Growth Rate, Biomass Production and Carbon Balance of Pseudomonas aeruginosa at ph Extremes in a Carbon-Limited Medium

Transcription

1 Jap. J. Limnol. 43, 4, , Growth Rate, Biomass Production and Carbon Balance of Pseudomonas aeruginosa at ph Extremes in a Carbon-Limited Medium Masayuki SET0 and Masahiro NODA Abstract 1. Introduction Microbes in an ecosystem act both as a decomposer of organic substances and as a producer of microbial biomass. The microbial biomass thus produced is fed by heterotrophic animals (MAGFADYEN, 1961; SETO and TAZAxr, 1971; TEZUKA, 1974), and in this respect, microbes occupy a pioneer niche in the detritus food chain (ODUM, 1962). Therefore, quantitative studies on the production of microbial biomass are essential for understanding the metabolism of an ecosystem. However, while many studies have been devoted to the role of microbes as decomposers, few have focussed on their role as producers, especially from a quantitative viewpoint. This may be attributable to the technological difficulties accompanying estimation of microbial biomass in situ. And these difficulties have made quantitative studies on the production of a microbial biomass in an ecosystem very difficult. Therefore, one approach would be to use a culture system in which the microbial biomass can be estimated with sufficient accuracy. Using a simple culture system, some quantitative studies from the physiological and biochemical viewpoints have been presented by M0N0D (1949), BAUCHOP and ELSDEN (1960), MAYBERRY et al. (1967) and PAYNE (1970) on the energy relationships between the substrate consumed and the bacterial biomass produced. However, an ecological approach was rarely used. Few of such studies dealt with the effects

2 264 Growth and Carbon Balance in a Bacterium of environmental factors such as temperature, osmotic pressure and ph on the production of microbial biomass. In a previous article (SETO and MISAWA, in press), it was shown that the efficiency of the biomass production and the balance of carbon of Pseudomonas aeruginosa in a glucose-limited medium were almost constant over a wide range of temperature and/or osmotic pressure of the medium. In the present study, using the same materials and methods as those mentioned above, the effects of the medium ph on the specific growth rate, the efficiency of the biomass production and the balance of carbon of the culture system were studied. 2. Materials and Methods The materials and methods were very similar to those used in a previous investigation (SETO and MISAWA, in press). Organism and culture method Pseudomonas aeruginosa was cultured aerobically at various ph values in a carbon-limited medium on a reciprocal shaker. The 1, 000 ml mineral solution of the carbon- limited medium consisted of the following: 3 mmol (NH4)2SO4, 0.1 mmol MgSO4.7H2O, 0.1 mmol CaC12.2H2O, 0.01 mmol MnCl2.4 H2O, 0.01 mmol H3B03, 0.01 mmol Nat Mo04.2H2O, mmol FeSO4.7H2O, trace CuSO4.5H2O, trace ZnSO4.7H2O, 0.1 mmol Na2SiOa and 30 ml 1M phosphate buffer solution. The phosphate buffer solution was made up using suitable ratios of KH2PO4 and Na2HPO4 to yield the various ph values. As the single substrate, 300 mg carbon of glucose, glutamate or glycerol was added to a 1, 000 ml mineral solution; and as the mixture of substrate, 100 mg carbon of glucose, glutamate and glycerol were added. The carbonlimited medium thus prepared was autoclaved at 120 C for 2.5 minutes except for the buffer solution. The buffer solution was autoclaved separately and added asceptically. As the seed source for the culture, Ps. aeruginosa was precultured twice in the same carbon-limited medium as that of the subsequent culture. Four ml of the bacterial suspension at the early stage of the stationary phase was inoculated into 200 ml of the carbon-limited medium in a 500 ml shaking flask. The temperature, osmotic pressure and ph of the medium for the preculture were 25 C, 2.9 bar and 7. 2, respectively. Extremely high or low ph change of the medium was effected both by changing the ratio of acid and basic phosphate in the mineral solution and by adding droplets of 1N KOH or 1N HCl solution. No precipitation was observed in the medium even at the highest medium ph. The change in the ph caused by metabolites excreted during culture was compensated by adding the 1N KOH or 1N HCl solution throughout the whole culture period. The measurement of the medium ph was conducted by using a glass electrode ph meter (Beckman-Toshiba, Lab-o-mate II). Determination of carbon amount The amount of biomass-c was calculated by subtracting the amount of the filtrate-c from that of the unfiltrate-c of the bacterial suspension. The amount of metabolite- C was calculated by subtracting the amount of residual substrate-c from that of the filtrate-c. The amount of respired C02-C was considered to be the difference between the organic carbon amounts in the medium before and after the culture. The organic carbon amount was determined by a wet combustion-ndir method (SETO, 1978) using 3.0 g potassium persulf ate and a modified oxidation vessel (SETO and TANGE, 1980). The amounts of residual glucose and glutamate were determined by the SOMOGYI-NELSON method referred to by Fuxul (1969) and by the COcKING-YEMM (1954) method, respectively. The amount of residual glycerol was not determined. When the glycerol was used as the substrate, the carbon balance in the early stage of the stationary phase was calculated on the assumption that all glycerol was consumed in this stage. The filtration of the culture medium was conducted using a membrane filter (Type, FM-45; pore size, 0.45 earn; Fuji Photo Film Co., Ltd.,

3 SETO arid NODA 265 Kyoto) which had been rinsed with doubledistilled water. The growth curve of the medium was plotted by measuring the optical density at 660 nm using a double-beam spectrophotometer (UV , Shimadzu Seisakusho Co., Ltd., Kyoto) and by referring to the standard curve relating to optical density and biomass-c To change the osmotic pressure of the medium, various amounts of sodium chloride were added. The mean of the two or three replicates was shown in the text. 3. Results The growth of Ps, aeruginosa in the glucose-limited medium was studied at various medium ph between 3.5 and 9.7. Some of the growth curves are shown in Fig. 1. In neutral, slightly alkaline and acid media (ph ), the growth curves were almost identical with each Fig. L The growth curves of Ps. aeruginosa in the glucose-limited media at various ph values. The numbers in the figure show the ph values of the media. The growth curves at ph between 6.2 and 7.6 were identical with that at 7.2. The culture temperature and osmotic pressure of the media were 25 C and 2.9 bar, respectively. other, and no lag times were observed. At ph above and below this range, longer lag times were conspicuous. At extremely high ph (9.4), no growth was observed during an incubation period of 68 hrs. When there were positive growths, exponential growth phases were observed, where the specific growth rates per hour were calculated. The relationships between the specific growth rates and ph values of the media was shown in Fig. 2 (A). Between ph 6.2 and 7. 6, the specific growth rates were almost constant, with a mean value of At ph above or below this range, the rate decreased linearly with the increase or decrease in ph, and no growth was observed at ph 3. 5, 3.8, 9.4 or 9.7. The balance of carbon of the culture system was determined in the early stage of the stationary phase. The relationship between the balances and ph values was shown in Fig. 2 (B). Between ph 5.6 and 8. 2, the balances were almost constant, i. e., 48% of the glucose-c consumed was produced as biomass-c, 7% was excreted as metabolite-c and 45% was respired as C02-C. At ph above or below this range, the amount of biomass-c decreased linearly with the increase or decrease in ph. On the contrary, the amount of C02-C respired increased remarkably with some increase in the amount of metabolite-c excreted. When there was positive growth, all glucose added was consumed completely. Therefore, the value for the amount of biomass-c in the early stage of the stationary phase was identical with the value for the efficiency of biomass production, expressed in terms of percentage of the ratio of biomass-c produced to glucose-c consumed. At ph 3.8 and 9.4, there was no positive growth, but there was still some glucose consumption. And 34% and 25% of added glucose had been lost, respectively, during an incubation period of 68 hrs. At ph 3.5 and 9. 7, no glucose was consumed. It is also noteworthy that the ph range, in which the balance of carbon was almost constant, was wider

4 266 Growth and Carbon Balance in a Bacterium Fig. 2. The specific growth rates per hour (A) of Ps. aeruginosa in the glucose-limited media, and the balances of carbon (B) of the culture system at various ph values. than the range in which the specific growth rate was almost constant. The studies cited thus far have indicated the growth of Ps. aeruginosa in the glucose-limited medium. The following describes the growth in the carbon-limited medium to which glutamate or glycerol was added as the single substrate. Growth was also studied in the medium in which a mixture of the three substrates was added. The growth curves of Ps, aeruginosa grown on various carbon sources at various ph had been ascertained to have almost the same pattern as those shown in Fig. 11 even in the media in which the mixture was added. Thus, in a certain ph range, growth curves were almost identical to each other with no appreciable lag times, and at ph above or below this range, longer lag times were conspicuous. When there was positive growth, the relationship between the specific growth rates and ph values of the media was shown in Fig. 3. In the figure, the relationship in the glucose-limited medium (Fig. 2 (A)) was also shown for reference. The relationship was the same as in the glucose-limited media in that at a certain ph range the specific growth rates were almost constant, and above or below this range, the rates decreased linearly with the increase or decrease in ph value; but the relationship was different in that both the specific growth rates and the ph ranges, in which the rates were constant, differed considerably among the media

5 SETO and NODA 267 Table 1. The balance of carbon of the culture system of Ps. aeruginosa grown on each or mixture of glucose, glutamate and glycerol at 25 C and 2.9 bar. containing different substrates. It is noteworthy that the wider ph range, in which the rate was constant, was obtained in the medium to which a mixture of the substrate was added. The balance of carbon in the early stage of the stationary phase, was shown in Table 1 of the culture system in which each or the mixture of the substrate was added. The balance of carbon in the glucose-limited medium (Fig. 2) was also shown for reference. The balance was almost the same among the media except for ph ranges with a constant balance. The widest ph range was obtained in the medium to which the mixture was added. When there was a positive growth, all glucose or glutamate added was consumed completely by the early stage of the stationary

6 268 Growth and Carbon Balance in a Bacterium Table 2. The effects of ph values on the specific growth rates and on the balances of carbon of the culture system of Ps. aeruginosa in the glucose-limited media at three different temperatures and two different osmotic pressures. 4. Discussion Some characteristics on the materials and methods used in the present study were discussed in a previous article (SETO and MISAWA, in press). One of the most conspicuous results in the present study is the constancy in the specific growth rate (Fig. 3) and in the balance of carbon (Table 1) over a wide ph range of the media. Moreover, a ph range, in which the constancy in a specific growth rate was observed, was narrower than the range, in which the constancy in the balance of carbon was observed. The specific growth rates in these ranges differed considerably among the media containing different substrates, whereas the balance of carbon was almost constant. This constancy was also observed in the efficiency of biomass-n production of Escherichia coli and in the balance of nitrogen over a wide ph range in a nitrogen-limited medium (SETO et al., 1979). HsUNG and HAUG (1975) observed that the intracellular ph of Thermoplasma acidophila grown at ph 2 was close to neutral (ph ) and that the cell could maintain the internal ph not by an energy-dependent process but by passive properties of the cell, suggesting a DQNNAN potential across the cell membrane. Cox et al. (1979) also observed that the intracellular ph of Thiobacillus ferro-

7 SETO arid NODA 269 oxidans was close to neutral (ph ) over a range of external ph from 1.0 to 8. 0, suggesting that the maintenance of the internal ph was not an energydependent process. In the light of these observations, the intracellular ph of growing Ps. aeruginosa cultured at various ph in the present study might be expected to be close to neutral. And the constancy in the biomass production may also suggest that the intracellular ph was maintained by an energy-independent process. Among the three substrates, the highest specific growth rate and the widest ph range, in which Ps. aeruginasa could grow, were observed when glutamate was the substrate (Fig. 3). The widest ph range, in which the constancy in the balance of carbon was observed, was also observed when glutamate was the substrate (Table 1). GALE (1951) observed that, when glutamic acid was consumed by E. coli, the chemical properties of the metabolite were affected by the ph of the medium, i. e., at a lower ph, the activity of decarboxylase increased to produce much j-aminobutyric acid and carbon dioxide, while at a higher ph, the activity of deaminase increased to produce cx-ketoglutarate and ammonium ion. These properties seem beneficial for E. coli to keep the intracellular ph constant over a wide range of external ph. If Ps aeruginosa had the same properties as E. coli, they seem also beneficial for Ps. aeruginosa to keep both the specific growth rate high (Fig. 3) and the balance of carbon constant (Table 1) over a wide range of external ph. The efficiency of biomass production of Ps. aeruginosa grown on glycerol (Table 1) was 46, expressed in terms of the percentage of the ratio of biomass-c produced to glycerol-c consumed. This efficiency, which was confirmed by repeated measurement, is quite small compared to the 59 for E. coli (SETO and TAZAKI, 1970) and the 61 calculated from "available electrons" (PAYNE, 1970). The reason for this low efficiency in the present study is not known. In the present study the residual amount of glycerol in the early stage of the stationary phase was not measured, and all glycerol was considered to have been consumed by this stage. Even if all the metabolites (6%, in Table 1) were residual glycerol, the efficiency would still be low at 49. Therefore, saving the measurement of residual glycerol does not account for the low efficiency. An exceptional property of a biochemical pathway in the bacterium used here might be a reason. The specific growth rate at slightly acid (ph 6.2) or alkaline (7.6) medium evidenced no difference with the one at close to neutral (7.2) medium at 25 C (Table 2). However, at 15 or 37 C, the ph 6.2 or 7.6 had some detrimental effects on the rate. In a previous study (SETO and MISAWA, in press), when Ps. aeruginosa was cultured both at various temperature and at varidous osmotic pressures, synergistic effects were observed. These results might show that the increase in temperature causes a decrease in the ph ranges where the bacterium could grow. When the mixture of three substrates was added, the ph range was widest, where the constancy in the balance of carbon was observed. Unpublished data (SETO and MISAWA) indicated that, when cultured at extremely high osmotic pressure (48.5 bar) in the glucose-limited medium, the specific growth rate of a pure culture of Ps. aeruginosa, E. coli or Bacillus megaterium was extremely small (0. 01, 0.03 or 0.06 per hour, respectively). And less than 50% of the glucose added was consumed during an incubation period of 160 hrs. On the other hand, the growth rate of the mixed population of these bacteria was relatively high at 0. 14, and 100% of the glucose added was consumed during an incubation period of 62 hrs. These results might suggest that the increase in the diversity of the substrate and species results in the increase in the ph range in which the bacteria could grow.

8 270 Growth and Carbon Balance in a Bacterium The addition of sodium chloride changes the osmotic pressure as well as the concentrations of sodium and chloride ions of the medium. Thus, the effect of the addition of sodium chloride is not necessarily the effect of osmotic pressure, but might be the effect of high concentrations of sodium and/or chloride ions. A preliminary study showed that the change in the specific growth rate of E. coli in the medium supplied with various amounts of sodium chloride was almost the same as the change in the medium supplied with various amounts of mannitol. However, since the organism in the present study was. Ps. aeruginosa and not E. coli, the effect of the addition of sodium chloride is still unsettled. Studies on the balance of materials using a simple culture system seem useful for the comprehension of microbes both as producer and decom poser. Nevertheless, many problems especially on the effect of environmental factors on the two roles of microbes remain unsolved. Acknowledgements We thank Prof. H. KURAISHI, Mr. MISAwA, Mr. YAMADA and other staff members of Tokyo Univ. of Agri. and Tech. for their helpful suggestions and discussion, and Assoc. Prof. T. U$HIJIMA and Miss H. YONEMITSU for their help in measuring the osmotic pressure. (in Japanese). GALE, E. F. (1951) : The Chemical Activities of Bacteria. Univ. Tutorial Press. London. HSUNG, J. C. and A. HAUG (1975) : Intracellular ph of Thermoplasma acidophila.eiochim. References BAUCHOP, T. and S. R. ELSDEN (1960) : The growth of micro-organisms in relation to their energy supply. J. gen. Microbiol., 23: COCKING, E. C. and E. W. YEMM (1954) : Estimation of amino acids by ninhydrin. Biochem. J., 58: 12. COX, J. C., D. G. NICHOLLS and W. J. INGLEDEW (1979) : Transmembrane electrical potential and transmembrane ph gradient in the acidophile Thiobacillus ferro-oxidans. Biochem. J., 178: FUKUI, S. (1969) : Quantitative Analysis of Reduced Sugars. Tokyo Univ. Press, Tokyo Biophys. Acta, 389: MACFADYEN, A. (1961) : Metabolism of soil invertebrates in relation to soil fertility. Ann. appl. Biol., 49: MAYBERRY, W. R., G. J. PROCHAZKA and W. J. PAYNE (1967) : Growth yields of bacteria on selected organic compounds. Appl. Microbiol., 15: MONOD, J, (1949) :The growth of bacterial cultures. Ann. Rev. Microbiol., 3: ODUM, E. P. (1962) : Relationships between structure and function in the ecosystem. Jap. J. Ecol., 12: PAYNE, W. J. (1970) : Energy yields and growth

9 SETO and NODA 271 of heterotrophs. Ann. Rev. Mierobiol., 24: SETO, M. and T. TAZAKI (1970) : Ecological studies on microbial growth. Balance of carbon and efficiency of yield in aerobic culture of Escherichia coli. Jap. J. Ecol., 20: SETO, M. and TAZAKI, T. (1971) : Carbon dynamics in the food chain system of glucose- Escherichia eoli-tetrahymena vorax. Jap. J. Ecol., 21: SETO, M. (1978) : Rapid and sensitive method for determination of total organic carbon by wet oxidation-nondispersive infrared gas analyzer. Japan Analyst, 27: (in Japanese). SETO, M., S. YAZAWA and T. TAZAKI (1979) Efficiency of biomass production and nitrogen balance of Escherichia coli in nitrogen-limited medium. Jap. J. Ecol., 29: SETO, M. and I. TANCE (1980) : Rapid and sensitive method for the determination of total organic carbon in soil by potassium persulfate-nondispersive infrared gas analyzer. J. Sci. Soil Manure, Japan,, 51: (in Japanese). SETO, M. and K. MISAWA (in press) : Growth rate, biomass production and carbon balance of Pseudomonas aeruginosa in a glucoselimited medium at temperature and osmotic pressure extremes. Jap. J Ecol. TEZUKA, Y. (1974) : An experimental study on the food chain among bacteria, Paramecium and Daphnia. Int. Rev. ges. Hydrobiol., 59: NODA, Fac. of Agri., Tokyo University of Agriculture and Technology, Fuchu City, Tokyo 183). Accepted: 9 August 1982