AEM Accepted Manuscript Posted Online 22 January 2016 Appl. Environ. Microbiol. doi:10.1128/AEM.04052-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Comparative toxicity of salts to microbial processes in soil
2
Kristin M. Rath*,1,2, Arpita Maheshwari1, Per Bengtson1, and Johannes Rousk1
3 4
*
5
Biology, Ecology Building, Lund University, 22362 Lund, Sweden. Email:
6
[email protected], phone: +46 46-222 37 63, Fax: n/a.
Corresponding author: Kristin M. Rath, Section of Microbial Ecology, Department of
7 8
1
Section of Microbial Ecology, Department of Biology, Lund University
9
2
Centre for Environmental and Climate research (CEC), Lund University
10 11
Full-length paper for Applied and Environmental Microbiology
12 13
Section: Microbial Ecology
14 15
Running title: Comparative toxicity of salts to microbial processes (53/54).
1
16
Abstract (250/250 words)
17
Soil salinization is a growing threat to global agriculture and carbon (C) sequestration,
18
but to date it remains unclear how microbial processes will respond. We studied the acute
19
response to salt exposure of a range of anabolic and catabolic microbial processes,
20
including bacterial (leucine incorporation) and fungal (acetate incorporation into
21
ergosterol) growth rates, respiration and gross N mineralization and nitrification rates.
22
To distinguish effects of specific ions from those of overall ionic strength, we compared
23
the addition of four salts frequently associated with soil salinization (NaCl, KCl, Na2SO4,
24
K2SO4) to a non-saline soil. To compare the tolerance of different microbial processes to
25
salt, and to interrelate the toxicity of different salts, concentration-response relationships
26
were established. Growth-based measurements revealed that fungi were more resistant to
27
salt exposure than bacteria. Effects by salt on C and N mineralization were
28
indistinguishable and, in contrast with previous studies, nitrification was not found to be
29
more sensitive to salt exposure than other microbial processes. Ion specific toxicity of
30
certain salts could only be observed for respiration, which was less inhibited by salts
31
containing SO42- than Cl- salts, in contrast with the microbial growth assessments. This
32
suggested that the inhibition of microbial growth was solely explained by total ionic
33
strength, while ionic specific toxicity should also be considered for effects on microbial
34
decomposition. This difference resulted in an apparent reduction of microbial growth
35
efficiency in response to exposure to SO42- salts but not to Cl- salts; no evidence was
36
found to distinguish K+ and Na+ salts.
37 38
Keywords: Soil salinization, Microbial ecology, Ecotoxicology, tolerance, respiration, nitrogen transformation, Fungal to Bacterial dominance.
2
39
Introduction
40
Soil salinization affects a large area of land globally and has become a major
41
threat to agricultural productivity and food security (1). Due to the wide distribution of
42
salt-affected soils around the world (2, 3), it is important to understand the influence of
43
salinity on the soil microbial community. The soil microbial decomposer community
44
plays an essential role in the decomposition and stabilization of soil organic matter
45
(SOM), as well as the cycling of nutrients vital for plant growth. How substrate during
46
decomposition is allocated to either microbial biomass production or respiration
47
determines the microbial growth efficiency (MGE), which is an important parameter for
48
the C sequestration potential of a soil (4). The potential for soil C storage could be
49
compromised by disturbances or unfavorable environmental conditions that reduce
50
microbial growth efficiencies due to the metabolic burden they place on microbial cells
51
(5).
52
It is generally held that fungal-dominated communities have a higher MGE than
53
communities dominated by bacteria (4). Changes in the relative contribution of bacteria
54
and fungi to the soil microbial community are thus thought to reflect changes in
55
ecosystem processes such as decomposition, C sequestration potential and nutrient
56
cycling (6, 7). It is unclear whether fungi and bacteria are affected by salt exposure to a
57
similar degree or if there are differences in salt sensitivity between these two major
58
decomposer groups. It has been shown that fungi are more resistant to osmotic pressure,
59
illustrated by their higher tolerance to high concentrations of low molecular weight
60
organic compounds (8, 9). In addition, fungi have also been found to be more resistant to
61
low water potentials brought about by decreasing soil moisture than most bacteria (10,
3
62
11). In soils exposed to salinity, both higher (12, 13) and lower (14-17) contributions of
63
fungi to the microbial community have been observed
64
Often the influence of soil salinity on the soil microbial community has been
65
studied using total microbial biomass measurements. However, the connection between
66
the total microbial biomass and microbial contribution to soil processes is tenuous at best
67
(6, 18, 19), rendering biomass a poor predictor for process rates carried out by the
68
microbial community. Instead, responses in processes carried out by the active and
69
growing part of the microbial community can be employed to detect inhibition by
70
exposure to salts. For instance, salt additions have been found to influence and reduce
71
microbial activity, measured as respiration (12, 20-23) or N transformation rates (22, 24).
72
To date, there is a lack of comparative studies on the degree of sensitivity of a
73
comprehensive range of different microbial processes. If processes show differential
74
sensitivity to salinity this could have implications for soil biogeochemical cycles and the
75
ecology of microorganisms, as well as the identification of informative endpoints for
76
toxicity assessments. In addition, not all salts associated with soil salinization have the
77
same effect on the microbial community. Differences in toxicity have been found
78
between e.g. SO42- and Cl- salts (25-30), as well as K+ and Na+ salts (28). However, few
79
studies have been designed to explicitly compare the toxicity of different salts using a
80
range of processes.
81
The aim of this study was to conduct a comparative analysis of the sensitivity of a
82
range of different microbial processes to short-term salt exposure in a non-saline soil. In
83
the first part of the study soil was exposed to a range of NaCl concentrations. The acute
84
growth responses of bacteria and fungi were compared to assess differences in their
4
85
tolerance to salinity. In addition, growth processes were compared to catabolic processes
86
including C and N mineralization and nitrification to investigate the potential for salts to
87
induce a shift in SOM dynamics and nutrient cycling. Considering the predicted higher
88
tolerance of fungi to osmotic pressure we hypothesized that fungal growth would show a
89
higher tolerance to salts associated with soil salinization than bacterial growth. Further,
90
we predicted that, as a symptom of the cost of physiological measures to cope with high
91
osmotic potentials, microorganisms allocate substrate away from biomass production
92
towards maintenance functions, leading to a situation where catabolic processes would be
93
less inhibited by salt exposure than anabolic or growth-related processes. Incubation
94
times were kept short to ensure that the measured responses are direct responses to salt
95
exposure, rather than inhibition confounded by the recovery due to a shift towards a more
96
tolerant community. In the second part of the study we conducted a comparative
97
assessment of the toxicity of salts common in saline soils (NaCl, KCl, K2SO4, Na2SO4)
98
on respiration, as well as fungal and bacterial growth. We hypothesized that Cl- salts
99
would be more toxic than SO42- salts, and that Na+ salts would be more toxic than K+
100
salts. We also predicted that irrespective of the type of salt used, fungi would be more
101
resistant than bacteria, and respiration less inhibited than growth.
102 103 104
Material and Methods
105 106
Soil sampling and characterization. Soil was collected from a grassland site situated in
107
Vomb, Southern Sweden (55° 40' 27" N, 13° 32' 45" E). The soil is a well-drained sandy
5
108
grassland soil. Multiple soil samples were collected with a spade from pits dug to a depth
109
of ca. 20 cm and combined into composite samples, homogenized, and sieved (
1
Comparative toxicity of salts to microbial processes in soil
2
Kristin M. Rath*,1,2, Arpita Maheshwari1, Per Bengtson1, and Johannes Rousk1
3 4
*
5
Biology, Ecology Building, Lund University, 22362 Lund, Sweden. Email:
6
[email protected], phone: +46 46-222 37 63, Fax: n/a.
Corresponding author: Kristin M. Rath, Section of Microbial Ecology, Department of
7 8
1
Section of Microbial Ecology, Department of Biology, Lund University
9
2
Centre for Environmental and Climate research (CEC), Lund University
10 11
Full-length paper for Applied and Environmental Microbiology
12 13
Section: Microbial Ecology
14 15
Running title: Comparative toxicity of salts to microbial processes (53/54).
1
16
Abstract (250/250 words)
17
Soil salinization is a growing threat to global agriculture and carbon (C) sequestration,
18
but to date it remains unclear how microbial processes will respond. We studied the acute
19
response to salt exposure of a range of anabolic and catabolic microbial processes,
20
including bacterial (leucine incorporation) and fungal (acetate incorporation into
21
ergosterol) growth rates, respiration and gross N mineralization and nitrification rates.
22
To distinguish effects of specific ions from those of overall ionic strength, we compared
23
the addition of four salts frequently associated with soil salinization (NaCl, KCl, Na2SO4,
24
K2SO4) to a non-saline soil. To compare the tolerance of different microbial processes to
25
salt, and to interrelate the toxicity of different salts, concentration-response relationships
26
were established. Growth-based measurements revealed that fungi were more resistant to
27
salt exposure than bacteria. Effects by salt on C and N mineralization were
28
indistinguishable and, in contrast with previous studies, nitrification was not found to be
29
more sensitive to salt exposure than other microbial processes. Ion specific toxicity of
30
certain salts could only be observed for respiration, which was less inhibited by salts
31
containing SO42- than Cl- salts, in contrast with the microbial growth assessments. This
32
suggested that the inhibition of microbial growth was solely explained by total ionic
33
strength, while ionic specific toxicity should also be considered for effects on microbial
34
decomposition. This difference resulted in an apparent reduction of microbial growth
35
efficiency in response to exposure to SO42- salts but not to Cl- salts; no evidence was
36
found to distinguish K+ and Na+ salts.
37 38
Keywords: Soil salinization, Microbial ecology, Ecotoxicology, tolerance, respiration, nitrogen transformation, Fungal to Bacterial dominance.
2
39
Introduction
40
Soil salinization affects a large area of land globally and has become a major
41
threat to agricultural productivity and food security (1). Due to the wide distribution of
42
salt-affected soils around the world (2, 3), it is important to understand the influence of
43
salinity on the soil microbial community. The soil microbial decomposer community
44
plays an essential role in the decomposition and stabilization of soil organic matter
45
(SOM), as well as the cycling of nutrients vital for plant growth. How substrate during
46
decomposition is allocated to either microbial biomass production or respiration
47
determines the microbial growth efficiency (MGE), which is an important parameter for
48
the C sequestration potential of a soil (4). The potential for soil C storage could be
49
compromised by disturbances or unfavorable environmental conditions that reduce
50
microbial growth efficiencies due to the metabolic burden they place on microbial cells
51
(5).
52
It is generally held that fungal-dominated communities have a higher MGE than
53
communities dominated by bacteria (4). Changes in the relative contribution of bacteria
54
and fungi to the soil microbial community are thus thought to reflect changes in
55
ecosystem processes such as decomposition, C sequestration potential and nutrient
56
cycling (6, 7). It is unclear whether fungi and bacteria are affected by salt exposure to a
57
similar degree or if there are differences in salt sensitivity between these two major
58
decomposer groups. It has been shown that fungi are more resistant to osmotic pressure,
59
illustrated by their higher tolerance to high concentrations of low molecular weight
60
organic compounds (8, 9). In addition, fungi have also been found to be more resistant to
61
low water potentials brought about by decreasing soil moisture than most bacteria (10,
3
62
11). In soils exposed to salinity, both higher (12, 13) and lower (14-17) contributions of
63
fungi to the microbial community have been observed
64
Often the influence of soil salinity on the soil microbial community has been
65
studied using total microbial biomass measurements. However, the connection between
66
the total microbial biomass and microbial contribution to soil processes is tenuous at best
67
(6, 18, 19), rendering biomass a poor predictor for process rates carried out by the
68
microbial community. Instead, responses in processes carried out by the active and
69
growing part of the microbial community can be employed to detect inhibition by
70
exposure to salts. For instance, salt additions have been found to influence and reduce
71
microbial activity, measured as respiration (12, 20-23) or N transformation rates (22, 24).
72
To date, there is a lack of comparative studies on the degree of sensitivity of a
73
comprehensive range of different microbial processes. If processes show differential
74
sensitivity to salinity this could have implications for soil biogeochemical cycles and the
75
ecology of microorganisms, as well as the identification of informative endpoints for
76
toxicity assessments. In addition, not all salts associated with soil salinization have the
77
same effect on the microbial community. Differences in toxicity have been found
78
between e.g. SO42- and Cl- salts (25-30), as well as K+ and Na+ salts (28). However, few
79
studies have been designed to explicitly compare the toxicity of different salts using a
80
range of processes.
81
The aim of this study was to conduct a comparative analysis of the sensitivity of a
82
range of different microbial processes to short-term salt exposure in a non-saline soil. In
83
the first part of the study soil was exposed to a range of NaCl concentrations. The acute
84
growth responses of bacteria and fungi were compared to assess differences in their
4
85
tolerance to salinity. In addition, growth processes were compared to catabolic processes
86
including C and N mineralization and nitrification to investigate the potential for salts to
87
induce a shift in SOM dynamics and nutrient cycling. Considering the predicted higher
88
tolerance of fungi to osmotic pressure we hypothesized that fungal growth would show a
89
higher tolerance to salts associated with soil salinization than bacterial growth. Further,
90
we predicted that, as a symptom of the cost of physiological measures to cope with high
91
osmotic potentials, microorganisms allocate substrate away from biomass production
92
towards maintenance functions, leading to a situation where catabolic processes would be
93
less inhibited by salt exposure than anabolic or growth-related processes. Incubation
94
times were kept short to ensure that the measured responses are direct responses to salt
95
exposure, rather than inhibition confounded by the recovery due to a shift towards a more
96
tolerant community. In the second part of the study we conducted a comparative
97
assessment of the toxicity of salts common in saline soils (NaCl, KCl, K2SO4, Na2SO4)
98
on respiration, as well as fungal and bacterial growth. We hypothesized that Cl- salts
99
would be more toxic than SO42- salts, and that Na+ salts would be more toxic than K+
100
salts. We also predicted that irrespective of the type of salt used, fungi would be more
101
resistant than bacteria, and respiration less inhibited than growth.
102 103 104
Material and Methods
105 106
Soil sampling and characterization. Soil was collected from a grassland site situated in
107
Vomb, Southern Sweden (55° 40' 27" N, 13° 32' 45" E). The soil is a well-drained sandy
5
108
grassland soil. Multiple soil samples were collected with a spade from pits dug to a depth
109
of ca. 20 cm and combined into composite samples, homogenized, and sieved (
