- Email: [email protected]
thawing event
Soil Biology & Biochemistry 100 (2016) 229e232
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Soil bacterial growth after a freezing/thawing event Hannu T. Koponen a, Erland Bååth b, * a b
Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FI-70211, Kuopio, Finland Microbial Ecology, Department of Biology, Ecology Building, Lund University, SE-223 62, Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2016 Received in revised form 23 June 2016 Accepted 26 June 2016 Available online 30 June 2016
Bacterial growth after freezing/thawing was studied in two soils with a history of annual freezing/ thawing events. Soil samples were frozen for 1 week at 3 C or 18 C, thawed at þ4 C, and respiration and bacterial growth (estimated using leucine incorporation) were compared with reference soils kept at þ4 C. There were no major differences between soils. A respiration pulse, peaking within 9 h, was found, but after 30e100 h respiration had decreased to that in the reference. Freezing at 18 C resulted in 2.2e2.5 times higher cumulative respiration than the reference, while at 3 C 1.6e1.8 times higher respiration was found. Bacterial growth rates immediately after thawing were 43e44% of the reference in the 3 C and 23e26% in the 18 C treatment. Growth rates then increased linearly, recovering after 36 h and around 50 h in the 3 C and 18 C freezing, respectively. Growth rates then increased further in the 18 C, but remained lower or similar to the reference in the 3 C treatment. The microbial response to freezing/thawing thus appeared similar to mild drying/rewetting (type 1 response sensu Meisner et al. (2015)). © 2016 Elsevier Ltd. All rights reserved.
Keywords: Freezing/thawing Drying/rewetting Bacterial growth Leucine incorporation Type 1 response
Freezing/thawing is a common phenomenon in temperate and cold climat soils; a perturbation that may occur several times during the year. Freezing/thawing results in a pulse of respiration (Skogland et al., 1988; Schimel and Clein, 1996; Koponen et al., 2006; Henry, 2007; Kim et al., 2012) and a decrease in microbial biomass (Skogland et al., 1988; Henry, 2007; Yanai et al., 2004) indicating freezing/thawing being detrimental to microorganisms. The microbial community composition will also change after a €nnisto € freezing/thawing event (Yergeau and Kowalchuk, 2008; Ma et al., 2009). These are similar effects as after drying/rewetting episodes (Skogland et al., 1988; Kim et al., 2012; Barnard et al., 2013). The actual mechanism may also be similar; freezing results in cells encountering altered osmotic potentials, eventually resulting in cells in a dry state. Thawing is thus a similar phenomenon as rewetting, although usually at much lower temperatures. Two bacterial growth response patterns are found after rewetting dry soil. The type 1 response results in fairly high growth rates directly after rewetting. Bacterial growth then directly starts to increase linearly and recover rapidly to similar or slightly higher growth rates as in moist soil (Iovieno and Bååth, 2008). Respiration
* Corresponding author. E-mail address: [email protected] (E. Bååth). http://dx.doi.org/10.1016/j.soilbio.2016.06.029 0038-0717/© 2016 Elsevier Ltd. All rights reserved.
is highest within hours after rewetting, and then decreases exponentially in the type 1 response. The type 2 response initially has very low growth rates after rewetting, followed by a lag period and an exponential increase in growth, resulting in slower recovery of growth compared to the type 1 response, but eventually in much €ransson et al., 2013). higher growth rates than in moist soils (Go Type 2 often has a second peak following the initial respiration burst upon rewetting. The type of response is soil dependent, but also depends on the extent of drying, where harsher treatments (longer times) result in a type 2 and milder treatments in a type 1 response (Meisner et al., 2013, 2015). Skogland et al. (1988) stated that the killing effect after drying/ rewetting appeared stronger than after freezing/thawing. Thus, we hypothesized that freezing/thawing, presumably similar to a mild drying/rewetting, would result in a type 1 response of bacterial growth. Freezing temperatures affects the killing effect (Elliott and Henry, 2009). Therefore, we compared the effect of freezing at 18 C and 3 C, hypothesizing that 18 C should result in a stronger killing effect and slower recovery of bacterial growth after thawing. We also predicted that 18 C would result in higher respiration due to more dead bacteria and eventually higher bacterial growth than freezing at 3 C. Two agricultural soils from Finland were used. The mull soil is a histosol (28% organic matter, pH(H2O) 5.7). The sandy soil is a medium textured dystric regosol (4.4% organic matter, pH 6.9).
230
H.T. Koponen, E. Bååth / Soil Biology & Biochemistry 100 (2016) 229e232
Mean annual temperature is 2e3 C (Maljanen et al., 2009; with more background data). Frost can appear in December and the soils can remain frozen until AprileJune. Thus, the soils and their organisms have a history of freezing/thawing as a normal part of the annual cycle. Twenty soil cores (5 cm diameter, 0e10 cm depth) were collected for each soil on 10th of October 2008, homogenized and kept at þ4 C (<2 months). Soil samples (25 g in 50 ml vials; 4 separate microcosms per soil and temperature) were frozen at 18 C and 3 C for seven days prior to thawing at þ4 C in a water bath. Soil samples at þ4 C were used as references. The use of a water bath resulted in fairly rapid thawing, within 2 h. The first sample was taken as frozen and bacteria immediately extract in þ4 C water (see below), but later samples were from thawed soil. Microbial activity was assessed by repeated sampling of the microcosms during a month (thus with n ¼ 4 each time), but with emphasis on the first four days for bacterial growth. The first 12 h after thawing, samples were taken every 3 h, the next 3 days twice a day, and at later time points on an even longer time scale. Bacterial growth was measured using leucine incorporation (Bååth et al., 2001) for 2 h at þ4 C (4 h after 7 days). Growth estimated as leucine incorporation into extracted bacteria per hour and g of wet soil will henceforth be denoted bacterial growth rate. Respiration was measured separately on a gas-chromatograph after thawing 20 g of soil in 550 ml bottles sealed with rubber septa using n ¼ 3 separate microcosms per treatment. At each measurement occasion (up to 168 h after thawing, see Fig. 1) CO2 concentration was measured in the beginning and at 3 times during 1.75 h periods. Respiration rate as CO2 released per hour and g of wet soil at þ4 C was calculated. Cumulative respiration during the first 100 h was calculated using the trapezoid method. The dynamics of microbial activities after thawing were similar for the two soils. Peak respiration was found after 9 h for the 3 C freezing, and after 1e5 h for 18 C (Fig. 1A, B). Maximum respiration was 6e9 times that of the þ4 C reference at 18 C freezing, and 3e6 times for soils at 3 C. Respiration became similar to the reference 30e100 h after thawing. Cumulative respiration for the 3 C treatment calculated for the first 100 h after thawing was 1.6 and 1.8 times the þ4 C reference in the mull and sandy soil, respectively, with higher values in the 18 C treatment (2.5 and
7
o
-18 C
4 3 2
B
o
+4 C
Relative respiration rate
Relative respiration rate
o
-3 C 5
10
A
o
+4 C
6
2.2 times the reference). Bacterial growth after freezing/thawing was very different from respiration (Fig. 2). Lowest values were found immediately after thawing. Growth rates in the 3 C treatment were 44 and 43% of the reference in the mull and sandy soil, respectively (Fig. 2C, D). Even lower values were found for the 18 C treatment, 23 and 26%. The survival of the bacteria, estimated as initial growth rates directly after thawing or rewetting, was thus higher for freezing/ thawing compared to drying/rewetting (<10%; Meisner et al., 2013). Cumulative respiration was also smaller (around 2 and 7 times the reference for freezing/thawing and drying/rewetting, respectively), also indicating less killing effects of freezing/thawing. Our results, especially after a 3 C freezing, therefore corroborates the suggestion by Skogland et al. (1988), that freezing/thawing is less detrimental for soil microorganisms compared to drying/rewetting. The bacterial growth rate increased linearly after thawing for both freezing temperatures and in both soils (Fig. 2C, D), with no indications of a long lag period. The dynamics of bacterial growth after freezing/thawing were thus similar to those found after drying/rewetting, resulting in a type 1 response (Iovieno and Bååth, 2008; Meisner et al., 2015). Noteworthy is that the earlier reported uncoupling between bacterial growth and respiration after drying/rewetting, here also for the first time is shown after freezing/thawing. Possible mechanisms explaining this uncoupling have been discussed earlier for drying/rewetting (Meisner et al., 2015) and it is likely that the same mechanism(s) will be true for freezing/thawing. The rate of increase was similar in all cases (slope of the regression line varying between 0.15 and 0.16; Fig. 2C, D). Therefore, the recovery time, i.e. the time point when bacterial growth rates after thawing were the same as in the reference at þ4 C, differed with freezing temperature, being shorter after 3 C freezing, 36 h in both soils, than after 18 C (50 h and 48 h in the mull and sandy soil, respectively). Meisner et al. (2013) reported that it took only 13 h after rewetting to recover bacterial growth rate to that in the moist reference soil. However, bacterial growth and recovery time are temperature dependent. The study by Meisner et al. (2013) was made at þ17 C, while we use þ4 C. Maienza and Bååth (2014) found that a recovery time after drying/ rewetting of a few hours at 25 C, corresponded to a recovery time
8
o
-3 C o
-18 C 6
4
2
1 0
0
50
100
Time after thawing (h)
150
0
0
50
100
150
Time after thawing (h)
Fig. 1. Respiration rates at þ4 C after freezing/thawing A) a mull soil and B) a sandy soil. Filled blue symbols and thick line ¼ frozen at 3 C before thawing, open red symbols and stippled line ¼ frozen at 18 C before thawing, triangles and thin black line ¼ soils kept at þ4 C as references. Data were standardized to 1 for the þ4 C reference for each soil and at every time point. Time indicates middle point of measurements. Bars indicate SE (n ¼ 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
H.T. Koponen, E. Bååth / Soil Biology & Biochemistry 100 (2016) 229e232
2
A
1.4
Relative bacterial growth rate
Relative bacterial growth rate
1.6
1.2 1 0.8
o
+4 C o
-3 C
0.6
o
-18 C 0.4 0.2
0
100
200
300
400
500
600
1.5
1 o
+4 C o
-3 C
0.5
o
-18 C 0
700
B
0
Time after thawing (h)
Relative bacterial growth rate
Relative bacterial growth rate
2
R = 0.72
1 0.8 0.6
o
+4 C o
-3 C
0.4
o
-18 C
Y = 0.016X + 0.23
0.2
2
0
10
20
300
400
500
600
700
D
Y = 0.016X + 0.43
1.4
2
R = 0.78
1.2 1 0.8 0.6
o
+4 C o
-3 C
0.4
o
-18 C
Y = 0.015X + 0.26
0.2
2
R = 0.89 0
200
1.6
C
Y = 0.016X + 0.44
1.2
100
Time after thawing (h)
1.6 1.4
231
R = 0.61 30
40
50
Time after thawing (h)
0
0
10
20
30
40
50
Time after thawing (h)
Fig. 2. Bacterial growth rates (measured as leucine incorporation) at þ4 C after freezing/thawing A) a mull soil and B) a sandy soil. Data for the first 50 h, with a linear regression using only data between 1 and 30 h, are shown for C) the mull and D) the sandy soil. Filled blue symbols and thick line ¼ frozen at 3 C before thawing, open red symbols and stippled line ¼ frozen at 18 C before thawing, triangles and thin black line ¼ soils kept at þ4 C as references. Data were standardized to 1 for the þ4 C reference at every time point. Time indicates middle point of growth measurements. Bars indicate SE (n ¼ 4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of around 10e20 h at 15e20 C, and around 200 h at 5 C. Thus, a recovery time of 36e50 h at þ4 C is actually rapid compared to 13 h at 17 C for drying/rewetting, also suggesting freezing/thawing to be a mild perturbation compared to drying/rewetting. After recovery to the level of the þ4 C reference, growth rates increased further in the 18 C treatments, reaching levels 1.5e2 times the reference (Fig. 2A) and staying high (mull soil) or decreasing over time (sandy soil). In the 3 C treatments the growth rate oscillated around the level found in the reference (Fig. 2B), staying similar as in the reference after 150 h (sandy soil) and 250 h (mull soil). Freezing at low temperatures was a harsher treatment since a larger respiration pulse, indicating extent of killed biomass, was found at 18 C than at 3 C (Fig. 1). This is commonly found (Elliott and Henry, 2009; Gao et al., 2015). Initially lower bacterial growth, indicating harshness of the treatment, was also found in the 18 C compared to the 3 C treatment directly after thawing (Fig. 2C, D). Still, differences between the freezing treatments were small, and the harsher treatment did not change the response from type 1 to type 2, as found when harsher drying conditions are
applied before rewetting (Meisner et al., 2015). Thus, even freezing/ thawing at 18 C, with a type 1 response, could be considered a mild perturbation compared with drying/rewetting conditions, resulting in a type 2 response. However, the differences in the microbial response between 3 C and 18 C freezing still emphasize recommendations (Henry, 2007), that freezing/thawing experiments should be performed at realistic freezing temperatures. This is the first time that the bacterial growth response after freezing/thawing has been followed at high time resolution. The bacterial growth response, a type 1 response, indicated that this perturbation is very similar to a drying/rewetting, although being milder. However, drying/rewetting can induce different response types of bacterial growth in different soils. If this is also the case for freezing/thawing is unknown. Furthermore, soils with different history of freezing/thawing may have different susceptibility to further freezing/thawing (Stres et al., 2010). Since the soils studied here frequently encounter freezing/thawing, it is possible that the microbial community had adapted to such conditions. Freezing/ thawing events in soils not commonly subjected to frost are thus
232
H.T. Koponen, E. Bååth / Soil Biology & Biochemistry 100 (2016) 229e232
predicted to be more detrimental to soil bacteria, with slower recovery of growth. The length of the freezing period may also be of importance, similarly to the extent of the drying period being important for microbial dynamics after rewetting (Meisner et al., 2013, 2015). Acknowledgements The authors acknowledge Prof P.J. Martikainen, University of Eastern Finland, for his support. References € derberg, K.H., 2001. Adaptation of a rapid and Bååth, E., Pettersson, M., So economical microcentrifugation method to measure thymidine and leucine incorporation by soil bacteria. Soil Biol. Biochem. 33, 1571e1574. Barnard, R.L., Osborne, C.A., Firestone, M.K., 2013. Responses of soil bacterial and fungal communities to extreme desiccation and rewetting. ISME J. 7, 2229e2241. Elliott, A.C., Henry, H.A.L., 2009. Freeze-thaw cycle amplitude and freezing rate effects on extractable nitrogen in a temperate old field soil. Biol. Fert. Soils 45, 469e476. Gao, Y., Zeng, X., Xie, Q., Ma, X., 2015. Release of carbon and nitrogen from alpine soils during thawing periods in the eastern Qinghai-Tibet plateau. Water Air Soil Poll. 226 article No 209. €ransson, H., Godbold, D.L., Jones, D.L., Rousk, J., 2013. Bacterial growth and Go respiration responses upon rewetting dry forest soil: impact of drought-legacy. Soil Biol. Biochem. 57, 477e486. Henry, H.A.L., 2007. Soil freeze-thaw experiments: trends, methodological weaknesses and suggested improvements. Soil Biol. Biochem. 39, 977e986. Iovieno, P., Bååth, E., 2008. Effect of drying and rewetting on bacterial growth rates
in soil. FEMS Microb. Ecol. 65, 400e407. Kim, D.-G., Vargas, R., Bond-Lamberty, B., Turetsky, M.R., 2012. Effects of soil rewetting and thawing on soil gas fluxes: a review of current literature and suggestions for future research. Biogeosciences 9, 2459e2483. €nen-Toivola, M.M., Kaipanen, S., Tuomainen, J., Koponen, H.T., Jaakola, T., Keina Servomaa, K., Martikainen, P.J., 2006. Microbial communities, biomass and activities in soils as affected by freeze thaw cycles. Soil Biol. Biochem. 38, 1861e1871. Maienza, A., Bååth, E., 2014. Temperature effects on recovery time of bacterial growth after rewetting dry soil. Microb. Ecol. 68, 818e821. € €nen, J., Oquist, Maljanen, M., Virkaj€ arvi, P., Hyto M., Sparrman, T., Martikainen, P.J., 2009. Nitrous oxide production in boreal soils with variable organic matter content at low temperature e snow manipulation experiment. Biogeosciences 6, 2461e2473. €nnisto € , M.K., Tiirola, M., H€ Ma aggblom, M.M., 2009. Effect of freeze-thaw cycles on bacterial communities of arctic tundra soil. Microb. Ecol. 58, 621e631. Meisner, A., Rousk, J., Bååth, E., 2013. Microbial responses upon rewetting soil dried for four days or one year. Soil Biol. Biochem. 66, 188e192. Meisner, A., Rousk, J., Bååth, E., 2015. Prolonged drought changes the bacterial growth response to rewetting. Soil Biol. Biochem. 88, 314e322. Schimel, J.P., Clein, J.S., 1996. Microbial response to freeze-thaw cycles in tundra and taiga soils. Soil Biol. Biochem. 28, 1061e1066. Skogland, T., Lomeland, S., Goksøyr, J., 1988. Respiratory burst after freezing and thawing of soil: experiments with soil bacteria. Soil Biol. Biochem. 6, 851e856. Stres, B., Philippot, L., Faganeli, J., Tiedje, J.M., 2010. Frequent freeze-thaw cycles yield diminished yet resistant and responsive microbial communities in two temperate soils: a laboratory experiment. FEMS Microb. Ecol. 74, 323e335. Yanai, Y., Toyata, K., Okazaki, M., 2004. Effects of successive soil freeze-thaw cycles on soil microbial biomass and organic matter decomposition potential of soils. Soil Sci. Plant Nutr. 50, 821e829. Yergeau, E., Kowalchuk, G.A., 2008. Responses of Antarctic soil microbial communities and associated functions to temperature and freeze-thaw cycle frequency. Env. Microbiol. 10, 2223e2235.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Soil bacterial growth after a freezing/thawing event Hannu T. Koponen a, Erland Bååth b, * a b
Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FI-70211, Kuopio, Finland Microbial Ecology, Department of Biology, Ecology Building, Lund University, SE-223 62, Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2016 Received in revised form 23 June 2016 Accepted 26 June 2016 Available online 30 June 2016
Bacterial growth after freezing/thawing was studied in two soils with a history of annual freezing/ thawing events. Soil samples were frozen for 1 week at 3 C or 18 C, thawed at þ4 C, and respiration and bacterial growth (estimated using leucine incorporation) were compared with reference soils kept at þ4 C. There were no major differences between soils. A respiration pulse, peaking within 9 h, was found, but after 30e100 h respiration had decreased to that in the reference. Freezing at 18 C resulted in 2.2e2.5 times higher cumulative respiration than the reference, while at 3 C 1.6e1.8 times higher respiration was found. Bacterial growth rates immediately after thawing were 43e44% of the reference in the 3 C and 23e26% in the 18 C treatment. Growth rates then increased linearly, recovering after 36 h and around 50 h in the 3 C and 18 C freezing, respectively. Growth rates then increased further in the 18 C, but remained lower or similar to the reference in the 3 C treatment. The microbial response to freezing/thawing thus appeared similar to mild drying/rewetting (type 1 response sensu Meisner et al. (2015)). © 2016 Elsevier Ltd. All rights reserved.
Keywords: Freezing/thawing Drying/rewetting Bacterial growth Leucine incorporation Type 1 response
Freezing/thawing is a common phenomenon in temperate and cold climat soils; a perturbation that may occur several times during the year. Freezing/thawing results in a pulse of respiration (Skogland et al., 1988; Schimel and Clein, 1996; Koponen et al., 2006; Henry, 2007; Kim et al., 2012) and a decrease in microbial biomass (Skogland et al., 1988; Henry, 2007; Yanai et al., 2004) indicating freezing/thawing being detrimental to microorganisms. The microbial community composition will also change after a €nnisto € freezing/thawing event (Yergeau and Kowalchuk, 2008; Ma et al., 2009). These are similar effects as after drying/rewetting episodes (Skogland et al., 1988; Kim et al., 2012; Barnard et al., 2013). The actual mechanism may also be similar; freezing results in cells encountering altered osmotic potentials, eventually resulting in cells in a dry state. Thawing is thus a similar phenomenon as rewetting, although usually at much lower temperatures. Two bacterial growth response patterns are found after rewetting dry soil. The type 1 response results in fairly high growth rates directly after rewetting. Bacterial growth then directly starts to increase linearly and recover rapidly to similar or slightly higher growth rates as in moist soil (Iovieno and Bååth, 2008). Respiration
* Corresponding author. E-mail address: [email protected] (E. Bååth). http://dx.doi.org/10.1016/j.soilbio.2016.06.029 0038-0717/© 2016 Elsevier Ltd. All rights reserved.
is highest within hours after rewetting, and then decreases exponentially in the type 1 response. The type 2 response initially has very low growth rates after rewetting, followed by a lag period and an exponential increase in growth, resulting in slower recovery of growth compared to the type 1 response, but eventually in much €ransson et al., 2013). higher growth rates than in moist soils (Go Type 2 often has a second peak following the initial respiration burst upon rewetting. The type of response is soil dependent, but also depends on the extent of drying, where harsher treatments (longer times) result in a type 2 and milder treatments in a type 1 response (Meisner et al., 2013, 2015). Skogland et al. (1988) stated that the killing effect after drying/ rewetting appeared stronger than after freezing/thawing. Thus, we hypothesized that freezing/thawing, presumably similar to a mild drying/rewetting, would result in a type 1 response of bacterial growth. Freezing temperatures affects the killing effect (Elliott and Henry, 2009). Therefore, we compared the effect of freezing at 18 C and 3 C, hypothesizing that 18 C should result in a stronger killing effect and slower recovery of bacterial growth after thawing. We also predicted that 18 C would result in higher respiration due to more dead bacteria and eventually higher bacterial growth than freezing at 3 C. Two agricultural soils from Finland were used. The mull soil is a histosol (28% organic matter, pH(H2O) 5.7). The sandy soil is a medium textured dystric regosol (4.4% organic matter, pH 6.9).
230
H.T. Koponen, E. Bååth / Soil Biology & Biochemistry 100 (2016) 229e232
Mean annual temperature is 2e3 C (Maljanen et al., 2009; with more background data). Frost can appear in December and the soils can remain frozen until AprileJune. Thus, the soils and their organisms have a history of freezing/thawing as a normal part of the annual cycle. Twenty soil cores (5 cm diameter, 0e10 cm depth) were collected for each soil on 10th of October 2008, homogenized and kept at þ4 C (<2 months). Soil samples (25 g in 50 ml vials; 4 separate microcosms per soil and temperature) were frozen at 18 C and 3 C for seven days prior to thawing at þ4 C in a water bath. Soil samples at þ4 C were used as references. The use of a water bath resulted in fairly rapid thawing, within 2 h. The first sample was taken as frozen and bacteria immediately extract in þ4 C water (see below), but later samples were from thawed soil. Microbial activity was assessed by repeated sampling of the microcosms during a month (thus with n ¼ 4 each time), but with emphasis on the first four days for bacterial growth. The first 12 h after thawing, samples were taken every 3 h, the next 3 days twice a day, and at later time points on an even longer time scale. Bacterial growth was measured using leucine incorporation (Bååth et al., 2001) for 2 h at þ4 C (4 h after 7 days). Growth estimated as leucine incorporation into extracted bacteria per hour and g of wet soil will henceforth be denoted bacterial growth rate. Respiration was measured separately on a gas-chromatograph after thawing 20 g of soil in 550 ml bottles sealed with rubber septa using n ¼ 3 separate microcosms per treatment. At each measurement occasion (up to 168 h after thawing, see Fig. 1) CO2 concentration was measured in the beginning and at 3 times during 1.75 h periods. Respiration rate as CO2 released per hour and g of wet soil at þ4 C was calculated. Cumulative respiration during the first 100 h was calculated using the trapezoid method. The dynamics of microbial activities after thawing were similar for the two soils. Peak respiration was found after 9 h for the 3 C freezing, and after 1e5 h for 18 C (Fig. 1A, B). Maximum respiration was 6e9 times that of the þ4 C reference at 18 C freezing, and 3e6 times for soils at 3 C. Respiration became similar to the reference 30e100 h after thawing. Cumulative respiration for the 3 C treatment calculated for the first 100 h after thawing was 1.6 and 1.8 times the þ4 C reference in the mull and sandy soil, respectively, with higher values in the 18 C treatment (2.5 and
7
o
-18 C
4 3 2
B
o
+4 C
Relative respiration rate
Relative respiration rate
o
-3 C 5
10
A
o
+4 C
6
2.2 times the reference). Bacterial growth after freezing/thawing was very different from respiration (Fig. 2). Lowest values were found immediately after thawing. Growth rates in the 3 C treatment were 44 and 43% of the reference in the mull and sandy soil, respectively (Fig. 2C, D). Even lower values were found for the 18 C treatment, 23 and 26%. The survival of the bacteria, estimated as initial growth rates directly after thawing or rewetting, was thus higher for freezing/ thawing compared to drying/rewetting (<10%; Meisner et al., 2013). Cumulative respiration was also smaller (around 2 and 7 times the reference for freezing/thawing and drying/rewetting, respectively), also indicating less killing effects of freezing/thawing. Our results, especially after a 3 C freezing, therefore corroborates the suggestion by Skogland et al. (1988), that freezing/thawing is less detrimental for soil microorganisms compared to drying/rewetting. The bacterial growth rate increased linearly after thawing for both freezing temperatures and in both soils (Fig. 2C, D), with no indications of a long lag period. The dynamics of bacterial growth after freezing/thawing were thus similar to those found after drying/rewetting, resulting in a type 1 response (Iovieno and Bååth, 2008; Meisner et al., 2015). Noteworthy is that the earlier reported uncoupling between bacterial growth and respiration after drying/rewetting, here also for the first time is shown after freezing/thawing. Possible mechanisms explaining this uncoupling have been discussed earlier for drying/rewetting (Meisner et al., 2015) and it is likely that the same mechanism(s) will be true for freezing/thawing. The rate of increase was similar in all cases (slope of the regression line varying between 0.15 and 0.16; Fig. 2C, D). Therefore, the recovery time, i.e. the time point when bacterial growth rates after thawing were the same as in the reference at þ4 C, differed with freezing temperature, being shorter after 3 C freezing, 36 h in both soils, than after 18 C (50 h and 48 h in the mull and sandy soil, respectively). Meisner et al. (2013) reported that it took only 13 h after rewetting to recover bacterial growth rate to that in the moist reference soil. However, bacterial growth and recovery time are temperature dependent. The study by Meisner et al. (2013) was made at þ17 C, while we use þ4 C. Maienza and Bååth (2014) found that a recovery time after drying/ rewetting of a few hours at 25 C, corresponded to a recovery time
8
o
-3 C o
-18 C 6
4
2
1 0
0
50
100
Time after thawing (h)
150
0
0
50
100
150
Time after thawing (h)
Fig. 1. Respiration rates at þ4 C after freezing/thawing A) a mull soil and B) a sandy soil. Filled blue symbols and thick line ¼ frozen at 3 C before thawing, open red symbols and stippled line ¼ frozen at 18 C before thawing, triangles and thin black line ¼ soils kept at þ4 C as references. Data were standardized to 1 for the þ4 C reference for each soil and at every time point. Time indicates middle point of measurements. Bars indicate SE (n ¼ 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
H.T. Koponen, E. Bååth / Soil Biology & Biochemistry 100 (2016) 229e232
2
A
1.4
Relative bacterial growth rate
Relative bacterial growth rate
1.6
1.2 1 0.8
o
+4 C o
-3 C
0.6
o
-18 C 0.4 0.2
0
100
200
300
400
500
600
1.5
1 o
+4 C o
-3 C
0.5
o
-18 C 0
700
B
0
Time after thawing (h)
Relative bacterial growth rate
Relative bacterial growth rate
2
R = 0.72
1 0.8 0.6
o
+4 C o
-3 C
0.4
o
-18 C
Y = 0.016X + 0.23
0.2
2
0
10
20
300
400
500
600
700
D
Y = 0.016X + 0.43
1.4
2
R = 0.78
1.2 1 0.8 0.6
o
+4 C o
-3 C
0.4
o
-18 C
Y = 0.015X + 0.26
0.2
2
R = 0.89 0
200
1.6
C
Y = 0.016X + 0.44
1.2
100
Time after thawing (h)
1.6 1.4
231
R = 0.61 30
40
50
Time after thawing (h)
0
0
10
20
30
40
50
Time after thawing (h)
Fig. 2. Bacterial growth rates (measured as leucine incorporation) at þ4 C after freezing/thawing A) a mull soil and B) a sandy soil. Data for the first 50 h, with a linear regression using only data between 1 and 30 h, are shown for C) the mull and D) the sandy soil. Filled blue symbols and thick line ¼ frozen at 3 C before thawing, open red symbols and stippled line ¼ frozen at 18 C before thawing, triangles and thin black line ¼ soils kept at þ4 C as references. Data were standardized to 1 for the þ4 C reference at every time point. Time indicates middle point of growth measurements. Bars indicate SE (n ¼ 4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of around 10e20 h at 15e20 C, and around 200 h at 5 C. Thus, a recovery time of 36e50 h at þ4 C is actually rapid compared to 13 h at 17 C for drying/rewetting, also suggesting freezing/thawing to be a mild perturbation compared to drying/rewetting. After recovery to the level of the þ4 C reference, growth rates increased further in the 18 C treatments, reaching levels 1.5e2 times the reference (Fig. 2A) and staying high (mull soil) or decreasing over time (sandy soil). In the 3 C treatments the growth rate oscillated around the level found in the reference (Fig. 2B), staying similar as in the reference after 150 h (sandy soil) and 250 h (mull soil). Freezing at low temperatures was a harsher treatment since a larger respiration pulse, indicating extent of killed biomass, was found at 18 C than at 3 C (Fig. 1). This is commonly found (Elliott and Henry, 2009; Gao et al., 2015). Initially lower bacterial growth, indicating harshness of the treatment, was also found in the 18 C compared to the 3 C treatment directly after thawing (Fig. 2C, D). Still, differences between the freezing treatments were small, and the harsher treatment did not change the response from type 1 to type 2, as found when harsher drying conditions are
applied before rewetting (Meisner et al., 2015). Thus, even freezing/ thawing at 18 C, with a type 1 response, could be considered a mild perturbation compared with drying/rewetting conditions, resulting in a type 2 response. However, the differences in the microbial response between 3 C and 18 C freezing still emphasize recommendations (Henry, 2007), that freezing/thawing experiments should be performed at realistic freezing temperatures. This is the first time that the bacterial growth response after freezing/thawing has been followed at high time resolution. The bacterial growth response, a type 1 response, indicated that this perturbation is very similar to a drying/rewetting, although being milder. However, drying/rewetting can induce different response types of bacterial growth in different soils. If this is also the case for freezing/thawing is unknown. Furthermore, soils with different history of freezing/thawing may have different susceptibility to further freezing/thawing (Stres et al., 2010). Since the soils studied here frequently encounter freezing/thawing, it is possible that the microbial community had adapted to such conditions. Freezing/ thawing events in soils not commonly subjected to frost are thus
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predicted to be more detrimental to soil bacteria, with slower recovery of growth. The length of the freezing period may also be of importance, similarly to the extent of the drying period being important for microbial dynamics after rewetting (Meisner et al., 2013, 2015). Acknowledgements The authors acknowledge Prof P.J. Martikainen, University of Eastern Finland, for his support. References € derberg, K.H., 2001. Adaptation of a rapid and Bååth, E., Pettersson, M., So economical microcentrifugation method to measure thymidine and leucine incorporation by soil bacteria. Soil Biol. Biochem. 33, 1571e1574. Barnard, R.L., Osborne, C.A., Firestone, M.K., 2013. Responses of soil bacterial and fungal communities to extreme desiccation and rewetting. ISME J. 7, 2229e2241. Elliott, A.C., Henry, H.A.L., 2009. Freeze-thaw cycle amplitude and freezing rate effects on extractable nitrogen in a temperate old field soil. Biol. Fert. Soils 45, 469e476. Gao, Y., Zeng, X., Xie, Q., Ma, X., 2015. Release of carbon and nitrogen from alpine soils during thawing periods in the eastern Qinghai-Tibet plateau. Water Air Soil Poll. 226 article No 209. €ransson, H., Godbold, D.L., Jones, D.L., Rousk, J., 2013. Bacterial growth and Go respiration responses upon rewetting dry forest soil: impact of drought-legacy. Soil Biol. Biochem. 57, 477e486. Henry, H.A.L., 2007. Soil freeze-thaw experiments: trends, methodological weaknesses and suggested improvements. Soil Biol. Biochem. 39, 977e986. Iovieno, P., Bååth, E., 2008. Effect of drying and rewetting on bacterial growth rates
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