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Effects of oxygen and carbon dioxide pressures on bacterial cellulose production by Acetobacter in aerated and agitated culture
JOURNALOF FERMENTATION AND BIOENOINEERING Vol. 84, No. 2, 124-127. 1997
Effects of Oxygen and Carbon Dioxide Pressures on Bacterial Cellulose Production by Acetobacter in Aerated and Agitated Culture TOHRU KOUDA,* TAKAAK I NARITOMI, HISATO YANO, AND FUMIHIRO YOSHINAGA Bio-Polymer Research Co. Ltd., KSP R & D Business-Park Bldg. B-1015, 3-2-l Sakato, Takatsu-ku, Kawasaki 213, Japan Received 5 March 1997IAccepted 27 May 1997
With the alm of improving bacterial cellulose (BC) production in an aerated and agitated culture, the effects of the oxygen and carbon dioxlde pressures were investigated. The BC production rate was dependent on the oxygen transfer rate, which declined as the broth viscosity increased, accompanied by BC accumulation. Increasing the partial pressure of oxygen by sparging with oxygen-enriched air and/or raising the operating pressure improved the oxygen supply, while the agitation power required was lowered. Although the BC production rate was not affected by higher oxygen pressure, it was reduced as the operating pressure was raised. The reduction in the production rate was considered to be due to the high carbon dioxide pressure, because carbon dioxide-enriched air also reduced the BC production rate, while the reduction was canceled out by increasing the alr flow rate. [Key words: bacterial cellulose, aerated and agitated culture, oxygen, carbon dioxide, pressure, volumetric agitation power] Bacterial cellulose (BC) produced by Acetobacter is a material being developed for industrial use. It is of interest because the physical properties of BC differ from those of plant cellulose (1, 2). Although BC has
the gaseous phase in the fermentor) while the partial pressure of carbon dioxide in the gaseous phase (pCOZ) increases. High levels of operating pressure (8, 9), oxygen pressure, and carbon dioxide pressure (10, 11) have been reported to inhibit microbial growth and production of other materials such as amino acids, but there are no reports on the inhibitory effects of these pressures on BC production. Therefore, in this study, we examined the effects of the operating pressure, ~4, and pCOz on the OTR and BC production by Acetobacter in an aerated and agitated culture.
new
been produced by static culture, which yields cellulosic pellicles such as nata-de-coca, agitated culture is more suitable for commercial-scale production because higher production rates can be achieved. With the aim of improving BC production in agitated culture, the screening of suitable strains (3), improvement of the producing organism (4) and medium components (5) have been investigated. It is also important to determine an effective procedure for the supply of oxygen in order to improve BC production in agitated culture. One reason why this is necessary is that broth containing BC is difficult to mix, due to its high, non-Newtonian viscosity (6). A second reason is that the production rate and yield of BC are proportional to the oxygen transfer rate (OTR) and oxygen transfer coefficient (KLa) measured by the static gassing out method. Experiments on mixing, oxygen transfer, and cultivation using several different agitators revealed that two type of impellers-the Maxblend@ (Sumitomo Heavy Industries Ltd., Tokyo) and a gate with turbine-are suitable for BC production (7). A high KLa requires a high agitation speed, and the agitation torque increases with the BC accumulation. Thus, high agitation power is required for a high production rate, because agitation power is proportional to both the agitation speed and agitation torque. However, high agitation power should be avoided when scaling up the agitation culture process, not only because it requires a large motor and thereby increases the energy cost, but also because it causes exothermic reaction by shearing. On the other hand, the OTR should be improved by increasing the partial pressure of oxygen in the gaseous phase (PO,). The OTR is enhanced by increasing the PO,, which can be achieved by using oxygen-enriched air and/or
raising
the operating
pressure
(the pressure
MATERIALS
AND METHODS
A jar fermentor with a nomi50 I that was suitable for operating at 1.O2.5 atm was used. The diameter and working volume were 320 mm and 30 I, respectively. A Maxblend@ impeller as shown in Fig. 1 was used; its shape was modified to give a large KLa in a viscous BC suspension. The fermentor was also fitted with 3 baffles, a ring sparger, a thermocouple, a pH electrode, and a dissolved oxygen electrode (Polar0 type; Ingold, USA). The operating pressure was controlled by an automatic control valve in the exhaust, the gas flow rate was controlled by a control valve with a flow meter at the gas inlet, and the operating pressure was determined at 1 atm when the fermentor was not pressurized. The impeller was driven by a variable-speed motor, the agitation speed was measured by a tachometer-generator indicating system, and torque data were calculated from the inverter output. The volumetric agitation power (Pv, kw . m-9 was calculated from the agitation speed, the net torque, and the volume of culture broth (7). Medium and cultivation conditions A mutant strain, Acetobacter xylinum subsp. sucrofermentans BPR3OOlA, which showed the highest BC production rate among the strains screened (Takemura, H. et al., Abstr. Annu. Meet. Japan Sot. Biosci., Biotechnol., and Agrochem., Japan, p. 288, 1996), was used. The Cultivation
nal volume
of
* Corresponding author. 124
of
apparatus
EFFECT OF PRESSURES ON BC PRODUCTION
VOL. 84, 1997 f---
320 mm B
concentration at the cylinder outlet was about 100% (v/v). The p02 of the oxygen-enriched air and the pCOz of the carbon dioxide-enriched air were calculated as follows: pOz=p(21
Va/,fcO v,)/(V~+v,)/lOO
pCOz=p(O.O35
FIG. 1. impeller.
Diagram of the fermentor
125
fitted with a Maxblend@
effects of the operating pressure, ~4, and pCOz on BC production and agitation power were examined as follows. A l-ml sample of a frozen stock culture was inoculated in a Roux flask into 100 ml CSL-Fru medium containing the same constituents as reported previously (7) except that the amount of fructose and CSL were changed to 40 g . I- * and 20 ml. i- 1, respectively. After cultivation for 3 d at 28”C, cells attached to the pellicle that formed at the surface of the medium were removed by manual shaking to produce a cell suspension. A 12.5-ml aliquot of this cell suspension was inoculated into 112.5 ml CSLFru medium (except that again it contained 40 g .1-l fructose and 20 ml .I-’ CSL) in a baffled conical flask and cultivated for 3 d at 28°C on a rotary shaker rotating 180 rpm. Then, 1 I of the culture was blended to disperse the aggregated BC and inoculated into 291 CSL-Fru medium (7) in a 50-I jar fermentor and cultivated for 1 d. Finally, 3 1 of the culture in the late exponential phase was inoculated into 27 I CSL-Fru medium in a 50-l jar fermentor, and cultivated until all the fructose had been consumed. The cultivation conditions in each jar fermentor were maintained at pH 5.0 by adding HzS04 and ammonia gas, and 30°C by circulating temperaturecontrolled water. The dissolved oxygen concentration (CL) was kept at 10% of the saturated dissolved oxygen concentration (C*) by regulating the agitation speed in order to attain the maximum production rate (Toyosaki, H. et al., Abstr. Annu. Meet. Japan Sot. Biosci., Biotechnol., and Agrochem., Japan, p. 334, 1997). Preparation of oxygen- and carbon dioxide-enriched air Oxygen-enriched air was prepared by mixing air and oxygen supplied by pressure swing adsorption (PSA) and sparged into the fermentor. The oxygen concentration at the PSA outlet was over 95%(v/v). Carbon dioxide-enriched air was prepared by mixing air and carbon dioxide supplied from a cylinder. The carbon dioxide
v,+ 100 v,)/( V~+vv,)/lOO
(1) (2)
where p is the operating pressure (atm), V, the air flow rate (I.min-l), CO the concentration of oxygen (% v/v) supplied by PSA, v, the oxygen flow rate (I.min-I), and v, the carbon dioxide flow rate (1.min.-l). The oxygen and carbon dioxide concentrations of air were considered to be 21 and 0.035% (v/v), respectively. The BC concentration of the culture Analysis broth was calculated from the dry weight of purified BC. Fructose was determined by HPLC (7). The oxygen consumption rate (OCR) was regarded to be the same as the OTR, which was calculated from the flow rate, pressure, temperature, and the oxygen and carbon dioxide concentrations of the inlet and outlet gases (7). These concentrations were determined by an oxygen and carbon dioxide analyzer (Able Co., Tokyo). The viable cell concentration was measured by the modified colony counting method and expressed as the number of colony forming units per ml (cfu). A 0.5-ml sample of the culture broth was diluted lo-fold with 100 mM potassium acetate buffer (pH 5.0), 1.0 ml cellulase (10% w/v celluclast, Novo Nordisk A/S, Denmark) was added, and the mixture was incubated at 30°C for 2 h with shaking to dissolve the BC. This reacted cell suspension was diluted with 100mM potassium acetate buffer, spread on CSL-Fru agar medium, and cultivated at 28°C for 5 d. The conventional colony counting method had some uncertainty due to the adsorption of cells by BC (12). However, good reproducibility was obtained by dissolving BC with celluiase. The change in the number of cells during the cellulase reaction was considered negligible, as the viable cell concentrations after 2and 4-h reaction did not differ. RESULTS Time course of BC production in the culture Figure 2 shows the changes in the parameters with time during cultivation at an operating pressure of 1.5 atm and an air flow rate of 0.5 vvm. During cultivation for 42 h, P, rose to 50 times its initial value, while the viable cell and BC concentrations increased 20-fold and 40fold, respectively. Effects of p02 on BC production and P, required for The agitation speed decreased when oxygen wwl~ oxygen-enriched air was sparged into the culture broth. The maximum agitation speed was 360 rpm when air was sparged at 1.5 atm, but 240 rpm when oxygen-enriched air containing 5O%(v/v) oxygen was sparged at the same pressure. Figure 3 shows the BC production rate and P, changes plotted against the pOz of the inlet gas increased by sparging with oxygen-enriched air and/or raising the operating pressure. Each P, value shown is that of the maximum during the cultivation. As the ~02 increased, the P, required to maintain CL at 10% of C” decreased in both cases. However, although the BC production rate remained constant during sparging with oxygenenriched air, it declined as the operating pressure was raised.
126
J. FERMENT.BIOENG.,
KOUDA ET AL.
0
20
10
30
Cultivation
40
0
50
The pCOz is Effect of pCOz on the BC production proportional to the operating pressure, and inversely proportional to the gas flow rate, because the carbon dioxide concentration decreases as a result of the exhaustion of carbon dioxide produced by microbial metabolism. Therefore, the pC02 becomes high when the operating pressure is high and the air flow rate is low if the carbon dioxide production rate remains constant. Figure 4 shows the relationship between the BC production rate and the pCOz at air flow rates of 0.25 to 1.O wm and operating pressures of 1 to 2.5 atm, with all the other conditions kept constant. The BC production rate was high when the pCOz was low and vice versa. Table 1 shows the effect of the air flow rate on pOz, pCOa and the BC production rate. The p0~ and pC0~ values shown are the maximum values measured at 30min intervals and the BC production rate is the average increase in the BC concentration per hour during the cultivation. As the air flow rate increased, the pCOz 7
0.5
20
0.45
0.4
1 0.2
I
I
I
I
I
0.3
0.4
0.5
0.6
0.7
Partial pressure of oxygen
0.15
0.1
Partial pressure of carbon dioxide (atm)
time (h)
FIG. 2. Changes in volumetric agitation power, BC concentration, and viable cell concentration at an air flow rate of 0.5 wm and an operating pressure of 1.5 atm. Symbols: 0, volumetric agitation power (PJ; •I , BC concentration; A, viable cell concentration.
0.35
0.05
15 0.8
5
(atm)
FIG. 3. BC production rate and volumetric agitation power changes in response to altering the partial pressure of oxygen (PO,) by adding oxygen-enriched air and/or raising the operating pressure of the fermentor. Symbols: ( 0, m) BC production rate; (0, +) volumetric agitation power (pv). Open symbols, sparging with oxygenenriched air; filled symbols, raising the operating air pressure.
BC production rate changes in response to altering the partial pressure of carbon dioxide (PO,) in the outlet gas. FIG.
4.
decreased and the BC production rate was improved, but p02 and P,(14- 15 kW . mS3) were not affected. In order to clarify the relationship between the BC production rate and pCO%, carbon dioxide-enriched air was sparged during the cultivation at an operating pressure of 1.5 atm. Table 2 shows the BC production rates, fructose consumption rates, and OCR at different levels of pCOz in sparged carbon dioxide-enriched air. All three rates were low when the pCOz of the sparged carbon dioxide-enriched air was high. DISCUSSION The results of the above experiments demonstrated clearly that (i) increasing the pOa reduced the P, required to maintain C,_ at a constant level during the cultivation, (ii) a high pCOz caused a reduction in BC production, and (iii) increasing the air flow rate canceled the inhibitory effect of pCOz. mass transfer in a viscous broth is In general, problematic, as it is in a xanthan gum system. The productivity of xanthan gum has been reported to depend on the shear rate, the mass transfer around the cells being enhanced as the shear rate increased (13). Thus, even though the BC production rate has been shown to depend on the lYLa (7), and this depends on the agitation speed, another possible factor affecting the BC production rate could be the shear rate of the agitation. However, our results showed that the BC production rate was not reduced as the agitation speed decreased when oxygen-enriched air was sparged (Fig. 3), indicating that the BC production rate does not depend on the shear rate. This implies that the BC production rate in an aerated and agitated culture is not limited TABLE
1. Effect of air flow rate on the partial pressure of carbon dioxide and BC production rate under a constant partial pressure of oxygen
Air flow rate (vvm) Partial pressure of oxygen @Oz) (atm) Partial pressure of carbon dioxide WOZ) (atm) BC production rate* (g .I-‘. h-l)
0.25 0.315 0.10
0.5 0.315 0.05
1.0 0.315 0.03
0.41
0.45
0.47
a Average increase in the BC concentration cultivation.
per hour during the
VOL. 84, 1997 TABLE 2.
EFFECT OF PRESSURES ON BC PRODUCTION
Effect of partial pressure of carbon dioxide in sparged air on the bacterial cellulose production process
Partial pressure of carbon dioxide olCO2) (atm) BC production rate= (g. 1-r. h-r) Fructose consumption rateb (g-1-r. h-r) Oxygen consumption rateC (mmol./-r.h-r)
o.ocO5 0.7 2.9 47
0.075 0.5 2.5 43
0.12 0.3 2.0 37
a Maximum value of the increase in the BC concentration per hour measured at 6-hourly intervals. b Maximum value of the decrease in the fructose concentration per hour measured at 6-hourly intervals. c Equal to the maximum OTR, measured at 30-min intervals, with CL at a constant level during the cultivation.
by mass transfer around the cells. BC is considered to have little effect on the mass transfer around cells because it is not soluble in water, whereas xanthan gum is soluble. Therefore, it was concluded that the BC production rate increased with a high KLa, not because the mass transfer around cells was enhanced but because of oxygen transfer from the gaseous phase to the broth. In order to improve the OTR, we increased the p0~ by sparging oxygen-enriched air and/or raising the operating pressure. Although raising the operating pressure is practical and economical in a commercial plant, the results showed that the BC production rate decreased as the operating pressure was raised (Fig. 3). This decrease in the production rate is considered to be caused by the high pCOz (Fig. 4). On the other hand, the inhibitory effect of pCOz was canceled out by the ventilation effect of increasing the air flow rate (Table 1): pC0~ at less than 0.05 atm did not inhibit BC production, the change in the production rate being negligible when the air flow rate increased from 0.5 to 1 .O vvm. Generally, increasing the air flow rate is an effective means of supplying oxygen to a culture broth. However, this method was not effective in the BC culture broth because the P, needed to maintain CL at a constant level was not reduced. This is mainly because the excess sparged air is not dispersed as small bubbles in the viscous broth containing BC, but passes through it to the exhaust. The mechanism responsible for the inhibitory effect of pCOz appears to be complex, because, as shown in Table 2, a high pC0~ inhibited not only the BC production but also the fructose and oxygen consumption. We intend to carry out further studies to determine why a high pCOz reduces the BC production rate.
127
ACKNOWLEDGMENTS We thank Prof. Makoto Shoda, Research Laboratory of Resources Utilization, Tokyo Institute of Technology, for his critical comments on the manuscript. We also thank Mr. K. Kume, Mr. Y. Yamashita and Ms. M. Kubota of our laboratory for their technical assistance. REFERENCES 1. Ross, P., Mayer, R., and Benziman, M.: Cellulose biosynthesis and function in bacteria. Microbial. Rev., 55, 35-58 (1991). 2. Yoshinaga, F., Tononehi, N., and Watanabe, K.: Research progress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material. Biosci. Biotech. Biochem.. 59. 219-224 (1997). 3. Toyosaki, H., Naritomi, T.,’ Seto, A., ‘l&t&da, T., and Yoshinaga, F.: Screening of bacterial cellulose-producing Acetobacter strains suitable for agitation culture. Biosci. Biotech. Biochem., 59, 1498-1502 (1995). 4. tshikawa, A., Matsuoka, M., Tsuchida, T., and Yoshinaga, F.: Increase in cellulose production by sulfaguanidine-resistant mutants derived from Acetobacter xylinum subsp. sucrofermentans. Biosci. Biotech. Biochem., 59, 2259-2262 (1995). 5. Matsuoka, M., Tsucbida, T., Matsushita, K., Adachi, O., and Yoshinaga, F.: A synthetic medium for bacterial cellulose production by Acetobacter xylinum subsp. sucrofermentans. Biosci. Biotech. Biochem., 60, 575-579 (1996). 6. Kouda, T., Yano, H., Yoshinaga, F., Kaminoyama, M., and Kamiwano, M.: Characterization of non-Newtonian behavior in the mixing of bacterial cellulose in a bioreactor. J. Ferment. Bioeng., 82, 382-386 (1996). 7. Kouda, T., Yano, H., and Yoshinaga, F.: Effect of agitator configuration on bacterial cellulose productivity in aerated and agitated culture. J. Ferment. Bioeng., 83, 371-376 (1997). 8 Yabannavar, V., Singh, V., and Schaefer, E.: Effect of pressure on aminoglycoside fermentation mediated by dissolved oxygen. J. Ferment. Bioeng., 73, 66-69 (1992). 9. Dufresce, D., TubanIt, J., Leduy, A., and Lencki, R.: The effect of pressure on the growth of Aureobasidium pullulans and the synthesis of pullulan. Appl. Microbial. Biotechnol., 32, 526-532 (1990). 10. Gill, C. 0. and Tan, K. H.: Effect of carbon dioxide on growth of Pseudomonas Juorescens. Appl. Environ. Microbial., 38, 237-240 (1979). 11. Almshi, K., Shibai, H., and Hirose, Y.: Inhibitory effects of carbon dioxide and oxygen in amino acid fermentation. J. Ferment. Technol.. 57. 317-320 (1979). 12. Marx-Figini, M. and’ Pion, B.‘G.: Kinetics investigation on biosynthesis of cellulose by Acetobacter xylinum. Biochim. Biophys. Acta, 338, 382-393 (1974). 13. Funahashi, H., Maehara, M., Taguchi, H., and Yosbida, T.: Effects of agitation by flat-bladed turbine impeller on microbial production of xanthan gum. J. Chem. Eng. Japan, 20, 16-22 (1987).
Effects of Oxygen and Carbon Dioxide Pressures on Bacterial Cellulose Production by Acetobacter in Aerated and Agitated Culture TOHRU KOUDA,* TAKAAK I NARITOMI, HISATO YANO, AND FUMIHIRO YOSHINAGA Bio-Polymer Research Co. Ltd., KSP R & D Business-Park Bldg. B-1015, 3-2-l Sakato, Takatsu-ku, Kawasaki 213, Japan Received 5 March 1997IAccepted 27 May 1997
With the alm of improving bacterial cellulose (BC) production in an aerated and agitated culture, the effects of the oxygen and carbon dioxlde pressures were investigated. The BC production rate was dependent on the oxygen transfer rate, which declined as the broth viscosity increased, accompanied by BC accumulation. Increasing the partial pressure of oxygen by sparging with oxygen-enriched air and/or raising the operating pressure improved the oxygen supply, while the agitation power required was lowered. Although the BC production rate was not affected by higher oxygen pressure, it was reduced as the operating pressure was raised. The reduction in the production rate was considered to be due to the high carbon dioxide pressure, because carbon dioxide-enriched air also reduced the BC production rate, while the reduction was canceled out by increasing the alr flow rate. [Key words: bacterial cellulose, aerated and agitated culture, oxygen, carbon dioxide, pressure, volumetric agitation power] Bacterial cellulose (BC) produced by Acetobacter is a material being developed for industrial use. It is of interest because the physical properties of BC differ from those of plant cellulose (1, 2). Although BC has
the gaseous phase in the fermentor) while the partial pressure of carbon dioxide in the gaseous phase (pCOZ) increases. High levels of operating pressure (8, 9), oxygen pressure, and carbon dioxide pressure (10, 11) have been reported to inhibit microbial growth and production of other materials such as amino acids, but there are no reports on the inhibitory effects of these pressures on BC production. Therefore, in this study, we examined the effects of the operating pressure, ~4, and pCOz on the OTR and BC production by Acetobacter in an aerated and agitated culture.
new
been produced by static culture, which yields cellulosic pellicles such as nata-de-coca, agitated culture is more suitable for commercial-scale production because higher production rates can be achieved. With the aim of improving BC production in agitated culture, the screening of suitable strains (3), improvement of the producing organism (4) and medium components (5) have been investigated. It is also important to determine an effective procedure for the supply of oxygen in order to improve BC production in agitated culture. One reason why this is necessary is that broth containing BC is difficult to mix, due to its high, non-Newtonian viscosity (6). A second reason is that the production rate and yield of BC are proportional to the oxygen transfer rate (OTR) and oxygen transfer coefficient (KLa) measured by the static gassing out method. Experiments on mixing, oxygen transfer, and cultivation using several different agitators revealed that two type of impellers-the Maxblend@ (Sumitomo Heavy Industries Ltd., Tokyo) and a gate with turbine-are suitable for BC production (7). A high KLa requires a high agitation speed, and the agitation torque increases with the BC accumulation. Thus, high agitation power is required for a high production rate, because agitation power is proportional to both the agitation speed and agitation torque. However, high agitation power should be avoided when scaling up the agitation culture process, not only because it requires a large motor and thereby increases the energy cost, but also because it causes exothermic reaction by shearing. On the other hand, the OTR should be improved by increasing the partial pressure of oxygen in the gaseous phase (PO,). The OTR is enhanced by increasing the PO,, which can be achieved by using oxygen-enriched air and/or
raising
the operating
pressure
(the pressure
MATERIALS
AND METHODS
A jar fermentor with a nomi50 I that was suitable for operating at 1.O2.5 atm was used. The diameter and working volume were 320 mm and 30 I, respectively. A Maxblend@ impeller as shown in Fig. 1 was used; its shape was modified to give a large KLa in a viscous BC suspension. The fermentor was also fitted with 3 baffles, a ring sparger, a thermocouple, a pH electrode, and a dissolved oxygen electrode (Polar0 type; Ingold, USA). The operating pressure was controlled by an automatic control valve in the exhaust, the gas flow rate was controlled by a control valve with a flow meter at the gas inlet, and the operating pressure was determined at 1 atm when the fermentor was not pressurized. The impeller was driven by a variable-speed motor, the agitation speed was measured by a tachometer-generator indicating system, and torque data were calculated from the inverter output. The volumetric agitation power (Pv, kw . m-9 was calculated from the agitation speed, the net torque, and the volume of culture broth (7). Medium and cultivation conditions A mutant strain, Acetobacter xylinum subsp. sucrofermentans BPR3OOlA, which showed the highest BC production rate among the strains screened (Takemura, H. et al., Abstr. Annu. Meet. Japan Sot. Biosci., Biotechnol., and Agrochem., Japan, p. 288, 1996), was used. The Cultivation
nal volume
of
* Corresponding author. 124
of
apparatus
EFFECT OF PRESSURES ON BC PRODUCTION
VOL. 84, 1997 f---
320 mm B
concentration at the cylinder outlet was about 100% (v/v). The p02 of the oxygen-enriched air and the pCOz of the carbon dioxide-enriched air were calculated as follows: pOz=p(21
Va/,fcO v,)/(V~+v,)/lOO
pCOz=p(O.O35
FIG. 1. impeller.
Diagram of the fermentor
125
fitted with a Maxblend@
effects of the operating pressure, ~4, and pCOz on BC production and agitation power were examined as follows. A l-ml sample of a frozen stock culture was inoculated in a Roux flask into 100 ml CSL-Fru medium containing the same constituents as reported previously (7) except that the amount of fructose and CSL were changed to 40 g . I- * and 20 ml. i- 1, respectively. After cultivation for 3 d at 28”C, cells attached to the pellicle that formed at the surface of the medium were removed by manual shaking to produce a cell suspension. A 12.5-ml aliquot of this cell suspension was inoculated into 112.5 ml CSLFru medium (except that again it contained 40 g .1-l fructose and 20 ml .I-’ CSL) in a baffled conical flask and cultivated for 3 d at 28°C on a rotary shaker rotating 180 rpm. Then, 1 I of the culture was blended to disperse the aggregated BC and inoculated into 291 CSL-Fru medium (7) in a 50-I jar fermentor and cultivated for 1 d. Finally, 3 1 of the culture in the late exponential phase was inoculated into 27 I CSL-Fru medium in a 50-l jar fermentor, and cultivated until all the fructose had been consumed. The cultivation conditions in each jar fermentor were maintained at pH 5.0 by adding HzS04 and ammonia gas, and 30°C by circulating temperaturecontrolled water. The dissolved oxygen concentration (CL) was kept at 10% of the saturated dissolved oxygen concentration (C*) by regulating the agitation speed in order to attain the maximum production rate (Toyosaki, H. et al., Abstr. Annu. Meet. Japan Sot. Biosci., Biotechnol., and Agrochem., Japan, p. 334, 1997). Preparation of oxygen- and carbon dioxide-enriched air Oxygen-enriched air was prepared by mixing air and oxygen supplied by pressure swing adsorption (PSA) and sparged into the fermentor. The oxygen concentration at the PSA outlet was over 95%(v/v). Carbon dioxide-enriched air was prepared by mixing air and carbon dioxide supplied from a cylinder. The carbon dioxide
v,+ 100 v,)/( V~+vv,)/lOO
(1) (2)
where p is the operating pressure (atm), V, the air flow rate (I.min-l), CO the concentration of oxygen (% v/v) supplied by PSA, v, the oxygen flow rate (I.min-I), and v, the carbon dioxide flow rate (1.min.-l). The oxygen and carbon dioxide concentrations of air were considered to be 21 and 0.035% (v/v), respectively. The BC concentration of the culture Analysis broth was calculated from the dry weight of purified BC. Fructose was determined by HPLC (7). The oxygen consumption rate (OCR) was regarded to be the same as the OTR, which was calculated from the flow rate, pressure, temperature, and the oxygen and carbon dioxide concentrations of the inlet and outlet gases (7). These concentrations were determined by an oxygen and carbon dioxide analyzer (Able Co., Tokyo). The viable cell concentration was measured by the modified colony counting method and expressed as the number of colony forming units per ml (cfu). A 0.5-ml sample of the culture broth was diluted lo-fold with 100 mM potassium acetate buffer (pH 5.0), 1.0 ml cellulase (10% w/v celluclast, Novo Nordisk A/S, Denmark) was added, and the mixture was incubated at 30°C for 2 h with shaking to dissolve the BC. This reacted cell suspension was diluted with 100mM potassium acetate buffer, spread on CSL-Fru agar medium, and cultivated at 28°C for 5 d. The conventional colony counting method had some uncertainty due to the adsorption of cells by BC (12). However, good reproducibility was obtained by dissolving BC with celluiase. The change in the number of cells during the cellulase reaction was considered negligible, as the viable cell concentrations after 2and 4-h reaction did not differ. RESULTS Time course of BC production in the culture Figure 2 shows the changes in the parameters with time during cultivation at an operating pressure of 1.5 atm and an air flow rate of 0.5 vvm. During cultivation for 42 h, P, rose to 50 times its initial value, while the viable cell and BC concentrations increased 20-fold and 40fold, respectively. Effects of p02 on BC production and P, required for The agitation speed decreased when oxygen wwl~ oxygen-enriched air was sparged into the culture broth. The maximum agitation speed was 360 rpm when air was sparged at 1.5 atm, but 240 rpm when oxygen-enriched air containing 5O%(v/v) oxygen was sparged at the same pressure. Figure 3 shows the BC production rate and P, changes plotted against the pOz of the inlet gas increased by sparging with oxygen-enriched air and/or raising the operating pressure. Each P, value shown is that of the maximum during the cultivation. As the ~02 increased, the P, required to maintain CL at 10% of C” decreased in both cases. However, although the BC production rate remained constant during sparging with oxygenenriched air, it declined as the operating pressure was raised.
126
J. FERMENT.BIOENG.,
KOUDA ET AL.
0
20
10
30
Cultivation
40
0
50
The pCOz is Effect of pCOz on the BC production proportional to the operating pressure, and inversely proportional to the gas flow rate, because the carbon dioxide concentration decreases as a result of the exhaustion of carbon dioxide produced by microbial metabolism. Therefore, the pC02 becomes high when the operating pressure is high and the air flow rate is low if the carbon dioxide production rate remains constant. Figure 4 shows the relationship between the BC production rate and the pCOz at air flow rates of 0.25 to 1.O wm and operating pressures of 1 to 2.5 atm, with all the other conditions kept constant. The BC production rate was high when the pCOz was low and vice versa. Table 1 shows the effect of the air flow rate on pOz, pCOa and the BC production rate. The p0~ and pC0~ values shown are the maximum values measured at 30min intervals and the BC production rate is the average increase in the BC concentration per hour during the cultivation. As the air flow rate increased, the pCOz 7
0.5
20
0.45
0.4
1 0.2
I
I
I
I
I
0.3
0.4
0.5
0.6
0.7
Partial pressure of oxygen
0.15
0.1
Partial pressure of carbon dioxide (atm)
time (h)
FIG. 2. Changes in volumetric agitation power, BC concentration, and viable cell concentration at an air flow rate of 0.5 wm and an operating pressure of 1.5 atm. Symbols: 0, volumetric agitation power (PJ; •I , BC concentration; A, viable cell concentration.
0.35
0.05
15 0.8
5
(atm)
FIG. 3. BC production rate and volumetric agitation power changes in response to altering the partial pressure of oxygen (PO,) by adding oxygen-enriched air and/or raising the operating pressure of the fermentor. Symbols: ( 0, m) BC production rate; (0, +) volumetric agitation power (pv). Open symbols, sparging with oxygenenriched air; filled symbols, raising the operating air pressure.
BC production rate changes in response to altering the partial pressure of carbon dioxide (PO,) in the outlet gas. FIG.
4.
decreased and the BC production rate was improved, but p02 and P,(14- 15 kW . mS3) were not affected. In order to clarify the relationship between the BC production rate and pCO%, carbon dioxide-enriched air was sparged during the cultivation at an operating pressure of 1.5 atm. Table 2 shows the BC production rates, fructose consumption rates, and OCR at different levels of pCOz in sparged carbon dioxide-enriched air. All three rates were low when the pCOz of the sparged carbon dioxide-enriched air was high. DISCUSSION The results of the above experiments demonstrated clearly that (i) increasing the pOa reduced the P, required to maintain C,_ at a constant level during the cultivation, (ii) a high pCOz caused a reduction in BC production, and (iii) increasing the air flow rate canceled the inhibitory effect of pCOz. mass transfer in a viscous broth is In general, problematic, as it is in a xanthan gum system. The productivity of xanthan gum has been reported to depend on the shear rate, the mass transfer around the cells being enhanced as the shear rate increased (13). Thus, even though the BC production rate has been shown to depend on the lYLa (7), and this depends on the agitation speed, another possible factor affecting the BC production rate could be the shear rate of the agitation. However, our results showed that the BC production rate was not reduced as the agitation speed decreased when oxygen-enriched air was sparged (Fig. 3), indicating that the BC production rate does not depend on the shear rate. This implies that the BC production rate in an aerated and agitated culture is not limited TABLE
1. Effect of air flow rate on the partial pressure of carbon dioxide and BC production rate under a constant partial pressure of oxygen
Air flow rate (vvm) Partial pressure of oxygen @Oz) (atm) Partial pressure of carbon dioxide WOZ) (atm) BC production rate* (g .I-‘. h-l)
0.25 0.315 0.10
0.5 0.315 0.05
1.0 0.315 0.03
0.41
0.45
0.47
a Average increase in the BC concentration cultivation.
per hour during the
VOL. 84, 1997 TABLE 2.
EFFECT OF PRESSURES ON BC PRODUCTION
Effect of partial pressure of carbon dioxide in sparged air on the bacterial cellulose production process
Partial pressure of carbon dioxide olCO2) (atm) BC production rate= (g. 1-r. h-r) Fructose consumption rateb (g-1-r. h-r) Oxygen consumption rateC (mmol./-r.h-r)
o.ocO5 0.7 2.9 47
0.075 0.5 2.5 43
0.12 0.3 2.0 37
a Maximum value of the increase in the BC concentration per hour measured at 6-hourly intervals. b Maximum value of the decrease in the fructose concentration per hour measured at 6-hourly intervals. c Equal to the maximum OTR, measured at 30-min intervals, with CL at a constant level during the cultivation.
by mass transfer around the cells. BC is considered to have little effect on the mass transfer around cells because it is not soluble in water, whereas xanthan gum is soluble. Therefore, it was concluded that the BC production rate increased with a high KLa, not because the mass transfer around cells was enhanced but because of oxygen transfer from the gaseous phase to the broth. In order to improve the OTR, we increased the p0~ by sparging oxygen-enriched air and/or raising the operating pressure. Although raising the operating pressure is practical and economical in a commercial plant, the results showed that the BC production rate decreased as the operating pressure was raised (Fig. 3). This decrease in the production rate is considered to be caused by the high pCOz (Fig. 4). On the other hand, the inhibitory effect of pCOz was canceled out by the ventilation effect of increasing the air flow rate (Table 1): pC0~ at less than 0.05 atm did not inhibit BC production, the change in the production rate being negligible when the air flow rate increased from 0.5 to 1 .O vvm. Generally, increasing the air flow rate is an effective means of supplying oxygen to a culture broth. However, this method was not effective in the BC culture broth because the P, needed to maintain CL at a constant level was not reduced. This is mainly because the excess sparged air is not dispersed as small bubbles in the viscous broth containing BC, but passes through it to the exhaust. The mechanism responsible for the inhibitory effect of pCOz appears to be complex, because, as shown in Table 2, a high pC0~ inhibited not only the BC production but also the fructose and oxygen consumption. We intend to carry out further studies to determine why a high pCOz reduces the BC production rate.
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ACKNOWLEDGMENTS We thank Prof. Makoto Shoda, Research Laboratory of Resources Utilization, Tokyo Institute of Technology, for his critical comments on the manuscript. We also thank Mr. K. Kume, Mr. Y. Yamashita and Ms. M. Kubota of our laboratory for their technical assistance. REFERENCES 1. Ross, P., Mayer, R., and Benziman, M.: Cellulose biosynthesis and function in bacteria. Microbial. Rev., 55, 35-58 (1991). 2. Yoshinaga, F., Tononehi, N., and Watanabe, K.: Research progress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material. Biosci. Biotech. Biochem.. 59. 219-224 (1997). 3. Toyosaki, H., Naritomi, T.,’ Seto, A., ‘l&t&da, T., and Yoshinaga, F.: Screening of bacterial cellulose-producing Acetobacter strains suitable for agitation culture. Biosci. Biotech. Biochem., 59, 1498-1502 (1995). 4. tshikawa, A., Matsuoka, M., Tsuchida, T., and Yoshinaga, F.: Increase in cellulose production by sulfaguanidine-resistant mutants derived from Acetobacter xylinum subsp. sucrofermentans. Biosci. Biotech. Biochem., 59, 2259-2262 (1995). 5. Matsuoka, M., Tsucbida, T., Matsushita, K., Adachi, O., and Yoshinaga, F.: A synthetic medium for bacterial cellulose production by Acetobacter xylinum subsp. sucrofermentans. Biosci. Biotech. Biochem., 60, 575-579 (1996). 6. Kouda, T., Yano, H., Yoshinaga, F., Kaminoyama, M., and Kamiwano, M.: Characterization of non-Newtonian behavior in the mixing of bacterial cellulose in a bioreactor. J. Ferment. Bioeng., 82, 382-386 (1996). 7. Kouda, T., Yano, H., and Yoshinaga, F.: Effect of agitator configuration on bacterial cellulose productivity in aerated and agitated culture. J. Ferment. Bioeng., 83, 371-376 (1997). 8 Yabannavar, V., Singh, V., and Schaefer, E.: Effect of pressure on aminoglycoside fermentation mediated by dissolved oxygen. J. Ferment. Bioeng., 73, 66-69 (1992). 9. Dufresce, D., TubanIt, J., Leduy, A., and Lencki, R.: The effect of pressure on the growth of Aureobasidium pullulans and the synthesis of pullulan. Appl. Microbial. Biotechnol., 32, 526-532 (1990). 10. Gill, C. 0. and Tan, K. H.: Effect of carbon dioxide on growth of Pseudomonas Juorescens. Appl. Environ. Microbial., 38, 237-240 (1979). 11. Almshi, K., Shibai, H., and Hirose, Y.: Inhibitory effects of carbon dioxide and oxygen in amino acid fermentation. J. Ferment. Technol.. 57. 317-320 (1979). 12. Marx-Figini, M. and’ Pion, B.‘G.: Kinetics investigation on biosynthesis of cellulose by Acetobacter xylinum. Biochim. Biophys. Acta, 338, 382-393 (1974). 13. Funahashi, H., Maehara, M., Taguchi, H., and Yosbida, T.: Effects of agitation by flat-bladed turbine impeller on microbial production of xanthan gum. J. Chem. Eng. Japan, 20, 16-22 (1987).