Chemostat Cultures of Bacillus subtilis KYA741

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Apr. 1982, p / $2./ Vol. 43, No. 4 Kinetics of Substrate Utilization in Adenine-Limited Chemostat Cultures of Bacillus subtilis KYA741 KATSUMI TSUCHIYA* AND TTSU KIMURA Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama, Japan Received 21 May 1981/Accepted 21 December 1981 The specific rates of limiting substrate utilization were investigated in adenineor glucose-limited chemostat cultures of Bacillus subtilis KYA741, an adeninerequiring strain, at 37C. With the glucose-limited cultures, the specific rate of glucose consumption versus dilution rate gave a linear relationship from which the true growth yield and maintenance coefficient were determined to be.9 mg of bacteria per mg of glucose and.2 mg of glucose per mg of bacteria per h, respectively. With the adenine-limited cultures, adenine as the limiting substrate was not completely consumed at lower dilution rates (e.g., D <.1), unlike in the glucose-limited cultures. When a linear relationship of specific rate of adenine consumption versus dilution rate was extrapolated to zero dilution rate, a negative value for the specific rate of adenine consumption, -.1 mg of adenine per mg of bacteria per h, was obtained, giving a true growth yield for adenine of 5.2 mg of bacteria per mg of adenine. On the other hand, the maintenance coefficient of oxygen uptake gave a positive value of 8.1 x 1-3 mmol/mg of bacteria per h. Based on previous results showing that adenine is resupplied by lysing cells, we developed kinetic models of adenine utilization and cell growth that gave a good estimation of the peculiar behavior of cell growth and adenine utilization in adenine-limited chemostat cultures. Many workers (1-1) have reported that the total rate of substrate utilization is divided into the rates for cellular maintenance and for cell growth when an energy source such as a carbon source is used as a growth-limiting substrate. Also, van Uden (14) has introduced a theoretical equation for substrate utilization for cases in which exponential cell death accompanies exponential growth. However, little is known about the relationship between the rates of substrate utilization and cell growth under conditions such as adenine-limited growth. As reported previously (12, 13), Bacillus subtilis KYA741, an adenine-requiring strain, showed the characteristic property of consuming adenine as a growthlimiting substrate, so that adenine remained at a constant level in the culture filtrate in even the stationary growth phase due to cell turnover; i.e., growth and lysis of cells balanced each other in this phase, and the lysing cells supplied adenine to the growing cells. In this paper, we describe the relationship between the rates of adenine utilization and cell growth and present a kinetic model for substrate consumption by B. subtilis KYA741 in adenine-limited chemostat cultures. MATRIALS AND MTHODS Microorganism. B. subtilis KYA741, which requires adenine, Casamino Acids, and biotin for growth, as described previously (12, 13), was used throughout. Culture conditions. Culture medium under adeninelimited conditions consisted of: soluble starch, 2 g; Casamino Acids, 6 g; Na2HPO4412H2O, 2.8 g; KH2PO4, 1.3 g; MgSO4-7H2O,.5 g; CaCl2 2H2O,.1 g; adenine, 3 mg; biotin,.1 mg; and water, 1 liter (ph 7.). Under glucose-limited conditions, soluble starch was replaced with 1.7 g of glucose, and the concentration of adenine was raised to 5 mg/liter. Seed culture was prepared in a 5-ml flask containing 1 ml of the culture medium and incubated for 2 h at 37 C on a reciprocating shaker. Fifty milliliters of the seed culture broth was inoculated into 8 ml of the culture medium in a 2-liter baffled flask and cultured at 37 C with vigorous agitation and aeration at a rate of 1 vol/vol per min. Under these aerating conditions, the dissolved oxygen (DO) concentration was obtained over 7% saturation during the experiments. When the batch culture reached the postexponential phase after about 1 h, the continuous culture was started by feeding the sterilized fresh medium into the culture vessel at constant flow rates with a peristatic pump. The culture volume was maintained at a constant level by an overflow method. Breakdown of foams was automatically carried out with silicone antifoamer (Toshiba Silicone YMA695) during cultivation. After reaching steady state, the cultures were continued for three generation times at various dilution rates. ach continuous run was assumed to be in the steady state if the cell concentration did not change over one generation time. Measurements of cell and substrate concentrations and of oxygen uptake. The culture broth was harvested 794

2 VOL. 43, 1982 after the steady state was established in the chemostat cultures. Cell concentration was measured by optical density at 6 nm, using a cell of 5-mm light path. One unit of optical density was equivalent to a cell concentration of.35 mg/ml. Adenine concentration in the culture broth was measured by the bioassay method. The culture broth was harvested and centrifuged at 7,5 x g for 1 min. Duplicate 1-ml samples of the supernatant liquid were collected in a 5-ml flask and sterilized at 121 C for 15 min. One loopful of cells from the stock culture of B. subtilis KYA741 was inoculated into the sterilized culture broth and incubated for 48 h at 37 C on a reciprocating shaker. The cell concentrations of these culture broths were measured, and.1 mg/ml was found to be equivalent to an adenine concentration of 8,ug/ml, as reported previously (13). The glucose concentration was measured with a glucose reagent (Sigma Chemical Co.). Oxygen uptake was measured with a Beckman oxygen analyzer (Fieldlab) in a closed system at 37 C. The culture broth harvested from the chemostat culture was immediately placed into a 15-ml vessel fitted with an oxygen probe, and oxygen was bubbled until the DO concentration was.47 mmol/liter. The vessel was closed so as to contain no bubbles. The DO concentration of the culture broth in the vessel was monitored with the oxygen analyzer. The oxygen uptake rate was estimated according to the method of Terui et al. (11), which is based on the following theory. The oxygen uptake rate is assumed to be expressed by the Michaelis-Menten equation. The following equation is obtained by integrating the Michaelis-Menten equation: t= -{[OJj(1 - r) - Kmlnr} (1) where V is the maximal rate of oxygen uptake (moles of 2 per liter per minute), [ 2]i is the dissolved oxygen concentration at the appropriate time (moles of 2 per liter), Km is the Michaelis constant (moles of 2 per liter), t is reaction time measured from the appropriate time, and r is the residual ratio of dissolved oxygen, [21i+u[ 21, at t. When r is 1/2, equation 1 gives the following equation; [21i = 2Vt1/ Km (2) where t1j2 is half-time (in minutes). Plots of [ 2]i versus tj,2, which can be calculated with the curve of DO monitored with a DO analyzer, give a straight line from equation 2. The respiration rate, Qo2, is obtained by dividing V given from the slope of the straight line with the cell concentration of the culture broth used for the measurement. RSULTS AND DISCUSSION Cell and substrate concentrations at steady state in chemostat cultures. When B. subtilis KYA741 grew in batch culture under adeninelimiting conditions, we found the peculiar behavior in utilization of the growth-limiting substrate reported previously (12, 13). Adenine, the SUBSTRAT UTILIZATION BY B. SUBTILIS 795 cm 4 3-O1 x S It-I a - 2 Af 6 x D (hr-') FIG. 1. Cell and substrate concentrations against dilution rate in adenine-limited chemostat cultures of B. subtilis KYA741. SAQ = 3,ug/ml. Symbols:, Cell concentration; A\, ademne concentration. growth-limiting substrate, remained at a constant level in the culture filtrate during the phase when no net increase in cell mass was observed. The concentration of adenine remaining in the culture broth in this phase was proportional to the initial concentration. These features of adenine consumption were based on cell turnover, a phenomenon whereby growth and lysis of cells balance each other in the stationary phase, and the lysing cells supply adenine to the growing cells. B. subtilis KYA741 with these characteristics was cultivated at various dilution rates in adenine-limited chemostat cultures. Figure 1 shows the steady-state concentrations of cells and adenine at various dilution rates. The cell concentration was high at lower dilution rates, decreasing gradually with increasing dilution rates. A washout state occurred at.8 h-1 of the dilution rate. Although adenine was used as a growthlimiting substrate, the adenine was not completely consumed, reaching a concentration of 4.7,ug/ml at lower dilution rates. When an energy source such as glucose is used as a limiting substrate, below a certain dilution rate (e.g., D <.1 h-1 in Fig. 2) the cell concentration falls as the dilution rate decreases; in addition, substrate is completely consumed at lower dilution rates (5, 15). In a glucose-limited chemostat culture of B. subtilis KYA741, we found the same behavior of cell and glucose concentrations as a function of the dilution rate (Fig. 2). The washout state occurred at a dilution rate of.45 h-1. In the batch culture under glucoselimiting conditions, the maximal specific growth rate was found to be.45 h-1, which was lower than that under adenine-limiting conditions in which soluble starch was used as a carbon source (Fig. 3). In general, of all carbon sources, glucose might be expected to support the highest

3 796 TSUCHIYA AND KIMURA APPL. NVIRON. MICROBIOL. (hr'1) D FIG. 2. Cell and substrate concentrations against dilution rate in glucose-limited chemostat cultures of B. subtilis KYA741. SG = 1.7 mg/ml. Symbols:, Cell concentration; A, gfucose concentration. growth rate. In the case of B. subtilis KYA741, however, soluble starch as the carbon source produced a higher growth rate than did glucose, xylose, or maltose under adenine-limiting conditions (K. Tsuchiya and T. Kimura, unpublished data). The cell concentration, expressed as milligrams of cells per milliliter, immediately decreased after the maximal cell concentration was observed, whereas under adenine-limiting conditions, the cell concentration remained at a constant level for a long period in the stationary phase (12). These results suggest that cell lysis occurs also under glucose-limiting conditions, but that cells cannot regrow because glucose has been completely consumed, and any usable carbon source might not be supplied by the lysing cells. Our results also suggest that the utilization mechanism of adenine is different from that of glucose. Substrate consumption rate in chemostat cultures. When an energy source such as a carbon source is used as a growth-limiting substrate, many workers (1-1) have found that the total rate of substrate utilization is divided into the rate for cellular maintenance and the rate for cell growth, as follows: 1 m 1,u ~=-+ - or-u=m+ -~ (3) Yxis Pux YG YG where YXlS is the total growth yield (milligrams of bacteria per milligram of substrate), pux is the specific growth rate (per hour), m is the maintenance coefficient (milligrams of substrate per milligram of bacteria per hour), YG is the true growth yield (milligrams of bacteria per milligram of substrate), and v is the specific rate of substrate utilization (milligrams of substrate per Time ( hr) FIG. 3. Growth of B. subtilis KYA741 in batch cultures under adenine- or glucose-limiting conditions. Initial concentrations of adenine and glucose were 3,ug/ml and 2 mg/ml, respectively. Symbols:, Cell growth in adenine-limited culture; A, cell growth in glucose-limited culture;, adenine utilization in adenine-limited culture; A, glucose utilization in glucoselimited culture. milligram of bacteria per hour). Since the specific growth rate (px,) equals to the dilution rate (D) at the steady state of continuous culture, the specific rate of substrate utilization (v) is calculated by the equation (S - S)DIX, in which So, 5, and X are substrate concentrations (milligrams per milliliter) of the influent medium and the culture broth and cell concentration (milligrams per milliliter), respectively. In the glucose-limited chemostat culture of B. subtilis KYA741, the specific rate of glucose utilization as a function of the dilution rate was expressed by equation 3 (Fig. 4), and the values of YG and m were found to be.9 mg of bacteria per mg of glucose and.2 mg of glucose per mg of bacteria per h, respectively. These values may be lower than those reported previously (1, 7-9). Little is known about the relationship between the rates of substrate and cell growth under conditions such as adenine-limited growth. In the case of B. subtilis KYA741, adenine should be taken up during the syntheses of not only nucleic acids but also energy carriers such as ATP. Therefore, we suggest that adenine, unlike other growth factors, may be similar in its manner of substrate utilization to energy sources. Also, equation 3, which is used generally for the utilization of carbon or energy sources (or both), may apply to adenine. Using the adenine-limited chemostat culture, we found the relationship between specific rate of adenine utilization and dilution rate shown in Fig. 5. A linear relation-

4 VOL. 43, 1982 SUBSTRAT UTILIZATION BY B. SUBTILIS cs N. 2)-- l l l C -o D (hr-1) FIG. 4. Specific rate of glucose consumption as a function of dilution rate in glucose-limited chemostat cultures of B. subtilis KYA a (5 tw2 (min)/ 1 I D (hr) FIG. 6. Specific rate of oxygen uptake as a function of dilution rate in adenine-limited chemostat cultures of B. subtilis KYA741. Insert shows the behavior of oxygen uptake by B. subtilis KYA741 according to the method of Terui et al. (11). 151 D (hr-1) FIG. 5. Specific rate of adenine consumption as a function of dilution rate in adenine-limited chemostat cultures of B. subtilis KYA741. ship was obtained as expressed in equation 3 However, interestingly, extrapolation to zerc dilution rate gave a negative value for the specif ic rate of adenine utilization. This negative main tenance coefficient shows apparent value, for ii may have resulted from cell turnover, resupplying of adenine to the medium by lysing cells (12) and incomplete utilization of adenine at loweir dilution rates. Applying equation 3 to the utiliza tion of adenine, the true growth yield and appar ent maintenance coefficient were found to be 5., 2 mg of bacteria per mg of adenine and -.1 mj of adenine per mg of bacteria per h, respective ly. On the other hand, the specific rate of oxyger uptake in the adenine-limited chemostat culture gave a positive maintenance value (Fig. 6), and this agreed with the relationship previously reported (7-9) showing that the respiration rate can be divided into terms of maintenance and growth as follows: = Qo2 mo + PxI YGO (4) where Qo is the specific respiration rate (millimoles of 62 per milligram of bacteria per hour), mo is the maintenance coefficient for respiration (millimoles of 2 per milligram of bacteria per hour), and YGO is the growth yield for oxygen (milligrams of bacteria per millimole of 2). The L values of YGO and m were found from Fig. 6 to 8 be 12.4 and 8.1 x 1-3, respectively. Although the value of YGO agrees with that of Nagai and Aiba (7), the value of mo is higher. This may have resulted from cell turnover occurring in the stationary phase of batch culture (12). These results indicate that the negative maintenance for adenine may be peculiar to adenine utilization by B. subtilis KYA741, an adenine-requiring strain. Kinetic model of adenine utilization. From assumption noted above, the mass balances for cell growth and adenine utilization in the adenine-limited chemostat culture is given as follows; dx = IL,XX - kx - DX dsa = D(SAO - SAX) - Y Ixx + rkx dt YGAP' (5) (6) where X is cell concentration (milligrams per milliliter), k is the lysing rate of cells (per hour), SAO and SA are adenine concentrations of inlet

5 798 TSUCHIYA AND KIMURA and outlet media, respectively (milligrams of adenine per milliliter), and r is the coefficient of resupplement of adenine by lysing cells (milligrams of adenine per milligram of bacteria). At the steady state, the following equations are obtained; ;Lx = D + K (7) -e x,- APPL. NVIRON. MICROBIOL. (8) where v = (SAO - SA)DIX. If r = (i.e., resupplement of substrate from lysing cells does not occur), equation 8 becomes: D v=y+ YG YG k k(9) The second term on the right equals the maintenance coefficient as described by Pirt (8). The specific growth rate of B. subtilis KYA741 is expressed by the following equation, as reported previously (12); I l.lmaxsa ( KAX + SA (1) where PLmax is the maximal specific growth rate (per hour) and KA is saturation constant (milligrams of substrate per milligram of bacteria). The concentrations of cell and adenine in steady-state continuous culture are obtained as following from equations 7, 8, and 9: SA = D /1 \ YG V YG I - D(SAO SA) (11) D-+ k 1--rj YG \YG D(D + k)kasao (D -D) [D + k(i - r + D(D + k)ka LYG \YG /J(12) where DW is the dilution rate of the washout state and equals P.max - k. The values of parameters in equations 11 and 12 were obtained from Fig. 1 and 5 and our previous report (12) as follows: SA =.3 mg of adenine/ml, YG = 5.2 mg of bacteria per mg of adenine, k(1/yg - r) = -.1 mg of adenine per mg of bacteria per h, k =.2 h-1, KA =.33 mg of adenine per mg of bacteria, and Dw =.8 h- 1. The values ofx and SA at each dilution rate were estimated from equations 11 and 12, using the above-given values, as shown in Fig. 7. Since the second term in the denominator of equation D ( hr-') FIG. 7. Comparison of calculations of equations 11 and 12 with adenine-limited chemostat culture data of B. subtilis KYA741. Solid lines are curves calculated from equations 11 and 12. Symbols:, Cell concentration; A, adenine concentration. 11, k(1iyg - r), is negative, D must be higher than - YGk(l/YG - r), about.5 h-1, to give a positive value of X. The calculated values represented well the behaviors of cell growth and adenine consumption in the adenine-limited chemostat culture of B. subtilis KYA741. Therefore, equation 6, which includes a term for substrate resupplement from lysing cells, is thought to be suitable for explaining the incomplete consumption of a growth-limiting substrate such as adenine by B. subtilis KYA741. LITRATUR CITD 1. Alba, S., S. Nagai, Y. Nishizawa, and M. Onodera nergetics and nucleic analysis of a chemostat culture of Azotobacter vinelandii. J. Gen. Appl. Microbiol. 13: Chen, Y. R., and A. G. Hashimoto Substrate utilization kinetic model for biological treatment process. Biotechnol. Bioeng. 22: Chiu, S. Y., L.. rickson, L. T. Fen, and I. C. Kao Kinetic model identification in mixed populations using continuous culture data. Biotechnol. Bioeng. 14: Doelle, H. W., and N. W. Hollywood Bioenergetic aspects of aerobic glucose metabolism of scherichia coli K-12 under varying specific growth rates and glucose concentrations. Microbios 21: Hitchener, B. J., A. F. gan, and P. J. Rogers nergetics of microbacterium thermosphactum in glucose-limited continuous culture. AppI. nviron. Microbiol. 37: Marr, A. G.,. H. Nilson, and D. J. Clark The maintenance requirement of scherichia coli. Ann. N.Y. Acad. Sci. 12: Nagai, S., and S. Aiba Reassessment of maintenance and energy coupling in the growth of Azotobacter vinelandii. J. Gen. Microbiol. 73: Pirt, S. J The maintenance energy of bacteria in growing cultures. Proc. R. Soc. London Ser. B 163: Schulze, K. L., and R. S. Lipe Relationship between substrate concentration, growth rate, and respira-

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