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Control of matric water potential by temperature differential
Journal of Microbiological Methods 6 (1987) 323- 326
323
Elsevier JMM 00214
Control of matric water potential by temperature differential R.J. Palmer Jr., J.A. Nienow and E.I. Friedmann Department of Biological Science, Florida State University, Tallahassee, FL (USA) (Received 19 December 1986) (Revised version received 16 April 1987) (Accepted 16 April 1987)
Summary A method for controlling relative humidity based on temperature differentials, rather than on salt solutions, is described. This method has the following advantages: (1) it does not exhibit the anomalous CO 2 solution effects that we have found to occur with salt solutions; (2) humidity is continuously adjustable without sample removal; (3) circulation of the atmosphere results in short equilibration times.
Key words: Matric water potential; Relative humidity; Water stress
Introduction The water potential of gaseous systems (matric water potential) is dependent upon the water vapor content (relative humidity) of the gas. In closed experimental systems, relative humidity is frequently controlled by equilibration over salt solutions [1], and this method has been used to study matric water stress in microorganisms (e.g., Refs. 2, 3). While performing experiments on photosynthetic 14CO2 uptake, we found that salt solutions interact with CO2, resulting in increased solubility and subsequent retention of this gas by solutions of high molality. Therefore we designed a system that circumvents this problem. Saturating the atmosphere at one temperature and subsequently pumping it to sample chambers held at an equal or higher temperature permits control of relative humidity by temperature difference.
Materials and Methods Absorption of CO 2 by salt solutions Freshly prepared NaC1 solutions (7.5 ml) of different molality were placed in 15 ml vials. The vials were capped with serum stoppers, and 50 tA of laCO2/air [4] Correspondence to: E.I. Friedmann, Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
0167-7012/87/$3.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
324 was injected into the h e a d s p a c e with a gas-tight syringe. A f t e r 24 h, the s e r u m s t o p p e r s were removed, a n d a 200/zl a l i q u o t o f salt s o l u t i o n was t a k e n f r o m each vial. T h e r e m a i n i n g s o l u t i o n was allowed to s t a n d u n c a p p e d a n d was s a m p l e d 24 a n d 48 h later to m e a s u r e the loss o f CO2 t h r o u g h diffusion. C O 2 a b s o r p t i o n in each a l i q u o t was d e t e r m i n e d by liquid scintillation s p e c t r o s c o p y [4] o n a P a c k a r d 2045 s c i n t i l l a t i o n counter.
Experimental system A d i a g r a m o f the system is shown in Fig. 1. A i r is p u m p e d t h r o u g h a s a t u r a t i n g c h a m b e r c o n t a i n i n g distilled water to e x p e r i m e n t a l c h a m b e r s held at a higher t e m p e r a t u r e . Temperatures in all c h a m b e r s are m a i n t a i n e d to w i t h i n 0.1 °C by m e a n s
,,~ K~ ,r
~
GLASS
~
TYGON
bF
.,e~]
A
RECORDER
~
AS VIEWED FROM ABOVE
. . . . . .
/
Fig. 1. 1, Vaisala humidity probe; 2, sample temperature (thermocouple); 3, bath temperature difference (differential thermocouple); A, water bath containing sample chambers and reference chamber; B, water bath (with styrofoam cover), maintains temperature of saturating bath; C, saturating bath; D, injection port closed by serum stopper; E - H , clamps. During equilibration clamps E and H are open, clamps F and G are closed, Air flow through chambers can be reversed by closing of clamps E and opening of clamps E To inject or withdraw gas, clamp H is closed and clamp G is opened.
325 of water baths. The atmosphere is circulated by a variable-speed peristaltic pump, and flow through the sample chambers can be reversed by opening and closing of appropriate clamps to provide uniform equilibration of samples. An injection port sealed by a rubber serum stopper allows introduction or withdrawal of gas during experiments. The temperature in an empty sample chamber (reference chamber) and the temperature difference between this chamber and the saturating water pool are continuously monitored by calibrated high-accuracy copper-constantan thermocouples (special-limit-of-error wire, Omega Engineering Inc., Stamford, CT). At equilibrium, the relative humidity in the sample chamber is equal to the quotient of the saturation vapor pressures of water at the two temperatures (Pbath/Psample). Values of saturation vapor pressures were obtained from the International Critical Tables [5]. Relative humidity in the reference chamber was monitored with a Humicap humidity metering system (Vaisala Corp., Helsinki, Finland). Results and Discussion
Measurements of 14CO2absorption and release by salt solutions (Table 1) show the discrepancy between solution of CO2 in NaC1 solutions and that in distilled water. The slow loss of radioactivity from one- and four-molal salt solutions, compared to that from distilled water and 0.25 molal-salt solutions suggests a chemical interaction between the CO2 and the salt solution. Therefore, because the concentration of salt in solution affects CO2 levels, salt solutions should not be used to control humidity in experiments involving photosynthetic organisms. In the system described, the range of relative humidities is limited by the temperatures of the saturating bath and the sample chambers. Using water as coolant in both baths, relative humidities from 26°70 to saturation (matric potentials from -183 000 to 0 kPa) are obtainable at an experimental chamber temperature of 20 °C. These values have been confirmed by the relative humidity probe, which has a reported accuracy of _+2070, constant over the entire range of humidity and temperature. The calculated accuracy of the system (based on measured temperature differentials) is + 1070at 98°70 RH (20°C). Accuracy increases with decreasing water
TABLE 1 EFFECT OF NaCI CONCENTRATION ON C O 2 SOLUBILITY 14CO2 is injected into vial headspace. Serum stopper remains in place during equilibration period and is subsequently removed to monitor diffusional loss. NaC1 concentration (molal)
0 0.5 2 4
% injected c p m i n solution after: 24 h equilibration
24 b equilibration 24 h diffusional loss
24 h equilibration 48 h diffusional loss
59,5 69.0 63,2 78,2
28.6 46.0 55.0 67.9
0 (background) trace ( < 2 × background) 6.6 48,5
326 p o t e n t i a l b e c a u s e o f the larger t e m p e r a t u r e differentials required. Therefore, once e q u i l i b r a t i o n times o f s a m p l e m a t e r i a l s have been established, the relative h u m i d i t y p r o b e b e c o m e s unnecessary. I f desired, h u m i d i t y c a n be varied over the course o f an e x p e r i m e n t w i t h o u t removal o f s a m p l e s b e c a u s e b a t h t e m p e r a t u r e s are c o n t i n u o u s l y adjustable. C i r c u l a t i o n o f the a t m o s p h e r e by the peristaltic p u m p results in s h o r t e r e q u i l i b r a t i o n times t h a n t h o s e in s t a g n a n t systems. W i t h o u r current e q u i p m e n t , flow rates c a n be a d j u s t e d f r o m 3 to 300 m l / m i n . In a t y p i c a l e x p e r i m e n t in which six s a m p l e c h a m b e r s a n d one reference c h a m b e r (50 ml each) are used, a m o d e r a t e flow rate o f 75 m l / m i n results in a t u r n o v e r t i m e o f a b o u t 7 rain. E q u i l i b r a t i o n times v a r y with the t y p e o f s a m p l e b u t are generally r a p i d even if wet m a t e r i a l is used. T h e system d e s c r i b e d here, rather t h a n salt solutions, is r e c o m m e n d e d for use in e x p e r i m e n t s requiring c o n s t a n t C O 2 levels a n d accurate c o n t r o l o f water potential. T h e versatility o f this m e t h o d can be seen in its varied a p p l i c a t i o n s . We have used the system to rehydrate d r y lichen m a t e r i a l p r i o r to 14CO2 u p t a k e studies [6]. E q u i l i b r a t i o n times o f d r y lichens to 99°70 relative h u m i d i t y (based on c o n s t a n t weight) were less t h a n 24 h in o u r system, whereas in s t a g n a n t systems several days m a y be required [7]. We also investigated the effect o f m a t r i c water stress o n 14CO2 i n c o r p o r a t i o n by the c r y p t o e n d o l i t h i c m i c r o b i a l c o m m u n i t y o f A n t a r c t i c a ( p a p e r in p r e p a r a t i o n ) . In these experiments, small pieces o f c o l o n i z e d s a n d s t o n e were inserted into the e x p e r i m e n t a l c h a m b e r s ; the t e m p e r a t u r e within the c h a m b e r s was m a i n t a i n e d at 8 °C. Also, m i c r o o r g a n i s m s f r o m liquid culture can be collected on m e m b r a n e filters a n d t r a n s f e r r e d to the c h a m b e r s . In this manner, we have e x a m i n e d the r e l a t i o n s h i p o f water stress to p h o t o s y n t h e s i s in a lichen p h y c o b i o n t (Trebouxia sp.) a n d in a c r y p t o e n d o l i t h i c c y a n o b a c t e r i u m (Chroococcidiopsis sp.) f r o m the Negev desert, Israel.
Acknowledgements This w o r k was s u p p o r t e d by N A S A grant 7337 a n d N S F grant D P P 83-14180 to E.I.E
References 1 Winston, P. W. and Bates, D.H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-237. 2 Potts, M. and Friedmann, E. I. (1981) Effects of water stress on cryptoendolithic cyanobacteria from hot desert rocks. Arch. Microbiol. 130, 267-271. 3 Potts, M., Bowman, M.A. and Morrison, N.S. (1984) Control of matric water potential in immobilised cultures of cyanobacteria. FEMS Microbiol. Lett. 24, 193 - 196. 4 Belly, R. and Brock, T.D. (1967) Ecology of iron-oxidizing bacteria in pyritic materials associated with coal. J. Bacteriol. 117, 726-732. 5 Washburn, E.W. (1928) The vapor pressures of water and ice up to 100 °C. In: International Critical Tables of Numerical Data, Physics, Chemistry and Technology 1928 (Washburn, E.W., editor-inchief) Vol. Ill, pp. 210-212, McGraw Hill, New York. 6 Palmer, R.J., Jr. and Friedmann, E.I. (1986) Effects of lowered water potential on photosynthesis in lichens and endolithic cyanobacteria from the Negev desert. Fourth International Symposium on Microbial Ecology, Ljubljana, Abstracts. p. 44. 7 Blum, O.B. (1973) Water Relations. In: The Lichens, 1973 (Ahmadjian, V., and Hale, M.E., eds.) pp. 381-400, Academic Press, New York.
323
Elsevier JMM 00214
Control of matric water potential by temperature differential R.J. Palmer Jr., J.A. Nienow and E.I. Friedmann Department of Biological Science, Florida State University, Tallahassee, FL (USA) (Received 19 December 1986) (Revised version received 16 April 1987) (Accepted 16 April 1987)
Summary A method for controlling relative humidity based on temperature differentials, rather than on salt solutions, is described. This method has the following advantages: (1) it does not exhibit the anomalous CO 2 solution effects that we have found to occur with salt solutions; (2) humidity is continuously adjustable without sample removal; (3) circulation of the atmosphere results in short equilibration times.
Key words: Matric water potential; Relative humidity; Water stress
Introduction The water potential of gaseous systems (matric water potential) is dependent upon the water vapor content (relative humidity) of the gas. In closed experimental systems, relative humidity is frequently controlled by equilibration over salt solutions [1], and this method has been used to study matric water stress in microorganisms (e.g., Refs. 2, 3). While performing experiments on photosynthetic 14CO2 uptake, we found that salt solutions interact with CO2, resulting in increased solubility and subsequent retention of this gas by solutions of high molality. Therefore we designed a system that circumvents this problem. Saturating the atmosphere at one temperature and subsequently pumping it to sample chambers held at an equal or higher temperature permits control of relative humidity by temperature difference.
Materials and Methods Absorption of CO 2 by salt solutions Freshly prepared NaC1 solutions (7.5 ml) of different molality were placed in 15 ml vials. The vials were capped with serum stoppers, and 50 tA of laCO2/air [4] Correspondence to: E.I. Friedmann, Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
0167-7012/87/$3.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
324 was injected into the h e a d s p a c e with a gas-tight syringe. A f t e r 24 h, the s e r u m s t o p p e r s were removed, a n d a 200/zl a l i q u o t o f salt s o l u t i o n was t a k e n f r o m each vial. T h e r e m a i n i n g s o l u t i o n was allowed to s t a n d u n c a p p e d a n d was s a m p l e d 24 a n d 48 h later to m e a s u r e the loss o f CO2 t h r o u g h diffusion. C O 2 a b s o r p t i o n in each a l i q u o t was d e t e r m i n e d by liquid scintillation s p e c t r o s c o p y [4] o n a P a c k a r d 2045 s c i n t i l l a t i o n counter.
Experimental system A d i a g r a m o f the system is shown in Fig. 1. A i r is p u m p e d t h r o u g h a s a t u r a t i n g c h a m b e r c o n t a i n i n g distilled water to e x p e r i m e n t a l c h a m b e r s held at a higher t e m p e r a t u r e . Temperatures in all c h a m b e r s are m a i n t a i n e d to w i t h i n 0.1 °C by m e a n s
,,~ K~ ,r
~
GLASS
~
TYGON
bF
.,e~]
A
RECORDER
~
AS VIEWED FROM ABOVE
. . . . . .
/
Fig. 1. 1, Vaisala humidity probe; 2, sample temperature (thermocouple); 3, bath temperature difference (differential thermocouple); A, water bath containing sample chambers and reference chamber; B, water bath (with styrofoam cover), maintains temperature of saturating bath; C, saturating bath; D, injection port closed by serum stopper; E - H , clamps. During equilibration clamps E and H are open, clamps F and G are closed, Air flow through chambers can be reversed by closing of clamps E and opening of clamps E To inject or withdraw gas, clamp H is closed and clamp G is opened.
325 of water baths. The atmosphere is circulated by a variable-speed peristaltic pump, and flow through the sample chambers can be reversed by opening and closing of appropriate clamps to provide uniform equilibration of samples. An injection port sealed by a rubber serum stopper allows introduction or withdrawal of gas during experiments. The temperature in an empty sample chamber (reference chamber) and the temperature difference between this chamber and the saturating water pool are continuously monitored by calibrated high-accuracy copper-constantan thermocouples (special-limit-of-error wire, Omega Engineering Inc., Stamford, CT). At equilibrium, the relative humidity in the sample chamber is equal to the quotient of the saturation vapor pressures of water at the two temperatures (Pbath/Psample). Values of saturation vapor pressures were obtained from the International Critical Tables [5]. Relative humidity in the reference chamber was monitored with a Humicap humidity metering system (Vaisala Corp., Helsinki, Finland). Results and Discussion
Measurements of 14CO2absorption and release by salt solutions (Table 1) show the discrepancy between solution of CO2 in NaC1 solutions and that in distilled water. The slow loss of radioactivity from one- and four-molal salt solutions, compared to that from distilled water and 0.25 molal-salt solutions suggests a chemical interaction between the CO2 and the salt solution. Therefore, because the concentration of salt in solution affects CO2 levels, salt solutions should not be used to control humidity in experiments involving photosynthetic organisms. In the system described, the range of relative humidities is limited by the temperatures of the saturating bath and the sample chambers. Using water as coolant in both baths, relative humidities from 26°70 to saturation (matric potentials from -183 000 to 0 kPa) are obtainable at an experimental chamber temperature of 20 °C. These values have been confirmed by the relative humidity probe, which has a reported accuracy of _+2070, constant over the entire range of humidity and temperature. The calculated accuracy of the system (based on measured temperature differentials) is + 1070at 98°70 RH (20°C). Accuracy increases with decreasing water
TABLE 1 EFFECT OF NaCI CONCENTRATION ON C O 2 SOLUBILITY 14CO2 is injected into vial headspace. Serum stopper remains in place during equilibration period and is subsequently removed to monitor diffusional loss. NaC1 concentration (molal)
0 0.5 2 4
% injected c p m i n solution after: 24 h equilibration
24 b equilibration 24 h diffusional loss
24 h equilibration 48 h diffusional loss
59,5 69.0 63,2 78,2
28.6 46.0 55.0 67.9
0 (background) trace ( < 2 × background) 6.6 48,5
326 p o t e n t i a l b e c a u s e o f the larger t e m p e r a t u r e differentials required. Therefore, once e q u i l i b r a t i o n times o f s a m p l e m a t e r i a l s have been established, the relative h u m i d i t y p r o b e b e c o m e s unnecessary. I f desired, h u m i d i t y c a n be varied over the course o f an e x p e r i m e n t w i t h o u t removal o f s a m p l e s b e c a u s e b a t h t e m p e r a t u r e s are c o n t i n u o u s l y adjustable. C i r c u l a t i o n o f the a t m o s p h e r e by the peristaltic p u m p results in s h o r t e r e q u i l i b r a t i o n times t h a n t h o s e in s t a g n a n t systems. W i t h o u r current e q u i p m e n t , flow rates c a n be a d j u s t e d f r o m 3 to 300 m l / m i n . In a t y p i c a l e x p e r i m e n t in which six s a m p l e c h a m b e r s a n d one reference c h a m b e r (50 ml each) are used, a m o d e r a t e flow rate o f 75 m l / m i n results in a t u r n o v e r t i m e o f a b o u t 7 rain. E q u i l i b r a t i o n times v a r y with the t y p e o f s a m p l e b u t are generally r a p i d even if wet m a t e r i a l is used. T h e system d e s c r i b e d here, rather t h a n salt solutions, is r e c o m m e n d e d for use in e x p e r i m e n t s requiring c o n s t a n t C O 2 levels a n d accurate c o n t r o l o f water potential. T h e versatility o f this m e t h o d can be seen in its varied a p p l i c a t i o n s . We have used the system to rehydrate d r y lichen m a t e r i a l p r i o r to 14CO2 u p t a k e studies [6]. E q u i l i b r a t i o n times o f d r y lichens to 99°70 relative h u m i d i t y (based on c o n s t a n t weight) were less t h a n 24 h in o u r system, whereas in s t a g n a n t systems several days m a y be required [7]. We also investigated the effect o f m a t r i c water stress o n 14CO2 i n c o r p o r a t i o n by the c r y p t o e n d o l i t h i c m i c r o b i a l c o m m u n i t y o f A n t a r c t i c a ( p a p e r in p r e p a r a t i o n ) . In these experiments, small pieces o f c o l o n i z e d s a n d s t o n e were inserted into the e x p e r i m e n t a l c h a m b e r s ; the t e m p e r a t u r e within the c h a m b e r s was m a i n t a i n e d at 8 °C. Also, m i c r o o r g a n i s m s f r o m liquid culture can be collected on m e m b r a n e filters a n d t r a n s f e r r e d to the c h a m b e r s . In this manner, we have e x a m i n e d the r e l a t i o n s h i p o f water stress to p h o t o s y n t h e s i s in a lichen p h y c o b i o n t (Trebouxia sp.) a n d in a c r y p t o e n d o l i t h i c c y a n o b a c t e r i u m (Chroococcidiopsis sp.) f r o m the Negev desert, Israel.
Acknowledgements This w o r k was s u p p o r t e d by N A S A grant 7337 a n d N S F grant D P P 83-14180 to E.I.E
References 1 Winston, P. W. and Bates, D.H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-237. 2 Potts, M. and Friedmann, E. I. (1981) Effects of water stress on cryptoendolithic cyanobacteria from hot desert rocks. Arch. Microbiol. 130, 267-271. 3 Potts, M., Bowman, M.A. and Morrison, N.S. (1984) Control of matric water potential in immobilised cultures of cyanobacteria. FEMS Microbiol. Lett. 24, 193 - 196. 4 Belly, R. and Brock, T.D. (1967) Ecology of iron-oxidizing bacteria in pyritic materials associated with coal. J. Bacteriol. 117, 726-732. 5 Washburn, E.W. (1928) The vapor pressures of water and ice up to 100 °C. In: International Critical Tables of Numerical Data, Physics, Chemistry and Technology 1928 (Washburn, E.W., editor-inchief) Vol. Ill, pp. 210-212, McGraw Hill, New York. 6 Palmer, R.J., Jr. and Friedmann, E.I. (1986) Effects of lowered water potential on photosynthesis in lichens and endolithic cyanobacteria from the Negev desert. Fourth International Symposium on Microbial Ecology, Ljubljana, Abstracts. p. 44. 7 Blum, O.B. (1973) Water Relations. In: The Lichens, 1973 (Ahmadjian, V., and Hale, M.E., eds.) pp. 381-400, Academic Press, New York.