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Comparative toxicity of salts to microbial processes in soil 1
Kristin M. Rath*,1,2, Arpita Maheshwari1, Per Bengtson1, and Johannes Rousk1 2
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*Corresponding author: Kristin M. Rath, Section of Microbial Ecology, Department of 4
Biology, Ecology Building, Lund University, 22362 Lund, Sweden. Email: 5
[email protected], phone: +46 46-222 37 63, Fax: n/a. 6
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1 Section of Microbial Ecology, Department of Biology, Lund University 8
2 Centre for Environmental and Climate research (CEC), Lund University 9
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Full-length paper for Applied and Environmental Microbiology 11
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Section: Microbial Ecology 13
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Running title: Comparative toxicity of salts to microbial processes (53/54). 15
AEM Accepted Manuscript Posted Online 22 January 2016Appl. Environ. Microbiol. doi:10.1128/AEM.04052-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Abstract (250/250 words) 16
Soil salinization is a growing threat to global agriculture and carbon (C) sequestration, 17
but to date it remains unclear how microbial processes will respond. We studied the acute 18
response to salt exposure of a range of anabolic and catabolic microbial processes, 19
including bacterial (leucine incorporation) and fungal (acetate incorporation into 20
ergosterol) growth rates, respiration and gross N mineralization and nitrification rates. 21
To distinguish effects of specific ions from those of overall ionic strength, we compared 22
the addition of four salts frequently associated with soil salinization (NaCl, KCl, Na2SO4, 23
K2SO4) to a non-saline soil. To compare the tolerance of different microbial processes to 24
salt, and to interrelate the toxicity of different salts, concentration-response relationships 25
were established. Growth-based measurements revealed that fungi were more resistant to 26
salt exposure than bacteria. Effects by salt on C and N mineralization were 27
indistinguishable and, in contrast with previous studies, nitrification was not found to be 28
more sensitive to salt exposure than other microbial processes. Ion specific toxicity of 29
certain salts could only be observed for respiration, which was less inhibited by salts 30
containing SO42- than Cl- salts, in contrast with the microbial growth assessments. This 31
suggested that the inhibition of microbial growth was solely explained by total ionic 32
strength, while ionic specific toxicity should also be considered for effects on microbial 33
decomposition. This difference resulted in an apparent reduction of microbial growth 34
efficiency in response to exposure to SO42- salts but not to Cl- salts; no evidence was 35
found to distinguish K+ and Na+ salts. 36
Keywords: Soil salinization, Microbial ecology, Ecotoxicology, tolerance, 37
respiration, nitrogen transformation, Fungal to Bacterial dominance. 38
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Introduction 39
Soil salinization affects a large area of land globally and has become a major 40
threat to agricultural productivity and food security (1). Due to the wide distribution of 41
salt-affected soils around the world (2, 3), it is important to understand the influence of 42
salinity on the soil microbial community. The soil microbial decomposer community 43
plays an essential role in the decomposition and stabilization of soil organic matter 44
(SOM), as well as the cycling of nutrients vital for plant growth. How substrate during 45
decomposition is allocated to either microbial biomass production or respiration 46
determines the microbial growth efficiency (MGE), which is an important parameter for 47
the C sequestration potential of a soil (4). The potential for soil C storage could be 48
compromised by disturbances or unfavorable environmental conditions that reduce 49
microbial growth efficiencies due to the metabolic burden they place on microbial cells 50
(5). 51
It is generally held that fungal-dominated communities have a higher MGE than 52
communities dominated by bacteria (4). Changes in the relative contribution of bacteria 53
and fungi to the soil microbial community are thus thought to reflect changes in 54
ecosystem processes such as decomposition, C sequestration potential and nutrient 55
cycling (6, 7). It is unclear whether fungi and bacteria are affected by salt exposure to a 56
similar degree or if there are differences in salt sensitivity between these two major 57
decomposer groups. It has been shown that fungi are more resistant to osmotic pressure, 58
illustrated by their higher tolerance to high concentrations of low molecular weight 59
organic compounds (8, 9). In addition, fungi have also been found to be more resistant to 60
low water potentials brought about by decreasing soil moisture than most bacteria (10, 61
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11). In soils exposed to salinity, both higher (12, 13) and lower (14-17) contributions of 62
fungi to the microbial community have been observed 63
Often the influence of soil salinity on the soil microbial community has been 64
studied using total microbial biomass measurements. However, the connection between 65
the total microbial biomass and microbial contribution to soil processes is tenuous at best 66
(6, 18, 19), rendering biomass a poor predictor for process rates carried out by the 67
microbial community. Instead, responses in processes carried out by the active and 68
growing part of the microbial community can be employed to detect inhibition by 69
exposure to salts. For instance, salt additions have been found to influence and reduce 70
microbial activity, measured as respiration (12, 20-23) or N transformation rates (22, 24). 71
To date, there is a lack of comparative studies on the degree of sensitivity of a 72
comprehensive range of different microbial processes. If processes show differential 73
sensitivity to salinity this could have implications for soil biogeochemical cycles and the 74
ecology of microorganisms, as well as the identification of informative endpoints for 75
toxicity assessments. In addition, not all salts associated with soil salinization have the 76
same effect on the microbial community. Differences in toxicity have been found 77
between e.g. SO42- and Cl- salts (25-30), as well as K+ and Na+ salts (28). However, few 78
studies have been designed to explicitly compare the toxicity of different salts using a 79
range of processes. 80
The aim of this study was to conduct a comparative analysis of the sensitivity of a 81
range of different microbial processes to short-term salt exposure in a non-saline soil. In 82
the first part of the study soil was exposed to a range of NaCl concentrations. The acute 83
growth responses of bacteria and fungi were compared to assess differences in their 84
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tolerance to salinity. In addition, growth processes were compared to catabolic processes 85
including C and N mineralization and nitrification to investigate the potential for salts to 86
induce a shift in SOM dynamics and nutrient cycling. Considering the predicted higher 87
tolerance of fungi to osmotic pressure we hypothesized that fungal growth would show a 88
higher tolerance to salts associated with soil salinization than bacterial growth. Further, 89
we predicted that, as a symptom of the cost of physiological measures to cope with high 90
osmotic potentials, microorganisms allocate substrate away from biomass production 91
towards maintenance functions, leading to a situation where catabolic processes would be 92
less inhibited by salt exposure than anabolic or growth-related processes. Incubation 93
times were kept short to ensure that the measured responses are direct responses to salt 94
exposure, rather than inhibition confounded by the recovery due to a shift towards a more 95
tolerant community. In the second part of the study we conducted a comparative 96
assessment of the toxicity of salts common in saline soils (NaCl, KCl, K2SO4, Na2SO4) 97
on respiration, as well as fungal and bacterial growth. We hypothesized that Cl- salts 98
would be more toxic than SO42- salts, and that Na+ salts would be more toxic than K+ 99
salts. We also predicted that irrespective of the type of salt used, fungi would be more 100
resistant than bacteria, and respiration less inhibited than growth. 101
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Material and Methods 104
105
Soil sampling and characterization. Soil was collected from a grassland site situated in 106
Vomb, Southern Sweden (55° 40' 27" N, 13° 32' 45" E). The soil is a well-drained sandy 107
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grassland soil. Multiple soil samples were collected with a spade from pits dug to a depth 108
of ca. 20 cm and combined into composite samples, homogenized, and sieved (<2.8 mm). 109
Samples were collected at several time points from the same site: September 2013, 110
October 2013, November 2014 and December 2014. 111
Following sieving and homogenization, the water content of the soil was 112
determined gravimetrically (105°C, 24 h) and the organic matter (OM) content was 113
measured as loss on ignition (600°C, 10 h). Electrical conductivity (EC) and pH 114
measurements were conducted in a 1:5 soil:water suspension. To measure NH4+ and NO3
- 115
concentrations diffusion traps were placed in a KCl soil extract. The total microbial 116
biomass was determined using substrate induced respiration (SIR) (31), by adding 6 mg 117
of glucose per g soil. After 2 h incubation, CO2 was measured using a gas chromatograph 118
(GC), equipped with a methanizer and a flame ionization detector. The measured 119
respiration rate was converted to microbial biomass-C using the relationship that 1 mg 120
CO2 h-1 corresponds to 20 mg biomass-C (31). These SIR-estimates of microbial biomass 121
matched previous chloroform-fumigation based assessment of biomass from the same 122
soils (Per Bengtson, unpublished). 123
Fungal biomass-C was estimated by extracting and quantifying the amount of 124
ergosterol in the soil sample. We assumed a fungal ergosterol content of 5 mg g-1 fungal 125
biomass and a fungal C content of 45% (32, 33). Bacterial biomass-C was estimated 126
using a mass balance of fungal biomass-C to total microbial biomass-C. 127
Soil samples were subsequently stored in gas-permeable polyethylene bags at 4°C until 128
further analysis. Before the start of the experiment soil was kept at 22°C for 2 days. The 129
study was divided into two parts, referred to as Experiment 1 and 2 below. 130
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131
Experiment 1. In the first part of the study, sensitivity of bacterial and fungal growth, 132
respiration and gross N mineralization and nitrification rates to sodium chloride (NaCl) 133
was determined. Bacterial growth measurements were repeated in samples from all four 134
sampling time points, fungal growth and respiration in samples collected in September 135
2013 and December 2014, whereas gross N mineralization and nitrification rates were 136
measured in the sample collected in October 2013. Before measuring microbial growth 137
and process rates NaCl was added to the soils in 8-12 different concentrations, which 138
covered a range from 0 to 3600 µmol NaCl per g soil in a series of 3-fold dilution steps, 139
together with 100 µl distilled water per g soil. The salt additions resulted in a range of 140
EC1:5 values from 0.06 to 65.6 dS m-1. Generally, a soil is described as saline if the 141
electrical conductivity measured in a saturated soil paste (ECe) is higher than 4 dS m-1 , 142
but studies of saline soils frequently include soils with a more than tenfold higher ECe 143
(23). Following the addition of NaCl, soils were incubated at room temperature for 1.5 h 144
before microbial variables were determined. 145
146
Experiment 2. In the second part of the study we compared the toxic effect of salts 147
common in saline soils, namely NaCl, potassium chloride (KCl), potassium sulfate 148
(K2SO4) and sodium sulfate (Na2SO4) on bacterial growth, fungal growth and respiration 149
rates (see sections below). For each salt the same molar concentrations were used (0, 150
0.06, 0.17, 0.52, 1.6, 4.7, 14.1, 42.3, 127, 380, 1140 and 3420 µmol salt per g soil). The 151
resulting electrical conductivity in the soil-salt combinations was measured in a 1:5 152
soil:water suspension, and covered a range of EC1:5 values from 0.01 dS m-1 to 75 dS m-1. 153
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Changes in soil pH following salt additions were small (from pH 6.4 to around 6.1 in the 154
treatment receiving the highest salt addition). Measurements were repeated on fresh 155
samples from the same soil to verify reproducibility. Bacterial growth measurements 156
were repeated in three independent experiments, while fungal growth and respiration 157
measurements were repeated in two independent experiments. 158
159
Bacterial and fungal growth. The bacterial growth rate was estimated by measuring the 160
incorporation of 3H-labeled leucine (Leu) into bacteria extracted from soil according to 161
(34) and (35). 2 g of soil were mixed with 20 ml of water followed by a 10 min 162
centrifugation step at 1000g. From the resulting bacterial suspension a 1.5 ml subsample 163
was used to measure bacterial growth. 2 µl [3H] Leu (37 MBq ml−1 and 5.74 TBq 164
mmol−1; Perkin Elmer, UK) was added to the suspension together with non-labeled Leu, 165
resulting in a final concentration of 275 nM Leu. After a 2 h incubation at 22 °C in the 166
dark bacterial growth was terminated by the addition of 100% trichloroacetic acid. After 167
a series of washing steps (34) the amount of incorporated radioactive label was measured 168
using liquid scintillation. To assess whether salt toxicity to bacterial growth rates could 169
be underestimated by measuring bacterial growth in a 20 ml soil suspension, we varied 170
the amount of water added to create the soil suspension (5, 10, 15 and 20 ml). When the 171
salt concentration was considered on a per g soil basis, the different dilutions associated 172
with the homogenization/centrifugation step had no influence on the dose-response 173
relationship (Fig. S1). Since the volume of water added to the soil had no influence on the 174
salt toxicity estimate (Fig. S1), the estimated bacterial response was to the salt 175
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concentration in the soil, prior to water addition, rather than the concentration of salt in 176
the bacterial suspension created. 177
Fungal growth was determined by measuring the incorporation of acetate into 178
ergosterol (32). 1 g of soil was transferred into glass tubes to which a mixture of 20 µl 1-179
[14C]acetic acid (Na+ salt, 2.04 GBq mmol−1, 7.4 MBq ml−1, Perkin Elmer, UK) and 180
unlabeled acetate, resulting in a final acetate concentration of 220 µM, was added 181
together with 1.95 ml distilled water. Samples were then incubated for 4 h at 22°C in the 182
dark, after which growth was terminated with the addition of 1 ml 5% formalin. 183
Ergosterol was then extracted, separated and quantified using high-performance liquid 184
chromatography and a UV detector (282 nm) and collected in a fraction collector (36). 185
The radioactivity incorporated into the ergosterol fraction was measured using liquid 186
scintillation. 187
188
Soil respiration. Basal soil respiration was measured by transferring 2 g of soil into a 20 189
ml vial and purging the headspace with pressurized air. After purging, the vials were 190
closed with crimp seals and incubated in the dark for approximately 16 h at 22 °C. 191
Afterwards the CO2 concentration in the headspace was analysed using a gas 192
chromatograph (GC), equipped with a methanizer and a flame ionization detector, and 193
background levels of CO2 in pressurized air were subtracted. 194
195
196
Nitrogen (N) transformation rates. Gross N mineralization and nitrification rates were 197
determined using the 15N pool dilution/enrichment technique (37). 10 g of soil were 198
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transferred to microcosms and mixed with 300 µl of NH4Cl containing 0.24 g N l-1 (16% 199
15N). One set of soil samples was extracted with 50 ml 1 M KCl within a few minutes 200
after the addition of the 15NH4+-label, a second set after an incubation period of 24 h at 201
room temperature. The extract was filtered through a Whatman GF/F-filter and the 202
concentration of 14NH4 +-N, 15NH4 +-N, 14NO3 −-N, and 15NO3 −-N in the filtrate 203
determined according to standard procedures using acidified diffusion traps containing a 204
filter disc (37). The amount of 15N/14N in the filter discs were measured at the stable 205
isotope facility at the Department of Biology, Lund University using a Flash 2000 206
elemental analyzer connected to a Delta V Plus isotope-ratio mass spectrometer via the 207
ConFlow IV interface (Thermo Scientific Inc., Bremen Germany). 208
209
Duration of toxic effect. Since the different endpoints chosen typically are measured 210
using different time-frames, we also included assessments for the duration of toxic effect 211
by the added salts. For bacterial growth and fungal growth, we covered the range 2 h to 212
96 h after salt addition, and for the effects on mineralization (respiration), we covered the 213
range 6 h to 96 h. 214
215
Calculations and data analysis. In order to compare the sensitivity of different 216
microbial processes to salts, dose-response relationships were determined. To do this, 217
values measured at different levels of salt additions were first normalized by dividing 218
them by the average of the values measured in the samples that received no or low levels 219
of salts and where no inhibition in process was observed. Normalized values would then 220
fall on a range between 1 (no inhibition of process) and 0 (complete inhibition of 221
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process). Values obtained in repeated runs of the experiment were combined to generate a 222
single curve. Log(IC50) values (the logarithm of the salt concentration resulting in 50% 223
inhibition of the process rate) were estimated using a logistic model (Eq. 1): 224
225
y = c / [1 + eb(x-a)] (Eq. 1). 226
227
where y is the relative normalized process rate, x is the logarithm of the salt 228
concentration, a is the value of log(IC50), b is a fitted parameter (slope) indicating the 229
inhibition rate and c is the process rate measured in the control without added salts (38). 230
Kaleidagraph 4.5.0 for Mac (Synergy Software, Reading, PA, USA) was used to fit 231
inhibition curves using the equation above. To compare toxicity, 95% confidence 232
intervals were estimated for the log (IC50) values based on the logistic model. Our used 233
criterion for significant differences was non-overlapping 95 % confidence intervals. This 234
is a conservative criterion, as non-overlapping 85 % confidence intervals correspond to 235
α=0.05 (39) to determine statistical significance. 236
Gross N mineralization and nitrification rates were estimated by the 15N pool 237
dilution/enrichment technique (40-43) using the equations in Table S1. 238
239
Results 240
241
Soil characteristics. The studied soil had a pH of 6.4 and an electrical conductivity of 242
0.09 dS m-1 (Table 1), classifying it as a non-saline soil. The SOM content was 19 mg C 243
g-1, while the NH4+
content was 3.26 µg N g-1, and the NO3- content 6.34 µg N g-1 (Table 244
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1). The total amount of microbial biomass in the soil was 137 µg biomass C g-1, of which 245
87 µg C g-1 were fungal and 50 µg C g-1 bacterial biomass (Table 1). 246
247
Acute toxicity of NaCl to microbial processes. All processes investigated showed clear 248
concentration-response relationships with salinity, with pronounced inhibition at high salt 249
concentrations (Fig. 1-2). These concentration-response relationships could be described 250
with a logistic model (R2 values ranging from 0.78 to 0.95 with an average R2 value of 251
0.88). The acute toxic effects for all these processes remained unchanged in the interval 1 252
h to 48 h after salt addition (Fig. S2). With a longer duration, bacterial growth started to 253
recover between 48 and 96 h , while mineralization and fungal growth remained 254
suppressed for the duration of this comparison. As such, the different standard time-255
frames used for the different endpoints (2 h to 24 h) did not bias the outcome of the 256
comparison. In addition, we also used soil samples sampled at different time points, to 257
investigate how robust our assessments were to generalize from. A formal comparison of 258
time-points showed no differences between soil samples run at different time-points. Log 259
IC50 values estimated using the model ranged from 1.74 for fungal growth, corresponding 260
to 54 µmol NaCl per g soil, to 2.94 for gross N mineralization, corresponding to 870 261
µmol NaCl per g (Table 2). If microbial responses were compared using the resulting 262
electrical conductivity measured in the salt additions, log IC50 values ranged from 0.10 263
for gross nitrification, corresponding to 1.26 dS m-1, to 0.96 for respiration, 264
corresponding to 9.05 dS m-1 (Table 2). 265
Bacterial growth, fungal growth and respiration were inhibited by NaCl exposure 266
to a similar degree, with log IC50 values of 1.80, 1.74 and 1.90, corresponding to NaCl 267
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concentrations of 6.3, 5.5 and 7.9 mol l-1 bacterial suspension, or 63, 55 and 79 µmol 268
NaCl per g soil, respectively. There is some indication that gross N mineralization (Fig. 269
2A), which had a log(IC50) value of 2.94, was less sensitive to NaCl than nitrification (log 270
(IC50) = 1.96) (Fig. 2C). However, there were no significant differences in sensitivity to 271
NaCl between these processes. Gross N mineralization was significantly less sensitive to 272
NaCl than bacterial growth rates. Increasing salinity had no discernible effect on the ratio 273
of C-to-N mineralization rates, which had an overall mass-ratio of 9 ± 1.3 (mean ± se). 274
275
Comparative toxicity of salts. In the second part of the experiment we compared the 276
toxicity of different salts on bacterial and fungal growth, as well as soil respiration (Table 277
2, Fig. 3, Fig. S3-S5). While we could not observe any significant differences between 278
bacterial growth, fungal growth and respiration in response to NaCl exposure, we found 279
that bacterial growth was more sensitive than fungal growth and respiration to KCl (log 280
(IC50) = 1.43) and Na2SO4 (log (IC50) = 1.19) and K2SO4 (log (IC50) = 1.68) (Table 2). 281
These log IC50 values correspond to 2.7, 1.5 and 4.8 mol l-1 bacterial suspension, or 27, 282
15 and 48 mg g-1. Fungal growth and soil respiration were affected by NaCl, KCl and 283
Na2SO4 to the same degree, while K2SO4 inhibited fungal growth (log (IC50) = 2.62, 284
corresponding to 420 mg g-1), but not respiration (Table 2). 285
No significant differences between the susceptibility of bacterial growth to the 286
different salts were found (Fig. 3A, Table 2). Fungal growth was significantly more 287
inhibited by NaCl than any of the other studied salts (Fig. 3B, Table 4). KCl, Na2SO4 and 288
K2SO4 did not differ in their effect on fungal growth rates. Of the salts included in the 289
study, NaCl was the most toxic to respiration (log(IC50) = 1.90) (Fig. 3C, Table 2). There 290
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was no observable inhibitory effect of K2SO4 on respiration rates, even at concentrations 291
that must have resulted in a saturation of the soil solution (Fig. 3C). 292
When salinities were expressed as electrical conductivities measured in 1:5 293
soil:water suspension, results were for the most part similar to using molar concentrations 294
of salts (Table 3, Fig. S6-S8). Of the few observed differences, we found that respiration 295
was significantly less affected by NaCl exposure (log(IC50) = 3.96) than bacterial growth 296
rates (log(IC50) = 3.26). Respiration was also less affected by Na2SO4 (log(IC50) = 4.48) 297
than both bacterial (log(IC50) = 3.05) and fungal growth rates (log(IC50) = 3.77). In 298
contrast to what was found using molar concentrations of salts, fungal growth rates were 299
not more sensitive to NaCl than other salts (Table 3). 300
301
Discussion 302
303
Microbial susceptibility to salts. Microbial growth responses, as well as respiration and 304
N transformation rates, were clearly inhibited by salinity in our experiment. The toxic 305
effects remained stable in the interval of 2-48 h after salt application, showing that the 306
window of opportunity to compare toxic effects was rather wide, and the toxic effects 307
were reproducible to repeated samplings of the same soil, between years and seasons, 308
highlighting the robustness of the assessments. The salinity responses of all microbial 309
processes could be well described with a logistic dose-response relationship (Fig. 1-3). 310
Our findings suggest that all of the above processes could be used as indicators for acute 311
salinity effects on soil microorganisms, and, as such, provide effective measures for 312
comparative toxicity assessments. 313
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In saline soil environments changes in salinity of course would be more gradual 314
than in our experiment. As a consequence microbial communities have more time to 315
respond to the changing conditions. Sensitive organisms would be outcompeted while 316
tolerant organisms would thrive, resulting in a community change. These adaptations and 317
community shifts could offset some of the inhibiting effect of salinity on the microbial 318
community we here document. Nevertheless, microbial activity remains reduced in soils 319
that experienced high salt concentrations for longer periods of time (12, 14, 20, 23), but it 320
is often unclear to which degree this observed reduction represents a direct effect of 321
salinity. Our results therefore represent the potential susceptibility of a process to a direct 322
salt exposure in the studied soil, while the degree to which microbial functions can 323
recover from these perturbations remains to be investigated. 324
We found indications that fungi are more tolerant to acute salt exposure than 325
bacteria for three out of four salts (Fig. 1, Table 2), with the exception of NaCl (Table 2). 326
It has been suggested that the chitinous cell walls of fungi offer better protection against 327
water loss, which makes them more resistant to changes in moisture (6, 44). It would be 328
reasonable that this would also offer protection against low water potential brought about 329
by increased osmotic concentration. Consistent with this prediction, it has been shown 330
that fungi are better able to cope with high osmotic pressure caused by high concentration 331
of organic substrates (8, 9). Moreover, the different localization of the proton gradient 332
used for energy generation in prokaryotic and eukaryotic cells could make fungi more 333
resistant to changes in the cation concentration outside the cell. In eukaryotic cells, post-334
glycolytic reactions take place in the mitochondria and the electrochemical potential used 335
to drive ATP-synthesis is established across the mitochondrial membrane, whereas in 336
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bacteria the potential needs to be maintained across the cell membrane and is therefore 337
more susceptible to environmental conditions outside the cell (45, 46). 338
The observation that fungal growth was less affected by acute salt exposure than 339
bacteria may, if the finding can be extrapolated, suggest that soil salinization could favor 340
fungi over bacteria, which could result in a shift in community composition towards a 341
more fungal-dominated community. In a recent study an increase in the abundance of 342
fungal biomarkers was observed in response to both increasing concentration of salts and 343
drying of soil (47). Important caveats for this extrapolation need to be carefully 344
considered, however. The microcosm systems we used were experimentally dispersal 345
limited, meaning that a number of halotolerant or halophilic microorganisms, that would 346
dominate the microbial community in naturally saline soils, were not present in the non-347
saline soil used in this study. While this would not affect the acute responses to salt, the 348
recovery after salt exposure could have been greatly affected. It is possible that bacteria 349
were particularly affected by this bias. 350
Previous literature reports on the relative dominance of fungi-to-bacteria in 351
naturally saline soils could be used to evaluate how the acute toxicity responses of 352
microorganisms can be translated to ecosystem effects. To date these reports are scarce, 353
however, and the few available studies do not unambiguously support the idea that saline 354
soils become more fungal-dominated. Saline soils have generally been found to contain 355
low microbial biomass, often with a decreasing ratio of fungal to bacterial biomass (14-356
17). However, high salinity often coincides with high alkalinity and it is possible that part 357
of the observed negative dependence of fungi to increasing salinity is driven by the well 358
known effect of pH (32). Consistent with this, a recent study using a salinity gradient not 359
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confounded by soil pH differences observed higher fungal biomass and growth rates in 360
highly saline soils than in non-saline soils (13). While our knowledge on the effects of 361
salinity is limited in soils, more systematic work is available for aquatic ecosystems. 362
Although hypersaline aquatic environments are dominated by prokaryotes, halophilic 363
aquatic fungi exist that can also grow under highly saline conditions (48). Additionally, 364
eukaryotic decomposers may be underrepresented in most aquatic systems for other 365
reasons than high salinity, e.g. the low availability of particulate organic substrate, which 366
is more abundant in terrestrial environments. 367
368
Comparative toxicity of Cl- and SO42- salts. The inhibitory effect of high concentrations 369
of salts on microbial processes is a combination of both the effects of highly negative 370
osmotic potential and of specific ion toxicity. In the comparison of the toxicity of 371
different salts we have the possibility to compare toxicity in terms of both total ionic 372
strength (electric conductivity) and molar concentrations of added salts, thus 373
disentangling osmotic potential and specific ion toxicity. Our comparison suggests a 374
lower toxicity of SO42- salts than Cl- salts at a comparable ionic strength for respiration 375
rates but not for microbial growth rates (Table 3). 376
High concentrations of salts in the cytoplasm of microbial cells can lead to 377
enzyme inhibition due to salting-out caused by high ionic strength. In addition specific 378
ion toxicities have been identified, e.g. some enzymes are particularly sensitive to Na+ 379
and Cl-, due to interactions of the ions with inhibitory binding sites (49). A lower toxicity 380
of SO42- ions than Cl- ions to soil microorganisms has been suggested previously (28), 381
e.g. cultured rhizobia strains were found to be less affected by SO42- salts than the 382
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corresponding Cl- salts (50). Chloride ions inside cells have been shown to inhibit protein 383
synthesis by preventing the binding of ribosomes to mRNA (51, 52). SO42- ions on the 384
other hand can be metabolized by many bacteria and fungi and have no ion-specific 385
toxicity on cellular processes. We could not find clear differences between salts 386
containing Na+ and K+ ions with regards to their toxicity to microbial processes, even 387
though K+ salts have previously been found to be less toxic than Na+ salts (28). 388
389
Responses of the microbial growth efficiency to exposure to salts. Respiration was 390
found to be less sensitive to exposure to SO42- salts than bacterial and fungal growth rates, 391
and affected to a similar degree as fungal growth rates by Cl- salts. K2SO4 was not 392
inhibitory to respiration rate and exerted only low inhibitory effects on growth rates 393
(Table 2, Table 3). This suggests that at high concentration of SO42-salts, microorganisms 394
are still actively respiring but no longer investing resources into biomass production, 395
supporting the hypothesis that microorganisms allocate substrate away from biomass 396
production towards maintenance functions in response to salt exposure, resulting in 397
decreased MGE. At high concentrations of Cl- salts, in contrast, both growth and 398
respiration were inhibited. 399
The methods we used to estimate microbial growth rates estimate protein 400
production (bacterial growth) or lipid synthesis (fungal growth). As such, resources used 401
for the synthesis of low molecular weight compounds for osmoregulation such as organic 402
solutes including betaine, ectoine and various sugars and amino acids would not be a 403
form of growth captured by these methods. These physiological adjustments should only 404
affect estimates of MGE to a minor degree, however (53). Lowered MGE of the 405
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microbial community in response to changes in environmental conditions is frequently 406
interpreted as indication of a stressed community (5, 54, 55). In our case, relating 407
respiration to newly synthesized biomass would lead to the interpretation that exposure to 408
SO42- salts is more stressful to the community than Cl- salt exposure. If these results of 409
acute responses to salinity can be extrapolated to predict longer term effects of salts in 410
soil, they would suggest that accumulation of SO42- salts in the soil can lead to a shift in C 411
allocation from microbial anabolism to catabolism. However, this interpretation is 412
problematically ambiguous. It is equally possible that Cl- salts actually exerted a stronger 413
effect on microorganisms leading to a higher rate of cell death than exposure to SO42- 414
salts. The lower number of surviving cells could have resulted in a stronger inhibition of 415
respiration together with growth, whereas during SO42- exposure more cells were still 416
alive to respire. This would give the misleading impression of a more stressed community 417
suggested by the reduced MGE, emphasizing that caution needs to be exercised in the 418
interpretation and extrapolation of this endpoint. 419
420
Sensitivity of nitrification. Nitrification rates were not found to have a lower log (IC50) 421
value to salt exposure than the other studied microbial processes (Fig. 2C; Table 2). This 422
contrasts with other studies where nitrification has been identified as a process that is 423
especially sensitive to salinity (24, 56). An important difference between our study and 424
many previous studies concerns the length of incubation after the addition of salts. We 425
measured the acute toxicity of salts shortly after salt exposure, whereas other studies 426
usually measure the response of nitrification and other processes after a longer incubation 427
time. This renders our assessment more directly interpretable than previous assessments. 428
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In a longer term assessment, the measured process is a product of two components: First, 429
the direct suppression or inhibition, and, second, the recovery of the process due to 430
sufficient time for a community shift with higher tolerance to salt to occur. The previous 431
reports of higher sensitivity of nitrification compared with other processes could thus be a 432
bias in the recovery of different processes, rather than differences in initial tolerance. The 433
steps involved in nitrification are carried out by only a small number of bacteria and 434
archaea. After addition of salts, microbes carrying out nitrification are probably affected 435
to a similar degree as microbes carrying out other less specialized processes, reflected in 436
a similar acute inhibition. However, a high functional redundancy allows alternative 437
groups to quickly take over reduced general processes, such as respiration, thereby 438
masking the inhibiting effect of salts. Conversely, nitrification remains impaired due to 439
the small species pool of nitrifying organisms. It therefore appears that nitrification is a 440
useful indicator of toxic effects of chemicals in recent history (57, 58), carrying a signal 441
of inhibition for a long duration after exposure. 442
443
Conclusion. Our results show that salinity exerts a strong inhibitory effect on a range of 444
microbial processes in soil, offering effective measures to assess comparative toxicity. 445
Acute toxic effects of added salts occurred immediately (within 2 h), and lasted for at 446
least 48 h, before tolerance induced via community changes led to a recovery of process 447
rates. Fungal growth was found to be less affected by salts than bacterial growth by three 448
of the salts included in the study (KCl, Na2SO4 and K2SO4). This difference in tolerance 449
could translate into ecological relevance by favoring fungi over bacteria at high salinities. 450
Nitrification was not found to be more sensitive to exposure to salts than other processes, 451
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in contrast to previous findings, probably due to the longer experimental time-frame in 452
earlier assessments. Although salinity initially inhibits all microbial processes, the 453
recovery of microbial processes with high functional redundancy, such as respiration, 454
should be significantly faster than more specialized processes, such as nitrification; an 455
imbalance that quickly would manifest into an apparent higher sensitivity of nitrification. 456
All studied salts inhibited microbial growth rates to a similar degree, suggesting that the 457
main factor affecting microbial growth rates is the total ionic strength of the soil solution. 458
In contrast, respiration rates were affected less by salts containing SO42- ions than Cl- 459
salts, indicating specific respiration inhibition by of Cl- ions. Respiration rates were 460
inhibited less than microbial growth rates at the same concentrations of SO42- salts, which 461
could lead to changes in MGE; however, alternative physiological interpretations stress 462
that this index must be interpreted with caution. 463
464
Acknowledgements 465
This work was supported by grants from the Swedish Research Council 466
(Vetenskapsrådet, grant no: 2015-04942), the Royal Physiographical Society of Lund 467
(Kungliga Fysiografiska Sällskapet) and by a PhD studentship awarded by the Centre for 468
Environmental and Climate research (CEC), Lund University. It was also part of the 469
Lund University Centre for studies of Carbon Cycle and Climate Interactions (LUCCI). 470
471
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Table 1: Overview of chemical and biological properties of the soil prior to salt
exposure.
Parameter Mean (SE1)
Water content (% of dry weight) 19.6 (0.1)
Organic matter content (mg C g-1
) 19.3 (0.1)
EC1:5 (dS m-1
)2 0.09 (0.004)
pH3 6.4 (0.03)
N-NH4+
(µg g-1
) 3.3 (0.20)
N-NO3- (µg g
-1) 6.3 (0.18)
Total microbial biomass (µg biomass-C g-1
)4 137 (8)
Fungal biomass (µg biomass-C g-1
) 5 87 (7)
Bacterial biomass (µg biomass-C g-1
) 6 50 (7)
Fungal growth (pmol Ac g-1
h-1
) 14.6 (0.2)
Bacterial growth (pmol Leu g-1
h-1
)
Respiration (µg CO2-C g-1
d-1
)
Gross N mineralization (µg N g-1
d-1
)
Gross nitrification (µg N g-1
d-1
)
42 (2.6)
12.0 (1.1)
1.4 (0.07)
2.0 (0.16)
1 Standard error of the mean
2 electrical conductivity in a 1:5 soil:water suspension
3 measured in a 1:5 soil:water suspension
4 based on substrate induced respiration (SIR) measurement
5 based on ergosterol concentration
6 mass balance of total microbial biomass and fungal biomass
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Table 2: Sensitivity of microbial processes to exposure to NaCl, KCl, Na2SO4 and
K2SO4 (in µmol salt g-1
).
NaCl KCl Na2SO4 K2SO4
logIC501 CI-
95%2
logIC501 CI-
95%2
logIC501 CI-
95%2
logIC501 CI-
95%2
bact.
growth3
1.80
(0.10)
1.60–
1.99
1.43(0.21) 1.02–
1.84
1.19(0.17) 0.85–
1.52
1.68(0.29) 1.11–
2.25
fung.
growth4
1.74
(0.20)
1.34–
2.13
2.53(0.12) 2.30–
2.77
2.56(0.11) 2.34–
2.77
2.62(0.17) 2.29–
2.96
resp.5 1.90
(0.18)
1.55–
2.25
2.66(0.16) 2.34–
3.00
3.12(0.19) 2.75–
3.50
no
inhibition
NH4+
prod.6
2.94
(0.36)
2.16-
3.72
NH4+
cons.7
2.21
(0.14)
1.90–
2.52
NO32-
prod.8
1.96
(0.16)
1.61–
2.31
1IC50values(logµmolg-1)correspondingtothesaltconcentrationleadingtoa50%inhibitionof
processrate.Standarderrorisgivenwithinbrackets.
295%confidenceintervaloftheIC50value
3bacterialgrowthmeasuredas3H-leucineincorporationintobiomass
4fungalgrowthmeasuredas14C-actetateincorporationintoergosterol
5respiration
6NH4+productionrate
7NH4+consumptionrate
8grossnitrification
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Table 3: Sensitivity of microbial processes to the electrical conductivity in a 1:5
soil:water suspension following addition of NaCl, KCl, Na2SO4 and K2SO4.
NaCl KCl Na2SO4 K2SO4
logIC501 CI-
95%2
logIC501 CI-
95%2
logIC501 CI-
95%2
logIC501 CI-
95%2
bact.
growth3
3.26
(0.10)
3.06–
3.45
2.91(0.11) 2.69–
3.12
3.05(0.10) 2.86–
3.25
3.51(0.16) 3.20–
3.83
fung.
growth4
3.67
(0.15)
3.37–
3.96
3.74(0.17) 3.41–
4.07
3.77(0.11) 3.55–
3.98
4.02(0.24) 3.55–
4.49
resp.5 3.96
(0.18)
3.60–
4.31
3.68(0.30) 3.06–
4.30
4.48(0.21) 4.06–
4.89
no
inhibition
NH4+
prod.6
3.94
(0.35)
3.18-
4.70
NH4+
cons.7
3.32
(0.14)
3.01–
3.63
NO32-
prod.8
3.10
(0.15)
2.77–
3.43
1IC50values(logµScm-1)correspondingtothesaltconcentrationleadingtoa50%inhibitionof
processrate.Standarderrorisgivenwithinbrackets.
295%confidenceintervaloftheIC50value
3bacterialgrowthmeasuredas3H-leucineincorporationintobiomass
4fungalgrowthmeasuredas14C-actetateincorporationintoergosterol
5respiration
6NH4+productionrate
7NH4+consumptionrate
8grossnitrification
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