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Jap. J. Limnol. 43, 4, 263-271, 1982.
Growth Rate, Biomass Production and Carbon Balance of
Pseudomonas aeruginosa at pH Extremes in a
Carbon-Limited Medium
Masayuki SET0 and Masahiro NODA
Abstract
The aerobic growth of a baderium (Psmdomoms aeruginosa) in a carbon-limited medium was
studied from an ecological viewpoint with special reference to the effect of the medium pH.
(1) Ps. aeruginosa was cultured at pH 7.2 in a glucose-limited medium (glucose-C, 300 mg・l-1;
culture temperature,25℃;osmotic pressure,2.9 bar). The specific growth rate was 0.44・hr-1 at
ｔhe exponential phase. The efficiency of biomass production(biomass-C produced/glucose-C consumed)
was 0.48 in the early stage of the stationary phase. The balance of carbon at this stage was as
follows;48% of the glucose-C consumed was produced as biomass-C,7% was excreted as metabolite-
Cand 45% was respired as CO2-C.
(2).Ps, aeruginQSa was cultured at pH between 3.5 and 9.7 in the glucose-limited medium. The
specific growth rates at pH betweenl 6.2and 7.6, and the efficiencies of biomass production and the
balances of carbon between 5.6and 8.2 were almost constant, showing the same values as those
mentioned above. At pH above or below these ranges, the rate and the effciency decreased, and the
respired CO2-C increased with some increases in metabolite-C. Biomass production was not observed
at pH 3.8 or 9.4, although some glucose consumption was observed.
(3)The constant efficiency of the biomass production at between pH 5.6 and 8.2 suggests that
intracellular pH might be maintained by an energy-independent process in this pH range, On the
contrary, the decrease in the efficiency above ar below this pH range indicates that intracellular pH
might be maintained by an energy-dependent process.
(4) The pH range, in which either constant efficiency of biomass production or specific growth
rate at 25℃ was observed, decreased when culture temperature decreased(15℃)or increased(37℃).
On the contrary, this pH range increased when a mixed substrate was added or a mixed bacterial
population was cultured.
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 decom-
posers, 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 quantita-tive 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 relation-ships between the substrate consumed and the bacterial biomass produced. However, an ecological approach was rarely used. Few of such studies dealt with the effects
264 Growth and Carbon Balance in a Bacterium
of environmental factors such as tempera-ture, 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 con-stant over a wide range of temperature and/or osmotic pressure of the medium. In the present study, using the same materi-als 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 inves-tigation (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, 0.002 mmol FeSO4.7H2O, trace CuSO4.5H2O, trace ZnSO4.7H2O, 0.1 mmol Na2SiOa and 30 ml 1M phosphate buffer solution. The phos-
phate 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, gluta-mate or glycerol was added to a 1, 000 ml mineral solution; and as the mixture of substrate, 100 mg carbon of glucose, gluta-mate and glycerol were added. The carbon-limited medium thus prepared was auto-claved 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 bac-
terial 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 con-ducted 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 bacte-rial 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 sub-strate, 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.,
SETO arid NODA 265
Kyoto) which had been rinsed with double-distilled water.
The growth curve of the medium was
plotted by measuring the optical density at 660 nm using a double-beam spectropho-tometer (UV-140-02, 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 chlo-ride 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 6.2-7.6), 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, ex-
ponential 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 0.44. At pH above or below this range, the rate decreased linear-ly 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 station-ary 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, re-spectively, 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
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, aeru-
ginosa 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 identi-
cal 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 rela-tionship in the glucose-limited medium
(Fig. 2 (A)) was also shown for refer-ence. 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
SETO and NODA 267
Fig.3. The speci薮c growth rates of Ps. mm8imsa at various pH values grown on
91utanユate (①),91ucose (C・), 91ycero1 (㊦)or mixture of these colnpounds(●).
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 note-worthy 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 com-
pletely by the early stage of the stationary
phase. Although residual glycerol was not
measured, its amount if any will be
insignificant in this stage.
Studies so far have demonstrated growth
of Ps. aemginosa in the carbon・limited
medium at a definite temperature (25ｰC)
and at a definite osmotic pressure {2.9
bar). The fallowing describes pH effects
an the specific growth rates and carbon
balances of Ps. aerκginosa at three different
temperatures and two osmotic pt essures.
As shown in fiable 2, when Ps. aeYUginosa
was cultured at 25ｰC, the specific growth
rakes and the balallces of carbon were
almost invariable at two osmotic pressures,
and pK seems to have had na effect on
the rate anti the haiance except at pH 7.2.
At pH 7.2, some increases in the amounts
of both biomass and metabolites wαe
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.
observed.
When cultured at low temperature(150C),
the specific gr◎wth rates at pH 6.2 and
7.6were lower than those at pH 7.2.
High osmotic pressure may have some
detrimental effects an the rate. Still the
balance was almost constant except both
at a high pH (7.6) and high osmotic
pressure(12 bard, where some increase in
the biomass amount was observed. With
a high temperature (37。C), the results
were almost the same as when cultured
at law temperature except with a law pH.
At the low pH, no detrimental effect in
the rate was observed at high osmotic
pressure. Increase in the amount of
biomass-C was also observed at high
osmotic pressure, with the increase in the
amount of metabolites and decrease of
CO2.
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 dif-fered considerably among the media con-taining 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 nitro-
gen-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 6.4-6.9) 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 mem-brane. Cox et al. (1979) also observed that the intracellular pH of Thiobacillus ferro-
SETO arid NODA 269
oxidans was close to neutral (pH 6.0-7.0) over a range of external pH from 1.0 to 8. 0, suggesting that the maintenance of the internal pH was not an energy-dependent 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 proc-ess. 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 me-tabolite 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 aerugino-sa 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 pro-duced to glycerol-C consumed. This effi-
ciency, 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 mea-sured, 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 excep-tional 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 evi-denced 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, syn-ergistic 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 press-ure (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.
270 Growth and Carbon Balance in a Bacterium
The addition of sodium chloride changes the osmotic pressure as well as the con-centrations of sodium and chloride ions of the medium. Thus, the effect of the addi-tion 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.
摘 要
炭素制限培地における緑膿菌(Pseudomonas aeru-
ginosa)の 好気的な成長を,と くに培地のpHの 影
響 を中心 に,生 態学的な視点か ら調べた.
(1) Ps. aeruginosaを グル コースを唯一の炭 素
源 とした培地 を用いてpH 7.2で 培養 した.こ の と
き,グ ルコー スの炭素量 は300mg/l,温 度 は25℃ そ
して浸透庄 は2.9barで あ った.対 数期の比増殖速
度は0.44/時 であった.菌 体生産効率 (消費 した グ
ル コースに対す る生産 された菌体の炭素量の比)は 定
常期 の初期でほぼ0.48で あ った.こ のときの炭素収
支 は消費 した グル コースの48%が 菌体 とな り,7%
が 代謝産物 として排泄 され,そ して45%が 二酸化炭
素 として放 出 された.
(2) Ps.aeruginasaをpH3.5か ら9.7の グ
ル コース制限培地で培養す ると,比 増殖速 度 はpH
6.2か ら 7.6,菌 体生産効率 と炭素収支 はpH5.6
か ら8。2の 範囲ではほぼ一定で,上 記のおのお の と
同じであ った。これ らの範囲よ り高いある い は 低 い
pHで は,比 増殖速度 と菌体生産効率は減少 し,二 酸
化炭素の放 出量 と代 謝座物の 排泄量は増加 した・pH
3.8あ るいは9.4で は,菌 体量の増加は認 められなか
ったが,グ ル コースの消費は認 め られた.
(3)培 地のpHが5.6か ら8.2の 範囲では菌体
生産効率は一定 であった.こ の ことは菌体 内 のpH
の調節は,培 地 のpHが この範囲では,エ ネルギーに
依存 しない過程 であることを示唆す る.こ れに対 して,
培地のpHが この範囲 よ り高 いあるいは低 い と き,
菌体生産効率 は減少 した.こ の ときの菌体内のpHの
調節 はエネルギーに依存す る過程であ ることを示唆す
る.
(4)培 養iの温度 と浸 透 圧 が 高 くな る と,・Ps.
aemgimsα が生育で きる培地のpHの 範 甥が減少す
ると考察 した.こ れ に対 して,基 質 と種組成の多様性
が増加す ると,こ のpHの 範 囲が増大す ると考 察 し
た.
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(著 者:瀬 戸 昌之 ・野 田 呂弘,東 京 農 工 大 学 農 学部,
東 京 都 府 中市 幸町;Masayuki SETO and Masahiro
NODA, Fac. of Agri., Tokyo University of Agri-culture and Technology, Fuchu City, Tokyo 183).
Accepted: 9 August 1982
