World
Journal
of Microbiology
Polyol grown
and Biotechnology
9, 579-682
concentrations in Aspergillus under salt stress
repens
U.P. Kelavkar and H.S. Chhatpar* Na + , K + and the ratio of Na ’ /K + were higher in cells of the halotolerant Asperghs repens grown with 2 M NaCl than without NaCl. The osmolytes, proline, glycerol, betaine and glutamate, did not affect the Na + IK + ratio, nor the polyol content of cells under any conditions. The concentrations of polyols, consisting of glycerol, arabitol, erythritol and mannitol, changed markedly during growth, indicating that they have a crucial role in osmotic adaptation.
Key words: Aspergilltisrepens,
halotolerant, osmolytes, polyols, salt stress.
Microbes within extreme environments have developed various strategies to combat the stressesto which they are exposed. The ability to adapt to ~uc~ations in external osmolarity and the mechanism of osmoregulation have been elucidated in some organisms (Brown 1978; Ben-Amotz & Avron 1983; Csonka 1989), though little info~ation is available in fungi. Studies on marine fungi have tended to concentrate on their salinity requirements and some aspects of their physiology and biochemistry (Jennings 1983; Kelavkar et a!. 1993). Aspergilhs repens is tolerant up to 2 M NaCl but grows in medium without NaCl and thus is termed halotolerant. In this work, we describe the dynamic process of adaptation of Aspergillus repens under salt stress and characterize the specific events involved in the process of adaptation at the cellular level. It has been proposed that
polyols in living organisms act as carbohydrate reserves, sites for storage of reducing power and translocatory compounds,
and have
functions
in osmoregulation
and
coenzyme regulation (Lewis & Smith 1967; Spencer & Spencer 1978; Gadd et al. 1984; Jennings 1984). In this paper we report the occurrence and possible role of polyols in NaCl-tolerance in the halotoler~t ~pergi~~~ repens.
Materials Organism,
and Methods Culfure
Conditions
and Medium
The fungus Aspergillw repens, a salt-pan isolate, was grown in medium containing (g/I): asparagine, 10; (NH&SO,, 3.5; KH,PO,, U.P. Kelavkar and H.S. Chhatpar are with the Department of Microbiology and Biotechnology Centre, Faculty of Science, MS. University of Baroda, Baroda 390 002, India; fax: 91 265 336653. *Corresponding author. @I lg%? Rapid Communications
of Oxford
Ltd
10; MgSO, .7H,O, 2.0; sucrose, 85; CaCI,, 0.075; ZnSO, .5H,O, 0.01; MnCI,, 0.005; (NH,),Mo,O,, .4H,O, 0.002; Na,B,O, 0.002; and FeSO,. 7&O, 0.002. The pH was adjusted to 5.5 and then spores (lO’/ml) were added to 50ml medium in a 250-ml Erlenmeyer flask and shaken on a gyrotary shaker (180 rev/min) at 30 + 2% The mycelia were harvested by washing, until free of medium, under reduced pressure. Dry
weight
and Water
Content
Measurement
The procedure for dry weight and water content measurement was a modification of that described by Bidochka et al. (1990). Suction was applied to the washed mycelia for 5 min and the myceiial mat was weighed to give the wet wt. The mat was then dried first at 50°C in an oven and then in a desiccator to constant weight (dry wt). Subtracting dry wt from wet wt gave the water content of the mycelia.
Fungal mycelia were washed at 4°C with 2.5 M CaCl, to remove loosely associated extracellular ions. They were then held at 500’C for 4 h and then dissolved in 6 M HCI, prepared with de-ionized water, and then autoclaved at 121°C for 1 h. The ion contents in the supematant solutions were analysed with a flame photometer (Evans Electroselenium, UK), with independent blanks and appropriate standards. Polyol Extraction and Determination The procedures for polyol extraction with trichloroacetic acid and the determination of total polyols by the periodate method were as previously described (Adler & Gustafsson 1980). The different polyols were analysed by isocratic HPLC with a Ca’ + -based cation exchange column at 85”C, with de-ionized (de-gassed) water as a mobile phase at a flow rate of 1 mlimin and a pressure of 8106 W/m’. Independent standards of known concentrations (25 mg/ml) were run to obtain their retention times and peak areas. The concentrations of polyols in the samples were determined by comparing their peak areas with those of standards.
U.P. klavkar land H.S. Chhafpar ~~li~~ia~
under control conditions (data not shown). The mechanism by which the osmolytes brought about such an increase in growth in our system is not understood but they could either suppress the uptake of Na + and K + or enhance the leakage of accumulated Na ’ and K + . Changes in the ratio Na’/K+ would influence growth but there was no significant change in the Na $-/K + ratio, in either control or in stressed cells, when osmolytes were added. The osmoiytes were therefore not acting by changing the Na+/KS ratios, but might have increased the osmoticum of the cell (Table 2). Since high concentrations of salt are generally inhibitory to metabolic unctions, various kinds of compensatory mechanisms are found among salt-toIerant organisms (Brown 1978: Kushner 1978; Adler & Guslafsson 1980; Csonka 1989; Kelavkar et al. 1993). When Aspergillw repens was grown under salt stress, polyols accumulated significantly (Table 3). Glycerol, arabitol, mannitol and erythritol were detected, and their contents changed markedly with time (Table 4). However, in our earlier findings, anafyses of total free amino acids in AspergiIiz repensunder stressrevealed that khe pool of total free amino acids was smaller at TZ h than at 144 h of growth, and was always higher than that in unstressed controls (Kelavkar & Chhatpar 1992). The concentration of polyols at 72 h was I&fold higher in the stressed cultures than thak in the controls, demons~at~ng that amino acids are rapidIy used up by growth at 72 h and the pdyots are switched over to salinity control systems (Table 3). After 144 h growth, the polyol concentration decreased (Table 3) as the contents of total free amino acids increased (Kelavkar & Chhatpar 1992), suggesting that, after the polyols, the amino acids switch over to a salinity-control system. The decrease in polyols after 144 h may be due to their use as an endogenous carbon source or as storage carbohydrates at that stage, S~risingly, when proline, glycerol, betaine and glutamate were added to the growth medium there was no significant decrease in the accumulation of total polyols (Table 3) in either stressed or unstressed cultures but the concentrations of individual polyols changed (Table 4).
of ~pe~men~5
All the experiments otherwise.
Results
were
repeated
at least five times
unless
stated
and Discussion
repens was haiotoler~t and showed considerable growth in the presence of 2 M NaCl (Figure I). When it was grown in medium supplemented with 2 M NaCl (referred to as ‘stress’) the intracellular concentrations of Na + and K + and the ratio Na + IK + increased compared with control values in NaCI-free medium (Table I). Gadd ef at. (1984 found a similar increase in Na + iK + ratio in P~i~llium ochro~c~loron under salt stress. When the osmoiytes proline, glutamate, betaine and glycerol were added at 10 mM to the growth medium, there was a significant increase in growth under stress but not ~pergi~l~
0
72
96
120
144
Incubation
t68
192
(h)
Figure 1. Growth of Aspergillus repens in 2%ml shaken flask cultures at 30% in W-ml volumes of medium without NaGI (a), or with 1 (II), 2 (A,) or 3 (‘l) t!~ NaCI. Cell dry wt was determined by drying the mycelia at 50°C to a constant weight.
Teble
1. Concentratfons
Growth
of Na+ and K+ in
Cell water @t/m@ dry wt)
(h)
Aspergi&s (mg
repensgrown
In control
medium
My~l~urn dry wtfml)
Mycellat
(f&Cl-Free) Ion contents
and stress (nmol~mg
Control Control 72 96 144 168 * Values
0.9 + 0.05 1.2 & 0.06 2.4 +O.lO 2.5&O.lT are
means
Stress 0.3 0.4 0.7 0.7
* 0.02 f 0.01 f 0.04 kO.05
j, standard
Control
Stress
3.6 & 0.07 6.8+0.20 t3.0 t 1.10 I3.4& 1.20 deviations
of five
2.5 4.4 7.0 7.3
+ + & f
0.22 0.41 0.10 0.10
to seven
Na+ 3.0 f 0.1 3.2 +0.3 2.81 0.1 2.240.2 independent
K+ 43.7 44.3 42.6 42.3
io.1 & 2.f sf: 2.2 t3.3
experiments.
medium
(2 M NaCi).*
dry wt) Stress
Na+lK+ 0.07 0.07 0.06 0.05
Ha+ 32.9 43.4 648 63.2
i; rt + *
K+ 2.2 2.7 3.1 2.7
190.0 199.8 195.2 190.9
+ + f &
Na+lK* 8.1 9.2 9.2 7.6
0.17 0.21 0.33 0.33
Polyol concenfrafionsin Aspergillus repens Table 2. Concentrations with 10 mu osmolytes.” Osmolyte (at lOmu)
of Na+
and K+
In Asjrergillus
repens
grown
Mycetfal
Growth (h)
in control
ion contents
(NaCI-free)
(nmolimg
and stress
(2 M NaCI)
media
dry wt)
Control
Stress
K+
72 144
2.9 * 0.1 2.2 + 0.3
44.6 ) 2.3 31.4 & 1.2
0.065
31.0 + 1.4
193.8
_+ 8.6
0.16
0.075
Proline
72 144
3.0 * 0.2 2.7 + 0.4
42.8 * 2.2 45.0 + 2.2
0.07 0.06
64.1 & 2.4 32.3 F 1.7
200.0 190.0
* 6.3 k 8.4
0.32 0.17
Glycerol
72
3.1 + 0.1
186.0 203.7
+ 9.6 k 9.3
0.34 0.16
2.6 + 0.2 2.9 + 0.1 2.7 t 0.1
0.06 0.06
63.3 + 2.7 32.6 k 1.9
144 72 144
51.6 + 2.1 43.3 + 2.2 41.4 * 2.1 45.0 & 1.9
0.07 0.06
65.1 & 2.7 30.7 + 1.3
197.2 180.5
* 6.8 i 6.2
0.33 0.17
63.5 + 2.0
198.4
+ 6.9
0.32
Betaine
Glutamate
l
Values
are
means
Table 3. Concentration in control (NaCI-free) Growth
f
standard
of total
and stress
deviation
of five
Na+/K+
to seven
independent
polyols (mglg dry wt) in Aspergillus (2 M NaCi) media.*
No addition
K+
experiments.
repens
Betaine
Na+
Na+lK+
Na+
grown
in the presence
Prolfne
or absence
of osmolytee
Glycerol
(10 mM)
Glutamate
(h) c
s
C
s
c
S
60
32.2 k 1.1
39.8 k 1.4
31.9 + 1.2
39.7 + 1.5
31.8 j, 1.1
39.2 & 1.4
42.0
+ 1.1
47.6 + 1.6
32.1
k 1.3
39.4 + 1.9
72 84 96
32.5 33.4 34.1
f 1.1 * 1.2 + 1.4
41.0 + 2.3 44.7 + 3.1 46.6 + 2.4
31.9 f 1.1 33.0 _+ 1.3 34.2 f 1.4
40.8 46.0 43.2
31.9 + 1.1 33.2 + 1.7
41.6 f 2.7 44.8 rfr 2.6
42.5 43.3
k 1.1 + 1.9
58.9 & 2.0 63.6 * 1.9
32.7 + 1.7 33.6 f 1.8
40.8 + 1.8 46.4 + 2.6
132 144
31.8 28.6
& 1.4 + 1.3
52.6 k 3.2 27.2 f 2.6
31.6 k 1.6 28.3 k 1.9
52.1 + 2.7 27.5 + 2.4
34.2 & 1.9 31.5 * 1.4
46.7 & 2.3 50.9 If: 3.1
43.9 + 1.2 42.1 k 1.4
68.6 + 1.8 89.4 -t 2.7
33.8 * 1.2 32.3 f 1.5
48.5 + 2.0 51.7 + 2.7
'I56 168
19.5 + 1.6 18.8 * 1.2
23.1 & 1.1 20.3 f 1.3
19.2 + 1.7 18.7 + 1.1
22.9 + 1.7 18.0 f 1.2
29.0 + 1.2 19.0 + 1.2 18.6 + 1.4
26.4 f 2.7 22.6 2 1.4 20.6 + 1.3
29.3 + 1.9 28.3 & 1.8 27.1 5 1.2
26.7 & 1.4 22.6 & 1.3 20.2 + 1.3
27.4 f 1.0 19.0 & 1.2 18.0 + 1.2
26.5 k 1.9 22.7 + 1.4 21.9 T 1.4
independent
experiments.
*Values C-Control
are
means medium:
fable 4. HPLC (10 m) in control Growth
f
standard S--stress
deviations medium.
of five
t 2.0 + 3.4 + 2.1
to seven
analysis of different polyols (mglg dry wt) in Aspergillus (NaCI-free) and stress (2 mu NaCI) media.*
Polyal
Glycerol Erythritol Arabitol Mannitol Glycerol Erythritol Arabitol Mannitol
repens
No 8ddltlOn
c
s
0.9 & 0.03 ND 1.8 f 1.08 21.3 k 1.0 0.2 * 0.01 ND 1.2 * 0.06 18.2 f 0.9
12.5 & 0.6 ND 4.1 * 0.2 23.4 f 1.2 6.7 & 0.2 1.7 & 0.07 2.3 F 0.1 15.2 k 0.7
* Values are means + standard C-Control medium; S-stress
grown
S
in the
c
presence
S
or absence
of osmolytes
Proline
(h)
72 72 72 72 144 144 144 144
c
S
C
S
c
s
C
s
10.2 + 0.8 ND 3.8 k 0.2 25.2 * 1.1 6.2 * 0.3 1.6 + 0.08 2.1 & 0.2 15.4 * 0.6
0.4 5 0 01 ND 2.0 & 0.06 24.2 5 1.3 0.1 & 0.004 ND 1.1 * 0.05 18.3 + 0.8
10.7 k 0.8 ND 4.2 f 0.3 26.1 i 1.0 6.3 + 0.3 1.5 f 0.08 2.0 + 0.1 15.3 k 0.6
2.2 + 0.1 ND 1.9 f 0.07 36.8 f 1.0 0.7 i 0.03 ND 1.1 * 0.04 20.3 + 0.9
16.4 + 0.8 ND 3.7 f 0.2 17.2 i 1.2 7.4 + 0.4 1.7 f 0.05 2.4 k 0.1 13.7 * 0.5
0.3 * 0.01 ND 2.0 + 0.08 28.7 & 1.3 ND ND 2.1 + 0.2 17.3 * 0.7
10.4 * 0.5 ND 3.9 * 0.3 25.3 & 1.0 6.4 + 0.3 1.5 L- 0.04 2.0 + 0.2 15.2 + 0.5
C
1.4 21.6 0.1 1.3 17.9
ND ND & 0.07 * 1.2 + 0.005 ND rt: 0.05 + 0.8
Glutamate
deviations of three independent medium: ND-not detectable.
experiments.
World journal of Microbiology and Biotechnology. Vol 9. 1993
581
UP.
Kelavkar
and
H.S. Chhatpar
The possible functions of these individual polyols cannot be envisaged at present but it is clear that the osmolytes, besides increasing the osmoticum of the cell, probably also have an indirect sparing effect over individual polyols (Table 4). Polyhydric alcohols (polyols) like glycerol, mannitol, sorbitol (Ben-Amotz & Avron 1983; Jennings 1984) are known to have a protective role in the cytoplasm of various cells (Truper & Galinski 1986). Our studies reveal that intracellular amino acids and intracellular polyols are essential for the survival of A. repens under stress and are produced preferentially. Although addition of osmolytes to the medium enhanced the growth of cells under stress it did not decrease their Na + /K + ratio or their total polyols but probably increased the osmoticurn of the cell and thus had an indirect sparing effect over individual polyols. Thus, the total salt level in Aspergillus repens is not sufficient to counteract the osmotic potential of the medium; an additional osmoregulatory mechanism must, therefore, be involved in determining halotolerance.
References Adler, J. & Gustafsson, L. 1980 Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Deburomyces hansenii. Archives of Microbiology 124, 123-130. Ben-Amotz, A. & Avron, M. 1983 Accumulation of metabolites by halotolerant algae and its industrial potential. Annual Review of Micrabiology 37, 95-119. Bidochka, MJ., Low, N.H. & Khachatourians, G.G. 1990 Carbohydrate storage in the entomopathogenic fungus Beauveria bassiana.Applied and Environmental Microbiology 56,318~+3190.
Brown, A.D. 1978 Compatible solutes and extreme water stress in eucaryotic microorganisms. Advances in Microbial Physiology 17, 181-242. Csonka, N. 1989 Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews 53, 121-147. Gadd, G.M., Chudek, J.A., Foster, R. & Reed, R.H. 1984 The osmotic responses of Penicillium ochro-chloron: changes in internal solute levels in response to copper and salt stress.]oumal of General Microbiology 130, 1969-1975. Jennings, D.H. 1983 Some aspects of the physiology and biochemistry of marine fungi. Biological Rev&s 58, 423-459. Jennings, D.H. 1984 Polyol metabolism in fungi. Advances in Microbial Physiology 25, 149-193. Kelavkar, U.P. & Chhatpar, H.S. 1992 Role of amino acids in halotolerant Aspergillus repens. Fungal Genetics Navsletler 39, 28-31 Kelavkar, U.P., Pandya, S.N. & Chhatpar, H.S. 1993 Salt stress and respiration in Aspergillw repens. Current Microbiology 26, 23-29. Kushner, D.J. 1978 Life in high salt and solute concentrations: halophilic bacteria. In Microbial Life in Extreme Environments, ed Kushner, DJ. pp. 317-368. London and New York: Academic Press. Lewis, D.H. & Smith, D.C. 1967 Sugar alcohols (polyols) in fungi and green plants. I. Distribution, physiology and metabolism. Newsletter of Phytology 66, 143-184. Spencer, J.F.T. & Spencer, D.M. 1978 Production of polyhydroxy alcohols by osmotolerant yeasts. In Economic Microbiology, Vol. 2, Primary Products of Metabolism, ed Rose, A.H. pp 393-425. London, New York and San Francisco: Academic Press. Truper, H.G. & Galinski, E.A. 1986 Concentrated brines as habitats for microorganisms. Experientia 42, 1182-1187.
(Received
in revised
accepted 9 April
form 6 April
1993)
1993;
Journal
of Microbiology
Polyol grown
and Biotechnology
9, 579-682
concentrations in Aspergillus under salt stress
repens
U.P. Kelavkar and H.S. Chhatpar* Na + , K + and the ratio of Na ’ /K + were higher in cells of the halotolerant Asperghs repens grown with 2 M NaCl than without NaCl. The osmolytes, proline, glycerol, betaine and glutamate, did not affect the Na + IK + ratio, nor the polyol content of cells under any conditions. The concentrations of polyols, consisting of glycerol, arabitol, erythritol and mannitol, changed markedly during growth, indicating that they have a crucial role in osmotic adaptation.
Key words: Aspergilltisrepens,
halotolerant, osmolytes, polyols, salt stress.
Microbes within extreme environments have developed various strategies to combat the stressesto which they are exposed. The ability to adapt to ~uc~ations in external osmolarity and the mechanism of osmoregulation have been elucidated in some organisms (Brown 1978; Ben-Amotz & Avron 1983; Csonka 1989), though little info~ation is available in fungi. Studies on marine fungi have tended to concentrate on their salinity requirements and some aspects of their physiology and biochemistry (Jennings 1983; Kelavkar et a!. 1993). Aspergilhs repens is tolerant up to 2 M NaCl but grows in medium without NaCl and thus is termed halotolerant. In this work, we describe the dynamic process of adaptation of Aspergillus repens under salt stress and characterize the specific events involved in the process of adaptation at the cellular level. It has been proposed that
polyols in living organisms act as carbohydrate reserves, sites for storage of reducing power and translocatory compounds,
and have
functions
in osmoregulation
and
coenzyme regulation (Lewis & Smith 1967; Spencer & Spencer 1978; Gadd et al. 1984; Jennings 1984). In this paper we report the occurrence and possible role of polyols in NaCl-tolerance in the halotoler~t ~pergi~~~ repens.
Materials Organism,
and Methods Culfure
Conditions
and Medium
The fungus Aspergillw repens, a salt-pan isolate, was grown in medium containing (g/I): asparagine, 10; (NH&SO,, 3.5; KH,PO,, U.P. Kelavkar and H.S. Chhatpar are with the Department of Microbiology and Biotechnology Centre, Faculty of Science, MS. University of Baroda, Baroda 390 002, India; fax: 91 265 336653. *Corresponding author. @I lg%? Rapid Communications
of Oxford
Ltd
10; MgSO, .7H,O, 2.0; sucrose, 85; CaCI,, 0.075; ZnSO, .5H,O, 0.01; MnCI,, 0.005; (NH,),Mo,O,, .4H,O, 0.002; Na,B,O, 0.002; and FeSO,. 7&O, 0.002. The pH was adjusted to 5.5 and then spores (lO’/ml) were added to 50ml medium in a 250-ml Erlenmeyer flask and shaken on a gyrotary shaker (180 rev/min) at 30 + 2% The mycelia were harvested by washing, until free of medium, under reduced pressure. Dry
weight
and Water
Content
Measurement
The procedure for dry weight and water content measurement was a modification of that described by Bidochka et al. (1990). Suction was applied to the washed mycelia for 5 min and the myceiial mat was weighed to give the wet wt. The mat was then dried first at 50°C in an oven and then in a desiccator to constant weight (dry wt). Subtracting dry wt from wet wt gave the water content of the mycelia.
Fungal mycelia were washed at 4°C with 2.5 M CaCl, to remove loosely associated extracellular ions. They were then held at 500’C for 4 h and then dissolved in 6 M HCI, prepared with de-ionized water, and then autoclaved at 121°C for 1 h. The ion contents in the supematant solutions were analysed with a flame photometer (Evans Electroselenium, UK), with independent blanks and appropriate standards. Polyol Extraction and Determination The procedures for polyol extraction with trichloroacetic acid and the determination of total polyols by the periodate method were as previously described (Adler & Gustafsson 1980). The different polyols were analysed by isocratic HPLC with a Ca’ + -based cation exchange column at 85”C, with de-ionized (de-gassed) water as a mobile phase at a flow rate of 1 mlimin and a pressure of 8106 W/m’. Independent standards of known concentrations (25 mg/ml) were run to obtain their retention times and peak areas. The concentrations of polyols in the samples were determined by comparing their peak areas with those of standards.
U.P. klavkar land H.S. Chhafpar ~~li~~ia~
under control conditions (data not shown). The mechanism by which the osmolytes brought about such an increase in growth in our system is not understood but they could either suppress the uptake of Na + and K + or enhance the leakage of accumulated Na ’ and K + . Changes in the ratio Na’/K+ would influence growth but there was no significant change in the Na $-/K + ratio, in either control or in stressed cells, when osmolytes were added. The osmoiytes were therefore not acting by changing the Na+/KS ratios, but might have increased the osmoticum of the cell (Table 2). Since high concentrations of salt are generally inhibitory to metabolic unctions, various kinds of compensatory mechanisms are found among salt-toIerant organisms (Brown 1978: Kushner 1978; Adler & Guslafsson 1980; Csonka 1989; Kelavkar et al. 1993). When Aspergillw repens was grown under salt stress, polyols accumulated significantly (Table 3). Glycerol, arabitol, mannitol and erythritol were detected, and their contents changed markedly with time (Table 4). However, in our earlier findings, anafyses of total free amino acids in AspergiIiz repensunder stressrevealed that khe pool of total free amino acids was smaller at TZ h than at 144 h of growth, and was always higher than that in unstressed controls (Kelavkar & Chhatpar 1992). The concentration of polyols at 72 h was I&fold higher in the stressed cultures than thak in the controls, demons~at~ng that amino acids are rapidIy used up by growth at 72 h and the pdyots are switched over to salinity control systems (Table 3). After 144 h growth, the polyol concentration decreased (Table 3) as the contents of total free amino acids increased (Kelavkar & Chhatpar 1992), suggesting that, after the polyols, the amino acids switch over to a salinity-control system. The decrease in polyols after 144 h may be due to their use as an endogenous carbon source or as storage carbohydrates at that stage, S~risingly, when proline, glycerol, betaine and glutamate were added to the growth medium there was no significant decrease in the accumulation of total polyols (Table 3) in either stressed or unstressed cultures but the concentrations of individual polyols changed (Table 4).
of ~pe~men~5
All the experiments otherwise.
Results
were
repeated
at least five times
unless
stated
and Discussion
repens was haiotoler~t and showed considerable growth in the presence of 2 M NaCl (Figure I). When it was grown in medium supplemented with 2 M NaCl (referred to as ‘stress’) the intracellular concentrations of Na + and K + and the ratio Na + IK + increased compared with control values in NaCI-free medium (Table I). Gadd ef at. (1984 found a similar increase in Na + iK + ratio in P~i~llium ochro~c~loron under salt stress. When the osmoiytes proline, glutamate, betaine and glycerol were added at 10 mM to the growth medium, there was a significant increase in growth under stress but not ~pergi~l~
0
72
96
120
144
Incubation
t68
192
(h)
Figure 1. Growth of Aspergillus repens in 2%ml shaken flask cultures at 30% in W-ml volumes of medium without NaGI (a), or with 1 (II), 2 (A,) or 3 (‘l) t!~ NaCI. Cell dry wt was determined by drying the mycelia at 50°C to a constant weight.
Teble
1. Concentratfons
Growth
of Na+ and K+ in
Cell water @t/m@ dry wt)
(h)
Aspergi&s (mg
repensgrown
In control
medium
My~l~urn dry wtfml)
Mycellat
(f&Cl-Free) Ion contents
and stress (nmol~mg
Control Control 72 96 144 168 * Values
0.9 + 0.05 1.2 & 0.06 2.4 +O.lO 2.5&O.lT are
means
Stress 0.3 0.4 0.7 0.7
* 0.02 f 0.01 f 0.04 kO.05
j, standard
Control
Stress
3.6 & 0.07 6.8+0.20 t3.0 t 1.10 I3.4& 1.20 deviations
of five
2.5 4.4 7.0 7.3
+ + & f
0.22 0.41 0.10 0.10
to seven
Na+ 3.0 f 0.1 3.2 +0.3 2.81 0.1 2.240.2 independent
K+ 43.7 44.3 42.6 42.3
io.1 & 2.f sf: 2.2 t3.3
experiments.
medium
(2 M NaCi).*
dry wt) Stress
Na+lK+ 0.07 0.07 0.06 0.05
Ha+ 32.9 43.4 648 63.2
i; rt + *
K+ 2.2 2.7 3.1 2.7
190.0 199.8 195.2 190.9
+ + f &
Na+lK* 8.1 9.2 9.2 7.6
0.17 0.21 0.33 0.33
Polyol concenfrafionsin Aspergillus repens Table 2. Concentrations with 10 mu osmolytes.” Osmolyte (at lOmu)
of Na+
and K+
In Asjrergillus
repens
grown
Mycetfal
Growth (h)
in control
ion contents
(NaCI-free)
(nmolimg
and stress
(2 M NaCI)
media
dry wt)
Control
Stress
K+
72 144
2.9 * 0.1 2.2 + 0.3
44.6 ) 2.3 31.4 & 1.2
0.065
31.0 + 1.4
193.8
_+ 8.6
0.16
0.075
Proline
72 144
3.0 * 0.2 2.7 + 0.4
42.8 * 2.2 45.0 + 2.2
0.07 0.06
64.1 & 2.4 32.3 F 1.7
200.0 190.0
* 6.3 k 8.4
0.32 0.17
Glycerol
72
3.1 + 0.1
186.0 203.7
+ 9.6 k 9.3
0.34 0.16
2.6 + 0.2 2.9 + 0.1 2.7 t 0.1
0.06 0.06
63.3 + 2.7 32.6 k 1.9
144 72 144
51.6 + 2.1 43.3 + 2.2 41.4 * 2.1 45.0 & 1.9
0.07 0.06
65.1 & 2.7 30.7 + 1.3
197.2 180.5
* 6.8 i 6.2
0.33 0.17
63.5 + 2.0
198.4
+ 6.9
0.32
Betaine
Glutamate
l
Values
are
means
Table 3. Concentration in control (NaCI-free) Growth
f
standard
of total
and stress
deviation
of five
Na+/K+
to seven
independent
polyols (mglg dry wt) in Aspergillus (2 M NaCi) media.*
No addition
K+
experiments.
repens
Betaine
Na+
Na+lK+
Na+
grown
in the presence
Prolfne
or absence
of osmolytee
Glycerol
(10 mM)
Glutamate
(h) c
s
C
s
c
S
60
32.2 k 1.1
39.8 k 1.4
31.9 + 1.2
39.7 + 1.5
31.8 j, 1.1
39.2 & 1.4
42.0
+ 1.1
47.6 + 1.6
32.1
k 1.3
39.4 + 1.9
72 84 96
32.5 33.4 34.1
f 1.1 * 1.2 + 1.4
41.0 + 2.3 44.7 + 3.1 46.6 + 2.4
31.9 f 1.1 33.0 _+ 1.3 34.2 f 1.4
40.8 46.0 43.2
31.9 + 1.1 33.2 + 1.7
41.6 f 2.7 44.8 rfr 2.6
42.5 43.3
k 1.1 + 1.9
58.9 & 2.0 63.6 * 1.9
32.7 + 1.7 33.6 f 1.8
40.8 + 1.8 46.4 + 2.6
132 144
31.8 28.6
& 1.4 + 1.3
52.6 k 3.2 27.2 f 2.6
31.6 k 1.6 28.3 k 1.9
52.1 + 2.7 27.5 + 2.4
34.2 & 1.9 31.5 * 1.4
46.7 & 2.3 50.9 If: 3.1
43.9 + 1.2 42.1 k 1.4
68.6 + 1.8 89.4 -t 2.7
33.8 * 1.2 32.3 f 1.5
48.5 + 2.0 51.7 + 2.7
'I56 168
19.5 + 1.6 18.8 * 1.2
23.1 & 1.1 20.3 f 1.3
19.2 + 1.7 18.7 + 1.1
22.9 + 1.7 18.0 f 1.2
29.0 + 1.2 19.0 + 1.2 18.6 + 1.4
26.4 f 2.7 22.6 2 1.4 20.6 + 1.3
29.3 + 1.9 28.3 & 1.8 27.1 5 1.2
26.7 & 1.4 22.6 & 1.3 20.2 + 1.3
27.4 f 1.0 19.0 & 1.2 18.0 + 1.2
26.5 k 1.9 22.7 + 1.4 21.9 T 1.4
independent
experiments.
*Values C-Control
are
means medium:
fable 4. HPLC (10 m) in control Growth
f
standard S--stress
deviations medium.
of five
t 2.0 + 3.4 + 2.1
to seven
analysis of different polyols (mglg dry wt) in Aspergillus (NaCI-free) and stress (2 mu NaCI) media.*
Polyal
Glycerol Erythritol Arabitol Mannitol Glycerol Erythritol Arabitol Mannitol
repens
No 8ddltlOn
c
s
0.9 & 0.03 ND 1.8 f 1.08 21.3 k 1.0 0.2 * 0.01 ND 1.2 * 0.06 18.2 f 0.9
12.5 & 0.6 ND 4.1 * 0.2 23.4 f 1.2 6.7 & 0.2 1.7 & 0.07 2.3 F 0.1 15.2 k 0.7
* Values are means + standard C-Control medium; S-stress
grown
S
in the
c
presence
S
or absence
of osmolytes
Proline
(h)
72 72 72 72 144 144 144 144
c
S
C
S
c
s
C
s
10.2 + 0.8 ND 3.8 k 0.2 25.2 * 1.1 6.2 * 0.3 1.6 + 0.08 2.1 & 0.2 15.4 * 0.6
0.4 5 0 01 ND 2.0 & 0.06 24.2 5 1.3 0.1 & 0.004 ND 1.1 * 0.05 18.3 + 0.8
10.7 k 0.8 ND 4.2 f 0.3 26.1 i 1.0 6.3 + 0.3 1.5 f 0.08 2.0 + 0.1 15.3 k 0.6
2.2 + 0.1 ND 1.9 f 0.07 36.8 f 1.0 0.7 i 0.03 ND 1.1 * 0.04 20.3 + 0.9
16.4 + 0.8 ND 3.7 f 0.2 17.2 i 1.2 7.4 + 0.4 1.7 f 0.05 2.4 k 0.1 13.7 * 0.5
0.3 * 0.01 ND 2.0 + 0.08 28.7 & 1.3 ND ND 2.1 + 0.2 17.3 * 0.7
10.4 * 0.5 ND 3.9 * 0.3 25.3 & 1.0 6.4 + 0.3 1.5 L- 0.04 2.0 + 0.2 15.2 + 0.5
C
1.4 21.6 0.1 1.3 17.9
ND ND & 0.07 * 1.2 + 0.005 ND rt: 0.05 + 0.8
Glutamate
deviations of three independent medium: ND-not detectable.
experiments.
World journal of Microbiology and Biotechnology. Vol 9. 1993
581
UP.
Kelavkar
and
H.S. Chhatpar
The possible functions of these individual polyols cannot be envisaged at present but it is clear that the osmolytes, besides increasing the osmoticum of the cell, probably also have an indirect sparing effect over individual polyols (Table 4). Polyhydric alcohols (polyols) like glycerol, mannitol, sorbitol (Ben-Amotz & Avron 1983; Jennings 1984) are known to have a protective role in the cytoplasm of various cells (Truper & Galinski 1986). Our studies reveal that intracellular amino acids and intracellular polyols are essential for the survival of A. repens under stress and are produced preferentially. Although addition of osmolytes to the medium enhanced the growth of cells under stress it did not decrease their Na + /K + ratio or their total polyols but probably increased the osmoticurn of the cell and thus had an indirect sparing effect over individual polyols. Thus, the total salt level in Aspergillus repens is not sufficient to counteract the osmotic potential of the medium; an additional osmoregulatory mechanism must, therefore, be involved in determining halotolerance.
References Adler, J. & Gustafsson, L. 1980 Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Deburomyces hansenii. Archives of Microbiology 124, 123-130. Ben-Amotz, A. & Avron, M. 1983 Accumulation of metabolites by halotolerant algae and its industrial potential. Annual Review of Micrabiology 37, 95-119. Bidochka, MJ., Low, N.H. & Khachatourians, G.G. 1990 Carbohydrate storage in the entomopathogenic fungus Beauveria bassiana.Applied and Environmental Microbiology 56,318~+3190.
Brown, A.D. 1978 Compatible solutes and extreme water stress in eucaryotic microorganisms. Advances in Microbial Physiology 17, 181-242. Csonka, N. 1989 Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews 53, 121-147. Gadd, G.M., Chudek, J.A., Foster, R. & Reed, R.H. 1984 The osmotic responses of Penicillium ochro-chloron: changes in internal solute levels in response to copper and salt stress.]oumal of General Microbiology 130, 1969-1975. Jennings, D.H. 1983 Some aspects of the physiology and biochemistry of marine fungi. Biological Rev&s 58, 423-459. Jennings, D.H. 1984 Polyol metabolism in fungi. Advances in Microbial Physiology 25, 149-193. Kelavkar, U.P. & Chhatpar, H.S. 1992 Role of amino acids in halotolerant Aspergillus repens. Fungal Genetics Navsletler 39, 28-31 Kelavkar, U.P., Pandya, S.N. & Chhatpar, H.S. 1993 Salt stress and respiration in Aspergillw repens. Current Microbiology 26, 23-29. Kushner, D.J. 1978 Life in high salt and solute concentrations: halophilic bacteria. In Microbial Life in Extreme Environments, ed Kushner, DJ. pp. 317-368. London and New York: Academic Press. Lewis, D.H. & Smith, D.C. 1967 Sugar alcohols (polyols) in fungi and green plants. I. Distribution, physiology and metabolism. Newsletter of Phytology 66, 143-184. Spencer, J.F.T. & Spencer, D.M. 1978 Production of polyhydroxy alcohols by osmotolerant yeasts. In Economic Microbiology, Vol. 2, Primary Products of Metabolism, ed Rose, A.H. pp 393-425. London, New York and San Francisco: Academic Press. Truper, H.G. & Galinski, E.A. 1986 Concentrated brines as habitats for microorganisms. Experientia 42, 1182-1187.
(Received
in revised
accepted 9 April
form 6 April
1993)
1993;
