flask contained 30 ml of cell suspension with an OD of 1.0. Routinely, 1 pl. catalase/ml and 0.5% fructose were added to the suspension. I n controls under air the cells grew exponentially for 6 hr. When the space above the suspension was filled with OZ-free nitrogen, the cells did not grow. However, if a H202 solution of appropriate concentration was continuously added to the anaerobic suspension, the cells grew a t a rate almost identical to that under air. Usually, 2.5 ml of HzOz solution per hr were pumped into 30 ml of suspension; at the end of a 1 hr interval a n equal volume of suspension was taken for the determination of the optical density. Under these conditions, the growth rate was high (Figs. 3 and 4) and was approximately equal to that under air, provided the rate of the addition of H202was equal to the rate of oxygen consumption by the cells. A lower flow rate as well as a higher flow rate of hydrogen peroxide resulted in minor growth rates.
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
419
Alcaligenes eutrophus 6.0- H16-PHB-4
*
41)-
I
I
I
I
I
I
1
2
3
4
5
6
Time in hours Fig. 3. Growth of cells of Alcaligenes eutrophus H 16-PHB-4 under an oxygenfree nitrogen atmosphere during oxygen supply by continual supply of diluted peroxide solutions. Several vessels each with 30 ml of a cell suspension of an initial OD of 1.0 and an oxygen-free nitrogen atmosphere were shaken a t 30°C. A continuous flow of 35 (a),140 (A), and 700 pmol HzOz (in 2.5 ml solution) was delivered into the suspension per hour; the peroxide was cleaved by catalase (1 pl./ml suspension). Oxygen liberated enabled the cells t o grow at a rate approximately equal to the rate of cells shaken under air (0).
(m)
With the addition of a peroxide solution, the cells even grew in a nonaerated, magnetically stirred submerged culture with an average generation time of 3.2 hr (Fig. 5). The bacterial and yeast species so far studied grew as well under these conditions (Figs. 4 and 5). Growth was followed by turbidimetric measurements. The use of the strain PHB-4, a PHB deficient mutant of Alcaligenes eutrophus, guaranteed that the increase in turbidity was due to growth and not to the accumulation of the storage polymer. Pseudomonas putida does not form storage substances. I n order to avoid misinterpretation protein content was determined in the initial and final samples. The increase in total protein agreed well with the increase of the turbidity of the suspension. Variation of Catalase Concentration
The catalase added to the suspension has the function of cleaving the hydrogen peroxide t o provide oxygen and to keep the concentration of H202minimal. The commercial bovine liver catalase used has a specific activity of 50,000 u/mg protein. The amount of catalase usually added to 30 ml of cell suspension was 30 pl. (= 0.6 mg) and would be able to cleave 1.8 X 106 pmol H202(per hr) a t a 12.5mM H202 concentration. However, only 35 pmol H202 were
420
SCHLEGEL Pseudomonas putida
Fig. 4. Growth of Paracoccus denilrijicans (on fructose), Pseudomonas putida (on fructose), C . oleophila (on glucose), and S . cerm'siae (on glucose) with oxygen supplied by hydrogen peroxide. H2O2 (amount added per hour indicated in pmol) was supplied as described in Figure 3.
added per hour; thus, catalase was present in high excess. The high catalase concentration guaranteed a peroxide concentration in the suspension far below the toxicity threshold. Experiments carried out with 1/10 or 1/100 of the usual concentration of catalase indicated that the catalase concentration chosen on the basis of preliminary experiments was approximately optimal (Fig. 6). Lower concentrations of catalase resulted in decreased growth rates, whereas higher catalase concentrations did not result in increased growth rates. The response to decreased catalase concentrations was not correlated to the catalase activity of the cells. The intracellular catalase does not, apparently, contribute to the cleavage of the peroxide added to the medium.
OXYGEN SUPPLY BY HYDROGEN PEROXIDE L
- Alcaligenes 6.00- H16-PHB-4 m 5.0 4.0E
(D
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eutrophus
-
.em
I
-.=
3.0-
5
20-
.-u) U
421
, . '
,d' 500 - 2 000 p o l e s li2O2
1
,
I
,
I
,
2
3
4
5
6
Time in hours
Fig. 5. Growth of Alcaligenes eutrophus PHB-4 in submerged culture with oxygen supplied by hydrogen peroxide. Three-hundred ml cell suspension with an initial OD of 1.11 containing 300 pl. catalase were stirred a t 150 rpm in an Erlenmeyer flask; the air space was flushed with 0%free nitrogen. A 175mM hydrogen peroxide solution was pumped into the suspension and the flow rate Control did not receive H ~ O (0). I was increased from 2.5 to 11.0 ml per hr
(m).
Acinetobacter calcoaceticus
Time in hours Figure 6. Variation of catalase concentration. Suspensions of Acinetobacter calcwceticus and C . oleophila were grown as described in Figure 3, however, with continual addition of 420 pmol HzOzper hour and with varied concentrations of catalase in the suspension (0.001 to 1.0 pl. catalase/ml). Symbols: 0-0 control under air without H10,; 0--0 control under N2without H ~ O Zall; others under Nz in the presence of catalase (numbers indicate pl. catalase/30 ml suepension).
-
422
SCHLEGEL
Since during oxygen supply by HzOz the respiratory carbon dioxide is not removed (swept out) by a stream of inert gas, the influence of high COz tensions on growth had to be checked. Alcaligenes eutrophus PHB-4 and C . oleophila were grown in their respective nutrient media, fructose minimal medium and peptone-YE-NB medium. The gas atmosphere was SO% carbon dioxide and 20% oxygen; 0.42% sodium bicarbonate was added to the medium to keep the same p H as in the control experiment run under air. Minor changes of the p H were observed and corrected. There was a drastic effect caused by COz in the growth rate. The doubling times were significantly higher in the presence of COz: 325 min compared t o 140 min under air for Alcaliyenes eutrophus and 200 min compared to 75 min under air for C . oleophila. These experiments indicate that the respiratory carbon dioxide accumulated in the medium may have drastic effects on the growth as well as other metabolic activities such as accumulation of storage materials and excretion of primary or secondary metabolites.
DISCUSSION The experiments described show that the supply of oxygen t o cell suspensions by hydrogen peroxide is possible. I n the presence of catalase in the medium and at minimal agitation hydrogen peroxide is decomposed fast enough to keep the steady-state concentration of H202 below the threshold of toxicity. At low and medium cell densities the cell suspensions reach the growth rates of the controls aerated conventionally. Furthermore, the cell growth indicates that during catalytic cleavage of hydrogen peroxide in the nutrient medium no toxic byproducts and superoxide radicals, which are formed. The reaction of H202 are produced during aerobic metabolism, and the formation of the most aggressive hydroxyl radicals4 had a t least t o be taken into consideration. During cleavage of HzOz by catalase not even singlet oxygen arises; rather, only triplet oxygen is f ~ r m e d . The ~ growth experiments done with Alcaliyenes eutrophus indicate that commercial hydrogen peroxide neither contains toxic byproducts, which impair growth, nor gives rise to the formation of toxic, reactive species of oxygen during catalytic cleavage. Initially, changing growth responses were observed when oxygen was supplied through hydrogen peroxide. Two reasons have to
OXYGEN SUPPLY BY HYDROGEN PEROXIDE
423
be considered: 1) overdosage of oxygen and 2) the rise of carbon dioxide concentrations. 1). Aeration by oxygen supply through air is a self-regulating system. The steady-state concentration of oxygen in a cell suspension is a function of the oxygen absorption rate and the oxygen consumption rate. If the concentration of dissolved oxygen decreases due to increased cellular respiration, the oxygen absorption rate rises automatically as i t is proportional to the oxygen deficit in the solution. During conventional aeration, the maintenance of medium or low oxygen concentrations in the cell suspension is therefore guaranteed. The oxygen concentration cannot exceed that observed in water saturated with air and cannot reach high values exerting toxic effects. Oxygen supply by hydrogen peroxide is not a self-regulatory system. Continuous measurement of the PO, and controlled addition of HzOz is needed t o keep the oxygen concentration in the suspension approximately a t the optimal growth value. Therefore, H z 0 2 aeration requires that one know the oxygen concentration optimal for growth of the microorganism or tissue cells t o be grown. I n addition, the use of oxygen probes and autotitrators for peroxide supply is needed. 2). Conventional aeration fulfills two functions : oxygen supply and carbon dioxide removal. During HzOzaeration, the respiratory carbon dioxide-as well as possibly other volatile metabolic products-are not swept out,, as only oxygen and no inert gas is added. Carbon dioxide is of great influence on cellular metabolism; as is well known it cannot be dispensed with. However, the effects of high C 0 2 concentrations on cellular metabolism are known only from a few examples, and further studies are needed. Oxygen supply by hydrogen peroxide may have various advantages. Especially that those effects which aim a t the enlargement of phase boundaries and a t the increase of flow velocities can be restricted. Therefore, H202 aeration may prove to be the method of choice when microorganisms or animal or plant tissue cells are cultivated which do not tolerate high shearing forces. I n fermentors, the use of H202, in addition t o conventional aeration, may be a means to reach higher cell densities. I n research experiments, the supply of oxygen by H202 lends itself to all systems which require a homogeneous phase free from air bubbles. The excellent technical assistance of Miss Maria Meyer is gratefully acknowledged. The study followed discussions with Dr. R. Gabellieri at Solvay, Brussels.
424
SCHLEGEL
References 1. H. G. Schlegel, 5th International Fermentation Symposium; Abstracts of Papers, H. Dellweg, Ed. , Verlag Versuchs- und Lehranstalt fiir Spiritusfabrikation und Fermentationstechnologie im Institut fiir Giirungsgewerbe und Biotechnologie, Berlin, 1976. 2. H. G. Schlegel, R. Lafferty, and I. Krauss, Arch. Mikrobiol., 71, 283 (1970). 3. H. G. Schlegel, H. Kaltwasser, and G. Gottschalk, Arch. Mikrobiol., 38, 55 (1961). 4. G. Cohen and R. E. Heikkila, J . BioE. Chem., 249, 2447 (1974). 5. D. J. T. Porter and L. L. Ingraham, Biochim. Bzophys. Acta, 334.97 (1974).
Accepted for Publication October 25, 1976
