Inhibitory Effect of Carbon Dioxide on
the Fed-Batch Culture of Ralstonia
: Evaluation by CO Pulse
Injection and Autogenous CO Methods
Longan Shang,1* Min Jiang,1** Chul Hee Ryu,1 Ho Nam Chang,1
Soon Haeng Cho,2 Jong Won Lee3

1Department of Chemical and Biomolecular Engineering, Korea AdvancedInstitute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu,Daejeon 305-701, Korea; telephone: 82-42-869-3912; fax: 82-42-869-3910;e-mail: [email protected] 2Separation Process Research Center, Korea Institute of Energy Research,72-1 Jang-dong, Yuseong-gu, Daejeon 305-343, Korea 3Department of Biochemistry, School of Medicine, Catholic University ofDaegu, 3056-6, Daemyeong 4-dong, Nam-gu, Daegu, 705-034, Korea Received 7 October 2002; accepted 14 January 2003 Abstract: In order to see the effect of CO inhibition re-
PHB of 97.2 g/L was obtained, which corresponded to sulting from the use of pure oxygen, we carried out a 70% of the PHB production at 1.0 vvm O flow rate. This comparative fed-batch culture study of polyhydroxybu- new method measures the inhibitory effect of CO pro- tyric acid (PHB) production by Ralstonia eutropha using duced autogenously by cells through the entire fermen- air and pure oxygen in 5-L, 30-L, and 300-L fermentors.
tation process and can avoid the overestimation of CO2 The final PHB concentrations obtained with pure O were inhibition without introducing artificial CO into the fer- 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L mentor. 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83:
fermentor, which increased 2.9 and 6.2 times, respec- tively, as compared to those obtained with air. In the Keywords: CO inhibition; PHB synthesis; pure oxygen;
300-L fermentor, the fed-batch culture with air yielded Ralstonia eutropha; CO pulse; autogenous CO only 8.4 g/L PHB. However, the maximal CO concentra- tions in the 5-L fermentor increased significantly from4.1% (air) to 15.0% (pure O ), while it was only 1.6% in INTRODUCTION
the 30-L fermentor with air, but reached 14.2% in the caseof pure O . We used two different experimental methods There have been many reports regarding the inhibitory ef- fects of carbon dioxide on the growth and metabolism of a autogenous CO methods. A 10 or 22% (v/v) CO pulse wide range of microorganisms such as bacteria, yeast, and with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO filamentous fungi (Dixon and Kell, 1989; Jones and Green- affects the synthesis of biomass and PHB. CO inhibited field, 1982; McIntyre and McNeil, 1998). The previous the cell growth and PHB synthesis significantly. The in- studies focused on using CO to control the growth of hibitory effect became stronger with the increase of the pathogenic organisms or food spoilage organisms and pro- concentration and pulse duration. The new pro- tect the quality of food or water (Dixon and Kell, 1989; place microbial cells under different CO level environ- McIntyre and McNeil, 1998). Later, it was found that the ments by varying the gas flow rate. Introduction of O fermentation progress could be affected by the CO pro- gas at a low flow rate of 0.42 vvm resulted in an increase duced by the microorganism itself (Dixon and Kell, 1989; of CO concentration to 30.2% in the exit gas. The final McIntyre and McNeil, 1998; Onken and Liefke, 1989).
Many studies on the mechanisms were summarized byJones and Greenfield (1982). Among the several molecular *Current address for Longan Shang: Department of Chemical Engineer- species of CO in the liquid phase, the CO (aq) and HCO − ing, Northwest University, Xian 710069, China. E-mail: Lashan@ were generally responsible for the CO inhibition on micro- **Current address for Min Jiang: College of Pharmacy and Life Science, organisms (Dixon and Kell, 1989; Jones and Greenfield, Nanjing University of Technology, Nanjing 210009, China.
1982). However, it is still difficult to find a single primary mechanism to explain the observed results (McIntyre and Contract grant sponsor: the Korea Science and Engineering Foundation Contract grant number: R01-2000-000-00331-1 CO inhibition on cell growth and product formation in Contract grant sponsor: the Brain Korea21 project of the Ministry of fermentation has been demonstrated. Two research groups have reported the effects of dissolved CO in two important MATERIALS AND METHODS
industrial strains, Penicillium chrysogenum (Edwards andHo, 1988; Ho and Smith, 1986) and Aspergillus niger Microorganism and Medium
(McIntyre and McNeil, 1997a,b,c, 1998). Their morpholo-gies, being of importance in the evaluation of cell growth Ralstonia eutropha (formerly known as Alcaligenes eutro- and product formation, were apparently affected if the in- phus) NCIMB 11599 was used throughout this study. The fluent CO concentration was higher than 5%. Other organ- seed culture medium composition and the initial medium isms such as Escherichia coli (Lacoursiere et al., 1986; composition for fed-batch culture are given in Table I. Thetrace element solution contains per liter: 10 g FeSO Mori et al., 1983), Clostridium acetobutylicum (Mollah and (7H O), 2.25 g ZnSO (7H O), 1.2 g CuSO (5H O), 0.5 g Stuckey, 1992), Pseudomonas sp. (Gill and Tan, 1979; King and Nagel, 1975), Saccharomyces cerevisiae (Kuriyama et (7H O), 0.12 g (NH ) Mo O , and 10 ml 35% HCl.
al., 1993), and Zymomonas mobilis (Veeramallu and Agrawal, 1986) were also sensitive to CO . The reported effects were dependent on the CO concentration and the Culture Condition
organism employed in their experiments. In order to avoid Fed-batch cultures of R. eutropha were carried out in three the overestimation of CO effects, a pulse or pulses of CO - different scale fermentors (5, 30, and 300 L). At 30°C the enriched gas, rather than the continuous CO -enriched gas- gas flow-rates were maintained at 1.0 vvm based on the sing of batch cultures from time zero, was used in batch initial culture volume and pH was maintained at 6.7. Gas cultures (McIntyre and McNeil, 1997c). The estimated CO2 flow-rates and pressures were monitored and independently effects would depend on the CO concentration and pulse controlled with a gas mixer and a mass flow meter. When lasting time in a given experiment. Therefore, the results pure oxygen was used as the gas phase, the dissolved oxy- obtained from continuous or CO pulse technique may not gen concentration was maintained at higher than 25% of air be applicable to real fermentation conditions where autog- saturation by increasing the agitation speed. When air was enous CO evolution is quite different from the experimen- used as the gas phase, the dissolved oxygen concentration was maintained as high as possible by increasing the agita- A number of studies on the production of poly- tion speed up to its maximum value. The maximum agita- hydroxyalkanoates (PHAs) have been carried out (Kim et tion speeds of 5, 30, and 300 L fermentors were 900, 450, al., 1994; Lee et al., 2000; Hori et al., 2002; Park et al., 2001; Rehm and Steinbuchel, 1999; Ryu et al., 1997; Shang In checking the effects of carbon dioxide on the process et al., 2003; Van Wegen et al., 2001). Ralstonia eutropha is of PHB production, a 6-h pulse of CO with 22% (v/v) in one of the major producers of PHAs since it can grow fast pure oxygen was introduced into fermentor at a defined cell and accumulate higher amounts of PHAs than any other growth stage (lag, exponential, stationary). A 3-h pulse of microorganisms. The highest productivity (3.75 g PHB/L.h) CO with 22% (v/v), and another 3-h pulse of CO with was obtained in the fed-batch culture with the aid of pure 10% (v/v), were employed to examine the effects of CO2concentrations and CO pulse lasting time on the fermen- oxygen (Ryu et al., 1999). However, the use of oxygen is tation process of PHB production. The gas mixture of CO expensive and thus it becomes necessary to maximize the and pure oxygen was prepared with a gas-mixer (New use of pure oxygen by reducing the oxygen flow rate. This would result in a CO concentration in culture media much In the absence of oxygen limitation, the CO concentra- higher than with air supply only. This high CO concentra- tion in the exit gas from a fermentor would increase with the tion may inhibit the metabolic activities of microbial cells decrease of the gas flow rate and the microbial cells can be (Ho and Smith, 1986; McIntyre and McNeil, 1997c).
placed under different CO concentration environments, In this study we investigated the effects of CO on cell then the effect of CO produced autogenously by cells can growth and PHB synthesis in the fed-batch culture of R. be estimated by changing the gas flow rate. Based on this eutropha. We applied the CO pulse technique to see how CO concentration and pulse duration affect PHB synthesis and cell growth. Our new proposed approach is to vary the oxygen flow rate so that the autogenous evolution of CO2 from the microbial cells may change the CO concentration in the culture broth. In this case, the CO concentration will increase with the decrease of gas flow rate. The results of this study will shed light on temporal and integral CO2 inhibition patterns on the biosynthesis of PHB and the growth of R. eutropha. This can give us insight into how to suppress or promote metabolic activities of various other SHANG ET AL.: CO INHIBITION ON FED-BATCH CULTURE idea, a fed-batch culture was carried out at an O flow rate ducing high-purity oxygen, it would become possible to of 0.42 vvm to examine the effect of CO produced by R. achieve a high cell density in large-scale fermentors as well.
But the CO concentration in effluent gas will also increase in high cell density cultures. As shown in Table II, the ratioof exit CO concentrations in pure oxygen and air was just Analysis Methods
3.6 in the 5-L fermentor, but it increased to 8.9 in the 30-L Cell growth was monitored by measuring an optical density fermentor. In fact, the pure oxygen flow rate would be kept (OD) at 600 nm with a spectrophotometer (Beckman, Palo as low as possible to fully utilize O . This would result in a Alto, CA). Cell concentration was determined by measuring further increase in the concentration of CO . A highly dis- the dry cell weight (DCW) of a 2-ml culture broth. The solved CO concentration is more likely to occur in a large- broth sample was centrifuged, washed with distilled water, scale industrial fermentor than in a lab-scale fermentor due and dried in an oven at 60°C until no further decrease was shown in weight. PHB concentration was determined with agas chromatograph (Varian 3300, San Fernando, CA) withbenzoic acid as the internal standard (Braunegg et al., 1978; CO Concentrations in the Effluent Gas and in
the Culture Broth
Shang et al., 2003). The residual cell weight (RCW) wasdefined as the cell concentration less PHB concentration.
A linear relationship between the dissolved CO concentra- Carbon dioxide concentration in the influent and effluent tion in the culture broth and that in exit gas was found when gas was measured with a gas analyzer (Model LKM2000- the operation pressure is steady (McIntyre and McNeil, 03, LOKAS Automation Corp., Korea). Glucose concentra- 1997c). The liquid phase CO concentration can be moni- tion in the medium was automatically analyzed and con- tored with a dCO probe. Its performance may be affected trolled at 9 g/L with a glucose analyzer (Model 2730, Yel- by the presence of organic acids, but not significantly (Pu- low Springs Instruments, Youngstown, OH).
har et al., 1980; McIntyre and McNeil, 1997c). Consideringthat CO in the culture broth can exist in various forms, such RESULTS AND DISCUSSION
and they may affect the metabolic activity of microbial cellsdifferently, we chose to monitor the concentrations of CO2 High Cell Density Culture With Pure Oxygen and
in the influent and effluent gas with a gas analyzer. At the CO Production
current experimental pH of 6.7 the most dominant forms of A set of fed-batch cultures of R. eutropha was carried out with pure oxygen or air, as shown in Table II. When air wasused, the DCW and PHB concentration obtained in the 5-Lfermentor decreased by 54% and 66%, respectively, as com- CO Inhibition on the Biosynthesis and
Cell Growth
pared with those obtained with pure oxygen. In the 30-Lfermentor, the DCW and PHB concentration decreased by73% and 84%, respectively. In the 300-L fermentor, the Carbon Dioxide Pulses at Different Cell obtained DCW was just 23.4 g/L due to the oxygen limita- tion. The supply of pure oxygen can effectively eliminatethe oxygen limitation and achieve a higher cell density.
A series of experiments were designed to estimate the ef- High cell density culture (HCDC) technologies have at- fects of CO on the cell growth and PHB formation in tracted attention, as they may guarantee high product level fed-batch cultures of R. eutropha. A 6-h pulse of 22% (v/v) (Babu et al., 2000; Riesenberg and Guthke, 1999). It is CO in pure oxygen was introduced into fermentor at a relatively easy to achieve a very high cell density in a small- defined cell growth stage such as the lag phase, the expo- scale fermentation for several important industrial microor- nential phase, or the stationary phase to determine which is ganisms such as Escherichia coli, yeast, Bacillus, and R. the most sensitive stage to CO based on cell growth and eutropha. However, with the decrease in the cost of pro- PHB formation (Fig. 1a–c). As a reference, a fed-batch Table II.
Summary of fed-batch cultures with oxygen and air.
Time course of OD, DCW, RCW, PHB concentration, and exit CO levels in the fed-batch cultures with a 6-hour CO pulse (22%) at a defined cell growth stage. a: Stationary stage between 34.5 and 40.5 h. b: Early exponential stage between 11 and 17 h. c: Lag stage between 3 and 9 h. d: Standard
culture without CO pulse. OD (᭢), DCW (᭹), RCW (᭡), PHB conc. (᭿), and exit CO conc. (ࡗ).
SHANG ET AL.: CO INHIBITION ON FED-BATCH CULTURE culture of R. eutropha was done with no CO Figure 1a shows the time course of OD, DCW, RCW, PHB, and exit CO concentrations of the fed-batch culture A 3-h CO pulse was added into the fermentor at exponen- with a CO pulse introduced in the stationary stage. During tial phase to see the effect of CO pulse lasting time in the 6-h CO pulse, the OD and DCW increased only a little comparison with the 6-h CO pulse (Fig. 2). The early ex- in the beginning. The CO concentration in the exit gas was ponential phase was chosen to introduce the CO pulse about 33% (v/v) during the CO pulse and it maintained at since a large amount of CO would be produced at this about 9% (v/v) thereafter. The decrease of OD and DCW may be attributed to the damage of cells caused by the During the CO pulse, the CO concentration was about temporarily elevated CO concentration in the solution 23% (v/v), which is similar to the case of the 6-h CO pulse.
(Dixon and Kell, 1989; Jones and Greenfield, 1982; McIn- After 25 h, the CO concentration was almost constant at tyre and McNeil, 1997a, 1998). After the CO pulse, the cell 10% (v/v). The final DCW and PHB concentration reached metabolic activity did not recover in the following 4 h. As 175 and 117 g/L, respectively; concentrations that are much a result, the final DCW and PHB concentration amounted to higher than those obtained in the case of the 6-h CO pulse 176 and 118 g/L, respectively, which are lower than the (Fig. 1b). This shows that the pulse duration of CO has a DCW (208 g/L) and PHB concentration (138.7 g/L) ob- strong effect on cell growth and PHB accumulation. The longer the pulse duration, the stronger the inhibition of CO2 Figure 1b shows the inhibitory effect of a CO pulse at on cell growth and production formation was found to be. In the exponential phase. Although the maximal CO concen- the culture of A. niger, the CO inhibition on cell growth tration in the exit gas was just about 23% (v/v), lower than and critic acid synthesis also increased with the CO pulse the 33% (v/v) mentioned above (Fig. 1a), the cell growth and PHB accumulation were severely influenced. During Another 3-h pulse of 10% (v/v) CO in pure oxygen was the CO pulse, the DCW and PHB concentration only in- introduced at the early exponential phase to see the effect of creased from 6.5–26 g/L and 0.8–2 g/L, respectively, as CO concentration on the fermentation process (Fig. 3). The compared with the DCW increase of 7–48 g/L and PHB CO pulse lasted only for 3 h and the maximal CO con- change of 0.6–6 g/L in the standard culture. After the CO centration in the exit gas was about 11% (v/v). Finally, it pulse ceased, the DCW and PHB concentrations increased maintained at a constant value of 9% (v/v), and lasted about continuously for 20.5 h, but did not increase further. The 20 h, which is very similar to the result obtained with the RCW continued to increase to 69 g/L in 34 h, which was 3-h CO pulse with 22% (v/v) CO in pure oxygen (Fig. 2).
obtained generally in about 22–24 h without a CO pulse.
The final DCW (190 g/L) and PHB concentration (122 g/L) This means that the average cell growth rate decreased to were much higher than those obtained in the 3-h 22% (v/v) CO pulse. From the results shown in Table IV, we can see The lowest DCW and PHB concentrations were obtained the CO inhibition in the cultures of P. chrysogenum, Z. with a CO pulse in the lag stage between 3–9 h (Fig. 1c).
mobilis, and S. cerevisiae also increased with the increase of The final DCW and PHB concentration just reached 138 CO concentration. All these clearly showed that the higher and 78.8 g/L, respectively. In this case, it took more than 30 the CO concentration, the stronger the inhibitory effect of h when the RCW reached its maximal value. After the CO CO on cell growth and production formation was.
pulse ceased, the cell growth and PHB accumulation were The efficient way to prevent CO inhibition is to lower still slow. It can be said that the lag stage, in which the the exit CO concentration from a fermentor by either in- productivity of PHB is the lowest (Table III), is the stage creasing the gas flow rate or supplying inert gases into the most sensitive to the CO pulse with 22% (v/v) CO in the fermentor. In the former case a waste of pure oxygen is influent gas stream. A similar result was found in the culture expected and in the latter case it will result in a low O2 of A. niger (McIntyre and McNeil, 1997c).
Table III.
Summary of the experimental results in the fed-batch cultures with CO pulses.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 3, AUGUST 5, 2003 Figure 1d shows that the maximal CO concentration in exit gas from a 5-L fermentor was about 15% (v/v) when thegas flow-rate was 1.0 vvm based on the initial liquid vol-ume. In a 30-L fermentor, the maximal CO concentration in exit gas was 14.2% (v/v) (as shown in Table II). At thegas flow rate of 0.42 vvm the maximal CO concentration from a 5-L fermentor reached 30.2% (v/v). In a pure oxygensystem, enough oxygen is always present in the liquid mediaand Monod constant or critical oxygen concentration foroxygen is less than 1 mg O /L. It is unlikely that the oxygen uptake is transport-limited rather than reaction-limited. Inthis case a higher CO concentration will occur at a lower Autogenous CO on the PHB synthesis and cell growth.
In a large-scale industrial fermentor it is likely that the CO2 concentration would be higher than that in a small-scalefermentor because of increased hydrostatic pressure (Onkenand Liefke, 1989). It is very important to choose a suitablemethod to simulate the conditions in a large industrial fer-mentor for the estimation of CO effect on microorganisms.
McIntyre and McNeil (1997c) reported that the previousexperimental method of continuous CO -enriched gassing of batch cultures from time zero may lead to a systematicoverestimation of CO inhibitory effects on mycelia organ- isms and proposed a CO pulse method. The results ob- Figure 2.
Time course of OD, DCW, RCW, PHB concentration, and exit tained in the CO pulse method clearly show that two pa- CO levels in the fed-batch culture with a 3-h CO pulse (22%) in the early rameters, CO concentration and pulse duration, play a key exponential stage between 10 and 13 h. OD (᭢), DCW (᭹), RCW (᭡), role in the estimation of CO inhibitory effect. However, PHB conc. (᭿), and exit CO conc. (ࡗ).
these two parameters are chosen artificially, which may leadto no conclusive reasoning on how the result of a pulse testis related to real experimental conditions. Another problem Effect of CO Produced Autogenously by Cells in the CO pulse method is the sudden increase of the CO General CO production pattern during aerobic fermen-
concentration that could not occur in actual fermentation 2
tation. Microbial cells can release or utilize CO . Both CO
productions in the ethanol fermentation by Saccharomyces Here we propose a new method to place microbial cells cerevisiae or Zymomonas mobilis and CO utilization for under a higher CO concentration just by lowering the gas PHB synthesis by R. eutropha in the presence of H are prod- flow rate, without the necessity of introducing CO artifi- uct-related (Veeramallu and Agrawal, 1986; Ishizaki et al., cially. As shown in Figure 4, the maximal CO concentra- tion reached 30.2% as the gas flow rate was maintained at 2001). If it is respiration-related, one mole of CO is produced 0.42 vvm. The final DCW (168 g/L) and PHB concentration per mole of organic carbon oxidized. Since R. eutropha is (97.2 g/L) decreased by 18.8% and 29.9%, respectively, as aerobic, CO will come from metabolic oxidation of glucose.
compared with the results obtained in the standard culture CO production per liter of the culture broth depends on (Fig. 1d). Also, the cell growth rate decreased by 39% when cell concentration in terms of g/L (X) and its specific meta- the CO concentration was at its maximal value (30.2%).
These decreases in DCW, PHB concentration, and cell growth rate were attributed to the inhibitory effect of theCO produced by cells themselves. During the lag phase less inhibition occurred because of the low CO concentra- tion, but during the exponential and stationary phases severe During fermentation with a gas flow rate of 0.42 vvm, it exponentially. At some point of this logarithmic phase oxy- was clear that the inhibitory effect of CO was higher than gen supply from air is not sufficient and thus pure oxygen is that in the case of a 3-h 22% CO pulse, but lower than that will increase and reach a maximum at the end in the case of a 6-h 22% CO pulse. The maximal CO of this phase. As the fermentation enters the stationary concentration lasted for 6 h and showed a pattern of cell phase, microbial product synthesis are induced, Q growth and CO inhibition. This technique is essentially a natural combination of pulse and continuous injection tech- SHANG ET AL.: CO INHIBITION ON FED-BATCH CULTURE BIOTECHNOLOGY AND BIOENGINEERING, VOL. 83, NO. 3, AUGUST 5, 2003 Figure 3.
Time course of OD, DCW, RCW, PHB concentration and exit Figure 4.
Time course of OD, DCW, RCW, PHB concentration, and exit CO levels in the fed-batch culture with a 3-h CO pulse (10%) in the early CO levels in the fed-batch culture at a gas flow rate of 0.42 vvm without exponential stage between 10.5 and 13.5 h. OD (᭢), DCW (᭹), RCW (᭡), a CO pulse introduced. OD (᭢), DCW (᭹), RCW (᭡), PHB conc. (᭿), PHB conc. (᭿), and exit CO conc. (ࡗ).
niques of variable CO concentrations, while pulse and con- References
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