Microbial activity in soils frozen to below −39 °C

Soil Biology & Biochemistry 38 (2006) 785–794 www.elsevier.com/locate/soilbio

Microbial activity in soils frozen to below K39 8C N.S. Panikova,*, P.W. Flanaganb, W.C. Oechelc, M.A. Mastepanovd, T.R. Christensend a

Chemistry and Chemical Biology Department, Stevens Institute of Technology, Castle Point on Hudson, Hoboken, NJ 07030, USA b Global Environmental Enterprises, Inc., 12133 Winfree Street, Chester, VA 23831, USA c Global Change Research Group, San Diego State University, San Diego, CA 92182, USA d Department of Physical Geography and Ecosystem Analysis, Lund University, SE-22362, Lund, Sweden Received 4 August 2004; received in revised form 15 July 2005; accepted 15 July 2005 Available online 18 August 2005

Abstract Recent research on life in extreme environments has shown that some microorganisms metabolize at extremely low temperatures in Arctic and Antarctic ice and permafrost. Here, we present kinetic data on CO2 and 14CO2 release from intact and 14C-glucose amended tundra soils (Barrow, Alaska) incubated for up to a year at 0 to K398C. The rate of CO2 production declined exponentially with temperature but it remained positive and measurable, e.g. 2–7 ng CO2–C cmK3 soil dK1, at K39 8C. The variation of CO2 release rate (v) was adequately explained by the double exponential dependence on temperature (T) and unfrozen water content (W) (r2O0.98): vZA exp(lTCkW) and where A, l and k are constants. The rate of 14CO2 release from added glucose declined more steeply with cooling as compared with the release of total CO2, indicating that (a) there could be some abiotic component in the measured flux of CO2 or (b) endogenous respiration is more coldresistant than substrate-induced respiration. The respiration activity was completely eliminated by soil sterilization (1 h, 121 8C), stimulated by the addition of oxidizable substrate (glucose, yeast extract), and reduced by the addition of acetate, which inhibits microbial processes in acidic soils (pH 3–5). The tundra soil from Barrow displayed higher below-zero activity than boreal soils from West Siberia and Sweden. The permafrost soils (20–30 cm) were more active than the samples from seasonally frozen topsoil (0–10 cm, Barrow). Finding measurable respiration to K39 8C is significant for determining, understanding, and predicting current and future CO2 emission to the atmosphere and for understanding the low temperature limits of microbial activity on the Earth and on other planets. q 2005 Elsevier Ltd. All rights reserved. Keywords: Psychrophiles; Kinetic analysis; Winter emission; CO2 entrapment; Respiration; Unfrozen water, Arctic soil respiration

1. Introduction Winter emission of CO2 from northern soils is a significant source of atmospheric CO2 that can account for up to half of the annual emission of CO2 from Arctic and boreal/forest ecosystems (Sommerfeld et al., 1993; Zimov et al., 1993, 1996; Oechel et al., 1997; Fahnestock et al., 1998, 1999; Jones et al., 1999; Panikov and Dedysh, 2000; Welker et al., 2000; Mikan et al., 2002; Schimel et al., 2004). The net exchange of CO2 is the difference between photosynthetic CO2 uptake and ecosystem respiration. During the cold season, photosynthesis is near 0 and respiration is 3–7% of the mid-summer intensity. Although * Corresponding author. Tel.: C1 201 216 8193; fax: C1 201 216 8240. E-mail address: [email protected] (N.S. Panikov).

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.07.004

they are not very strong, per se, the winter processes can significantly affect the net annual CO2 emission, as during the summer, photosynthesis and respiration can be nearly equal. When winter flux is considered, Arctic ecosystems, which can be a net sink in the summer, are often a net source of CO2 to the atmosphere on an annual basis (Oechel et al., 1997). However, the mechanism for the observed cold season CO2 emission has been unclear and could conceivably result from a variety of processes. From the literature, we found the following hypothetical explanations of winter CO2 emission: (a) the physical release of summer-accumulated gases, accelerated by soil freezing fronts moving down or up, in fall or spring, respectively (Coyne and Kelley, 1971), (b) the biological activity occurring in a warm, unthawed soil layer (Zimov et al., 1993), and (c) the respiration of cold-adapted (i.e. /0 8C) microbes and plant roots within the bulk of the frozen soil (Oechel et al., 1997; Panikov, 1999).

786

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

Do microorganisms metabolize in the northern polar tundra when the winter soil surface temperature drops to K40 8C (Romanovsky and Ostercamp, 1995; Romanovsky et al., 2003)? Most textbooks on microbiology clearly deny demonstrable metabolism in the frozen state because of the absence of free water. However, microbial growth and activity well below the freezing point have been occasionally recorded in ‘frozen’ food (Larkin and Stokes, 1968), polar sea ice, frozen soil, and permafrost (Schroeter et al., 1994; Kappen et al., 1996). Studies on microbial growth below 0 8C (Mazur, 1980; Russell, 1990; Finegold, 1996; Geiges, 1996) confirm that growth ceases or becomes imperceptible, requiring prolonged incubation at about K12 8C. Earlier reports on microbial activity below K12 8C were not confirmed (Mazur, 1980). Growth of bacteria and fungi in frozen food is generally considered to be limited to temperatures above K8 8C (Geiges, 1996). Pure laboratory cultures of fungi or bacteria isolated from Arctic and Antarctic soils have been reported to stop growth and lose metabolic activity in the range of K7 to K128C (Straka and Stokes, 1960; Larkin and Stokes, 1968; Flanagan and Bunnell, 1980; Mazur, 1980). Soil fungi have been considered to be the main source of CO2 released below 0 8C in tundra (Flanagan and Veum, 1974; Flanagan and Bunnell, 1980) because their live biomass was estimated to be 10 times larger than that of co-occurring bacteria. The lowest temperature allowing continuous metabolism was reported for Antarctic lichens that photosynthesized down to K17 8C (Schroeter et al., 1994; Kappen et al., 1996). A significant part of the above data was obtained with isolated microbial cultures (an exception are lichens, in which in situ growth was observed macroscopically). However, most soil microorganisms ([90%) do not grow on laboratory media and among those could be species more resistant to cold than those successfully cultured to date (Amann et al., 1995). The temperature response of the whole soil (active layer or permafrost) community was measured in a number of laboratory incubation studies. The rate of 14C-acetate incorporation into lipids was used to quantify microbial growth in Siberian permafrost samples (Rivkina et al., 2000). The lowest temperature studied was K20 8C and incubation was as long as 600 d. However, data points below K12 8C were close to the detection limits of the technique used. The authors concluded that while C uptake at K20 8C could be measured transiently during the abnormal ‘log’ growth phase caused by substrate amendment, under natural stable permafrost conditions, the respiration would not be measurable (Rivkina et al., 2000). With Alaskan tundra soils, metabolic activity was assessed directly as observed CO2 evolution (which is respirationCdiffusion of any gas accumulated in the soil) from isolated soil samples incubated from 0 to K10 8C (Mikan et al., 2002). The rates of CO2 formation declined abruptly with freezing, producing abnormally high Q10 values (up to 237),

suggesting that factors other than temperature (e.g. limited water availability) have a significant effect on microbial activity. In a similar study of well-drained tundra heath from Northeastern Greenland (Elberling and Brandt, 2003), the release of CO2 was observed down to K18 8C. The authors confirmed the anomaly in Q10 and explained this phenomenon by uncoupling CO2 production in frozen soils from CO2 release into the atmosphere: they believe that soil organisms continuously produce CO2 during summer and winter seasons; however, up to 80% of the winter-produced CO2 is trapped in frozen soil to be released in the spring. Our objective was to test the hypothesis that the microbial communities are able to metabolize and produce CO2 as a final product of microbial respiration in soil frozen to well below 0 8C. These metabolic reactions should be distinguished from abiotic processes, such as liberation of previously respired CO2 trapped in frozen soil. To test this hypothesis, we used (a) advanced and reliable automated incubation techniques that allowed us to extend the longterm respiration measurements up to a year and to precisely maintain temperatures in the range from 0 to K40 8C, (b) laboratory incubations with intact and 14C-amended soils, and (c) kinetic analysis of dynamic data to differentiate metabolic respiration from abiotic CO2 release.

2. Materials and methods 2.1. Soils The samples of frozen soil were taken from three sites: (a) Barrow (71819 0 N, 156836 0 W), tussock tundra under Carex aquatilis, Eriophorum angustifolium, Arctophila fulva, and Dupontia fishcheri: The active layer is generally no more than 40 cm from the surface, the site is seasonally frozen from mid-October until June, the mean annual air temperature is K12 8C, and the soil temperature at 10 cm varies in January from K20 to K25 8C. Other site details are described elsewhere (Brown et al., 1980; Oechel et al., 1997). (b) West Siberia, Tomsk Area, Plotnikovo Field Station (57801 0 N, 82835 0 E), mesotrophic peat bog under Carex rostrata–Sphagnum majus plant community: The site is seasonally frozen from November until May, the mean annual air temperature is K0.2 8C, and the January soil temperature at 10 cm ranges from K9 to K12 8C, as described by Panikov and Dedysh (2000). (c) Koppara˚s mire, South-Central Sweden (5787.5 0 N, 14830 0 E), mesotrophic bog under Sphagnum magellanicum, S. papillosum, Eriophorum angustifolium, and Calluna vulgaris: This site is mainly unfrozen, with an average January soil temperature at 10 cm of around K2 8C. Malmer (1962) described the sampling area of the Koppara˚s site.

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

The frozen soils were extracted with a metal corer from the upper 0–30 cm of earth after snow removal. They were delivered unthawed to the laboratory and stored at K18 8C before the experiments. 2.2. Measurements of respiration In the walk-in cold room (K18 8C), the frozen soil was broken up into aggregates 3–5 mm in size to increase the gas transfer rate, and packed into airtight polypropylene columns, 40 g soil columnK1. The columns were incubated in a refrigerated circulator bath (Model FP40-HD, Julabo USA, Inc.) filled with absolute ethanol as antifreeze. The rate of CO2 release was measured automatically (Fig. 1) with regular (every 0.1–6 h, depending on activity) flushing of the soil columns with a synthetic, pre-cooled air (O2: N2Z20: 80, 370 ml CO2 lK1, at a flow rate of 50 ml minK1. The CO2 was determined using non-dispersive infrared (NDIR) gas analyzers (Li-800, LI-COR, Inc., Lincoln, NE, USA). Automatic respirometry was normally run with eight gas lines: one background, one calibration mixture (Scott Gas, USA), and six soil columns. Two sets of PC-controlled solenoid valves (KIP Inc., USA) directed airflow from the gas tanks to the soil columns via an electronic flow stabilizer (300-FID, California Analytical Instruments) and a digital flow meter (Alicat Scientific, Inc., USA). To log data to a PC, we developed software, ‘Profiler2000’, which allows the calculation of respiration rates from both the height and area of CO2 peaks produced after each soil flushing. The temperature was recorded continuously (HOBO XT Temperature Data Logger, Onset Computer Corporation, MA, USA), with a temperature sensor tip implanted in one of the soil columns.

787

The lower detection limit of the described procedure was about 1 ml CO2 lK1 dK1, which is equivalent to CO2 flux as low as 15 mg CO2–C mK2 dK1 (assuming emission from the upper, 30-cm soil layer). This is lower by a factor of 10K5– 10K6 than soil respiration typically measured at 15–20 8C. Field measurements of winter emission are reported to vary between 80 and 300 mg C mK2 dK1 (Sommerfeld et al., 1993; Zimov et al., 1993; Oechel et al., 1997; Panikov and Dedysh, 2000). (Summer soil respiration values would be well above the useful range of the equipment employed here.) 2.3. Differentiation of respiration and abiotic release of trapped CO2 The analyzed frozen soil samples contain some amount of trapped CO2. The neutral and alkaline soils also have CO23 and HCO3 releasing CO2 after metabolic acidification. Emission of CO2 derived from these sources contributes to the apparent CO2 flux from incubated soil interfering with the determination of true respiration rates. To account for this contribution, we assume the first-order kinetics of the release of CO2 and slower first- or zero-order CO2 evolution originating from respiratory oxidative reactions. Both components can be separated by fitting the experimental dynamic data to the following equation: Release of trapped CO2

Net CO2 flux

dp=dt

Z

k1 s

Microbial respiration

RðtÞ

C

Z k1 s0 expKðk1 tÞ C RðtÞ

(1)

where p is the CO2 concentration in the air headspace over the soil, s is the instantaneous pool size of CO2 in the soil, s0

4

2

6

1

IRGA

Alcohol Bath –40…+10 °C 4'

Air KOH

KOH 5

KOH 7

Fig. 1. The experimental set-up to determine respiration in frozen soil samples: (1) synthetic air flow control, (2) pre-cooling loop, (3) plastic container filled with 2–5 mm soil crumbles, with thermistor temperature sensor inside (the whole system was placed inside a Julabo thermostat with ethanol as antifreeze), (4) and (4 0 ) solenoid valves controlled by PC, (5) 14CO2 absorbers, (6) CO2 analyzer, and (7) data acquisition computer displaying CO2 recording peak.

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

is the s-value at zero time, tZ0, and k1 is the kinetic constant for CO2 mass-transfer. The respiration rate, R(t), was assumed to be a dynamic variable slowly declining, according to the exponential equation:

3.1. Dynamics of CO2 exchange during isothermal subzero soil incubation

(2)

where R 0 and k2 are constants and k2!k1. To assess directly the amount of trapped CO2 and to compare this with the calculated value s0, 5 g of frozen soil were placed into 30-ml capped vials and after injection of 3.0 ml of 1 M H3PO4, were incubated at room temperature for 1 h. The CO2 released to the headspace was measured with a GC (Shimadzu GC-14A gas chromatograph equipped with TCD, the Supelco 80/100 Carboxen 1004-column, 2-m!1.6-mm, He as a carrier gas) and was used to calculate 2K the total amount of soil mineral C: CO2 C HCOK 3 CCO3 . 2.4. Soil incubation with added substrates, 14C-counting To minimize the interference of trapped CO 2 on respiratory measurements, the crushed frozen soil was kept at K18 8C for 2–7 d in desiccators under soda lime before the incubation experiments began. Then the soil aggregates were allowed to warm to C1 8C in a temperature-controlled bath. Five mg of unlabelled D-glucose and 0.1 mCi of 14C-glucose (Sigma) were added gK1 of soil. Labeled CO2 was counted in 1 N KOH traps placed after the second set of solenoid valves and the total (12CC14C) CO2 evolution was recorded by IR gas analyzer (note that two sets of solenoid valves were needed to measure simultaneously labeled and unlabeled CO2 in several, usually six, soil samples and only one IR gas analyzer). The KOH solution was replaced every day and counted with a Beckman LS 5000 liquid scintillation analyzer. To ensure that low values were meaningful, each sample was counted three times for 10 min to yield a limit of error 2 s of less than 5%. We also added other substrates: unlabeled glucose, acetate, and Difco yeast extract to the frozen soils in the same way and at the same rates as described above. 2.5. Soil sterilization Soil was autoclaved 1 h at 121 8C and checked for sterility by plating soil suspensions on Difco yeast extract agar and aerobically incubating at 10 8C for 1 week. The efflux of 14CO2 and 12CO2 (soil respiration) was recorded as described above with the only exception being that the flushing air was preliminarily passed through standard air filters (New Brunswick Scientific, NJ, USA) normally used to preserve sterility of fermentors. 2.6. Other techniques The gas-filled pore space of the soil column, the water content, and the soil bulk density were determined using conventional methods of soil physics (Klute, 1986).

The dynamic pattern of gas exchange during isothermal incubation was typical of a system having a weak but steady CO2 source and high initial concentrations of accumulated CO2 (Fig. 2). For the first several days, the rates of CO2 evolution declined rapidly, from 0.5–1.5 mg C cmK3 soil dK1 to w0.1 mg C cmK3 dK1, and then progressively slowed down. The entire dynamic curve was approximated by Eqs. (1) and (2), which allowed us to distinguish a slow component, attributed to respiration, and a rapid component, interpreted as release of entrapped gas (continuous dotted curve 3 on Fig. 2). For example, the contribution of physical release was significant only in the first 1–2 d, when it accounted for up to 70% of the total CO2 flux and then rapidly declined to less than 10%, starting from d 3 of incubation (Fig. 2). Chemical analysis of accumulated CO2 and carbonates gave values very close to the kinetic term s0 determined using Eq. (1) (and results varied in different runs from 1 to 5 mg C kgK1 soil). The slow-released CO2 seems to be formed de novo, as it exceeded the amount of inorganic carbon present in the frozen soil before incubation. 10 CO2 evolution rate (µg C cm –3 soil d –1)

RðtÞ Z R 0 C R0 expðKk2 tÞ

3. Results

1 1 0.1

0.01 0

20

40

60

40

60

6 CO2 release (µg C cm –3 soil)

788

2 4 2

3

0 0

20 d

Fig. 2. The long-term dynamics of CO2 evolution from the frozen Barrow soil incubated at K21 8C. Soil was sampled from the depth 5–15 cm, bulk density 1.32 g (wet wt.) cmK3 and 1.06 g (dr. wt) cmK3. The upper panel shows the rate of CO2 evolution (1); the bottom panel displays the cumulative CO2 release (2) and the calculated release of trapped CO2 (dotted line 3). Curve 1 was calculated from Eq. (1) with the following parameters: s0Z1.01 mg C cmK3 (trapped CO2), kZ1.23 dK1 (trapped CO2 removal rate) and respiration dynamics R(t)Z0.08C 0.247 exp(K0.085t) mg C cmK3 dK1 with an initial respiration rate of 0.08C0.247Zmg C cmK3 dK1. Chemical determination of inorganic C gave close to the s0-value, 1.1G0.1 mg C cmK3. Note that CO2 release exceeded the total reserves of inorganic C in the soil after 2–3 d.

Lowering or raising the incubation temperature produced rapid changes in CO2 production rates at new steady states during the following days (Fig. 3, open circles). Inexplicably, temperature increases of w5 8C were accompanied by short-term overshoots in CO2 release. Amending the soil with 14C-glucose slightly increased the total rate of CO2 evolution and resulted in immediate release of labeled 14 CO2 without any significant overshoots (Fig. 3, closed circles). Generally, the production of total and labeled CO2 was synchronous, although a trend for the progressive decline of the 14C-to-total-C ratio at lower incubation temperatures was obvious. There were steady-state rates of CO2 release established 2 4 d after each temperature shift, versus respective temperature (Fig. 4). The respiration rates declined abruptly in the temperature range from 0 to K5 8C. Further cooling to K33 8C was accompanied by an almost linear, gradual decrease in soil respiration rate. The plot for 14 CO2 was similar but essentially steeper than the corresponding plot for total CO2 output (compare open and closed circles on Fig. 4), i.e. the oxidation of the added 14 C-glucose was more sensitive to cooling than the oxidation of the naturally occurring soil C-substrates. The relative amount of labeled 14CO2 (14CO2C12CO2)K1 declined exponentially with temperature (Fig. 5). Soil subsamples treated by autoclave and refrozen remained sterile and inactive (Fig. 3, squares). They produced, by three orders of magnitude, lower amounts of 14CO2 than untreated soils, irrespective of incubation temperature, down to at least K35 8C. 3.3. Effect of added unlabeled compounds The soil was amended with glucose, yeast extract, and acetate, which are used as C and energy sources by a wide range of known soil microorganisms. The experimental setup was the same as the one shown in Fig. 3: soil was briefly unfrozen, mixed with substrates and then refrozen and

789

100

6

10

5 1

1

4

0.1

3 2

0.01

2

Unfrozen water (%)

3.2. CO2 release rate after addition of 14C-glucose and stepwise change of incubation temperature

Respiration (µg CO2 – C cm –3 d –1)

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

3 0.001

1

0.0001 –35

0 –30

–25

–20 –15 –10 Temperature (°C)

–5

0

Fig. 4. The effect of incubation temperature on steady-state soil respiration and unfrozen soil water content. Respiration data points were taken from the stabilized parts of the dynamic curves shown in Fig. 3. The unfrozen soil water content (dashed line, 3) was calculated from Barrow data (Romanovsky and Ostercamp, 2000). The continuous lines are multiple regression curves calculated from the double exponential equation (Table 1). Note that oxidation of added 14C-glucose (line 2) declines more steeply with decreasing temperature than does soil/microbial endogenous carbon release. Soil is the same as shown on Fig. 2.

incubated with periodic temperature increases of 3–5 8C. Fig. 6 shows only final results: steady-state respiration rates versus incubation temperature. Energy supplements from yeast extract and glucose (not shown) stimulated the respiration rate of frozen soil compared to the control samples, receiving equivalent amounts of water. In contrast, acetate substantially inhibited CO2 production. For none of the tested compounds did we observe an increase in the CO2 production rate due to microbial growth on the added substrate, even during 2–3 months of isothermal incubations (data not shown). 0.4

20

1

1 0.1

10 0

2

–10

0.01

3

–20

4

0.001

–30 –40

0.0001 0

5

10

15

20

25

30

35

14 C/(12C + 14C)

30

10

Temperature (°C)

Respiration (µg CO2– C cm –3 d –1)

0.3 40

100

0.2

0.1

0 – 35

– 30

– 25 – 20 – 15 – 10 Temperature (°C)

–5

0

d 14

Fig. 3. Dynamics of the total CO2 (1) and CO2 (2) release from the Barrow soil amended with 14C-glucose in response to an abrupt change in incubation temperature (3). Line 4 is the 14CO2 release from autoclaved soil (sterile control). Soil is the same as shown on Fig. 2.

Fig. 5. The effect of temperature on the 14C/12C ratio in CO2 evolved from frozen soil. Calculation was based on the isotope dilution principle (dpm mgK1 of added glucose C at time 0) and not corrected for biological isotopic preferences. Note the dramatic decline in isotopic ratio at low temperature.

790

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

Respiration (µg CO2– C cm– 3 d–1)

1000 100 10 1 2

0.1 0.01

13

0.001 0.0001

0.00001 –40

– 30

– 20 – 10 Temperature (°C)

0

10

Fig. 6. The effects of substrates (yeast extract, line 2) and inhibitors (acetate, line 3) added to soil prior to freezing. Oxidizable substrate increased respiration while acetate inhibited respiration, providing further evidence for a biological origin of evolved CO2. Legend: (1) unamended control, (2) yeast extract, (3) acetate. Soil is the same as shown on Fig. 2.

3.4. Comparison of tundra and boreal soils We have not analyzed a wide range of soil samples but confined ourselves to a limited number of soil types. Fig. 7 shows the plot of CO2 release versus temperature for three soils. The CO2-generation in cold temperatures was significantly and consistently stronger in the Arctic soil from Barrow, Alaska, as compared with the activity found in the soils from the warmer boreal forest regions in West Siberia and Sweden. The lowest temperature with detectable CO2 production was K39 8C in Arctic soils, while the boreal soils did not display activity below K31 8C. It is also quite interesting that the Arctic soil sampled at the end of the warm season seemed to exhibit lower rates of Respiration (µg CO2–C cm–3 d–1)

10

1

Barrow-Winter Barrow-Fall W-Siberia-Fall Sweden-Fall

0.1

below-zero respiration than soil sampled during the winter months (e.g. 0.13 and 1.4 mg CO2–C cmK3 dK1, respectively, at an incubation temperature of K21 8C), while they had the same rate of respiration activity at an incubation temperature of 0K5 8C. These differences may reflect seasonal changes in the abundance of cold-active microorganisms, which could be inactive during the summer or have physiological acclimation through the summer to warmer temperatures. Moreover, under even mild warming (1–2 d incubation of the winter soil samples at C5 8C), the cold respiratory activity declined by 50–70%, which could be considered an indication of significant sensitivity to warming in these cold-adapted microorganisms. 3.5. Variation of respiration activity along the profile of Barrow soil Soil cores w30 cm deep were divided into eight depth classes (Fig. 8) and each of these was subsampled for testing at C22, K14, or K21 8C. At 22 8C, the maximum respiration was found in the upper soil layers, while at temperatures below K14 8C (near or below typical winter temperatures), the maximum absolute rates of respiration were found in soils from below 15 cm. These would be the coldest soils in summer and the deepest soils would be near freezing, even at the end of summer.

4. Discussion Although microbial cells can survive at temperatures well below 0 8C, respiration of Arctic soil microbes has not been previously demonstrated at temperatures lower than K12 8C in culture (Straka and Stokes, 1960; Flanagan and Bunnell, 1980; Mazur, 1980; Kushner, 1981) or below K18 8C in incubated soil samples (Elberling and Brandt, 2003). In this work, we measured respiration of frozen soil down to K39 8C under carefully controlled laboratory conditions, taking special care to differentiate microbial activity from abiotic CO2 release. 4.1. Reliability of CO2 evolution data as an indicator of respiration intensity

0.01

0.001 –40

–35

–30

–25 –20 –15 Temperature (°C)

–10

–5

0

Fig. 7. Respiration activity of tundra (Barrow) and boreal forest soils (WSiberian and Swedish sites) as dependent on incubation temperature. Note that Barrow soil (especially winter-sampled) showed significantly higher rates at any given temperature than boreal soils (as would be expected in cold adapted/acclimated organisms). All samples were taken from the depth 5–15 cm. The bulk density values for Barrow, W-Siberian, and Swedish sites were respectively 1.32, 1.09, and 1.08 g (wet wt.) cmK3. The moisture contents were, respectively, 19.8, 91.6, and 91.8 wt%.

Respiration can be estimated directly as (a) the rate of O2 uptake and (b) the rate of CO2 formation. The first approach is more accepted in hydrobiological research than in soil research because of its low measurement sensitivity accuracy against high background O2 concentrations and small variability in O2 concentrations. The detection of CO2 production is much more precise but has obvious limitations stemming from the ‘sticky’ nature of CO2: unlike O2, CO2 can be easily transformed into a non-volatile form (carbonates), dissolved in soil water, and absorbed by the soil solid phase (Monger and Wilding, 2002). Gas exchange in frozen soil is especially slow, leading to a significant

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

Bulk density (g cm –3)

Respiration (µg CO2– C cm –3 d –1)

Depth (cm)

0.1

1

10

100

0

1000

0

0

5

5 –14°C

10 15 20

791

0.5

1

g dry wt cm–3

1.5

2

g wet wt cm–3

10

–21°C

15 22°C

20

25

25

30

30

35

35

y = 53.763x – 68.715 r2 =1

Fig. 8. Vertical distribution of respiration activity at three incubation temperatures (left) and bulk density in Barrow soil. The bulk density was given in terms of both dry and wet weight cmK3 to facilitate recalculating the respiration activity into mg CO2–C gK1 dK1 of dry or wet soil. Note the log-scale for respiration activity. Soil was sampled in winter. The maximal active layer depth in summer season at this particular spot varied from year to year from 20–35 cm below the surface.

build-up of CO2, which interferes with the measurement of respiration rates. Our approach to this problem included: (a) mildly crushing frozen soil to increase the gas transfer rate, (b) removing CO2 during cold preincubation of soil over a CO2 absorber, (c) kinetic analysis of CO2 evolution curves to differentiate de novo CO2 formation versus release of previously accumulated CO2, and (d) recording labeled CO2 release from soil amended with labeled substrate. The kinetic analysis is based on the assumption that the release of entrapped CO2 should be fast enough and follow simple, first-order kinetics: the bigger the CO2 pool, the higher the release rate. In this case, the rate of gas release should rapidly decay because the size of the soil CO2 pool is limited and non-renewable. Contrary to physical release, microbial respiration is a slow and continuous conversion to CO2 of oxidizable substrates, which are not depleted in the course of the experiments. Therefore the fast, first-order component of the dynamic curve has been assigned to physical CO2 release, while the slow component is attributable to microbial respiration. The close correspondence between such kinetic calculations (parameter s0) and the actual chemical assay of inorganic C supports this assumption but does not prove it firmly. Needless to say, we cannot exclude other ways of interpreting dynamic data and more complex CO2 release kinetics (e.g. accounting for soil heterogeneity in CO2 retention). However, our estimates are likely conservative since we are excluding labile substrate made available when the soils are disturbed and lightly crushed. These labile pools would most readily be included in the physical component of CO2 release.

The nature of renewable substrates driving below-zero soil respiration is obscure. They could be intracellular constituents of soil microbes (see Section 4.3) and volatile or soluble compounds migrating from the soil active layer via air-filled microscopic pores or films of unfrozen water. These C-sources are renewable in situ but are limited under the artificial conditions of laboratory incubation when the supply is cut off. Therefore, it is not surprising that the slow component of the CO2 production curve was also decaying, indicating a gradual exhaustion of the oxidizable pool. Finally, respiration rates estimated by methods (c) and (d) agreed well when applied to frozen soil amended with 14 C-substrate (see Section 4.3). The CO2 and 14CO2 release rates were similar, although not identical because the labeled substrate was only part of the oxidizable pool. Importantly, release of 14CO2 was detected at the lowest temperatures tested, independently indicating that microbial respiration at this low temperatures was not an experimental artifact. 4.2. CO2 release rate as a function of subzero temperature A central problem in kinetic studies is the search for an adequate mathematical description of the temperature effect on the rate of the process studied. Such a relationship can shed light on the underlying mechanism and allows prediction of the process dynamics under a changeable environment. The experimental data (Fig. 4) were approximated by three alternative kinetic equations (Table 1). Two popular expressions, the Arrhenius and exponential

Table 1 Fitting of experimental data plotted on Fig. 3 to selected kinetic equations r2

Equation

12

Ea or Q10 (kcal/mol) 14

CC C

1. Arrhenius 2. Simple exponential 3. Double exponential

vZA exp[KEa/R(273CT)] vZA exp(T) vZA exp(TCkW)

0.857 0.889 0.987

14

12

0.905 0.934 0.987

EaZ19.2 Q10Z4.5 Q10Z2.1

C

CC14C

14

C

EaZ27.5 Q10Z8.5 Q10Z3.8

792

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

equations, gave reasonable agreement (r2 0.86–0.93), although a clear systematic deviation was seen above K5 8C. Another disadvantage was the abnormally high values of the kinetic parameters characterizing the slope of the temperature curve: the activation energy, Ea, was as large as 27 kcal molK1 versus the usual 4–6 kcal molK1. The Q10 temperature coefficient was as large as 8.5, as compared with the usual value of 2–3 for soil respiration above 0 8C (Lloyd and Taylor, 1994; Panikov, 1997). One of the reasons for these deviations is that below-zero temperatures affect microbial activity not only directly (changing the kinetic energy of reactants), but also indirectly, through changes in the status of soil water. It is known that the content of unfrozen water in frozen soil declines abruptly with cooling and is believed to be the major factor restricting microbial activity below 0 8C (Kushner, 1981; Hinkel and Outcult, 1994; Marion and Grant, 1997; Price, 2000). Indeed, when we tried the multiple exponential regression (Table 1, Eq. 3) of v versus T and unfrozen water content W (calculated from data of Romanovsky and Ostercamp, 2000, for Barrow), the agreement was significantly improved (r2 increased to 0.98–0.99) and the temperature parameter Q10 returned to values expected for known biological processes.

inhibition rather than activation of microbial respiration (Panikov et al., 1997). The suggested tentative mechanism is that in the soil solution at pH 4–5, the Na-acetate is converted to a free acid that is sufficiently hydrophobic to penetrate the cellular membrane in the majority of soil microorganisms. Typically bacterial cells and fungal mycelia have a neutral cytosol (pHw7.0) with KC being the major intracellular cation (Alberts et al., 2002). Therefore, the acetic acid transported into the cytosol undergoes dissociation (CH3COOH4CH3COOKCHC), which produces protons discharging the transmembrane electrochemical gradient and stopping oxidative phosphorylation. The response of the microbial communities to addition of stimulatory (glucose, yeast extract) versus inhibitory (acetate) compounds above and below 0 8C, is a very convincing argument supporting the biological origin of CO2 release. The small size of substrate-induced respiration, as well as the absence of a noticeable increase in its rate even during prolonged isothermal incubation, suggests that these added substrates are used only for maintenance respiration, not for cell growth at the temperatures tested (Panikov, 1995). 4.5. Variation in CO2 release rate as dependent on soil geographical location, sampling depth and time

4.3. Oxidation of added and endogenous C-compounds Another engaging observation was that the temperature dependence for 14CO2 was much steeper than for the corresponding plot of total CO2 output (compare open and closed circles, Fig. 4). Why was the oxidation of added 14Cglucose more sensitive to cooling than the oxidation of the soil substrates? We can safely assume that below-zero metabolism can be driven only by easily available substrates and we can completely exclude from consideration soil humus and other recalcitrant compounds. Moreover, localization of substrates at microscale relative to microcolonies and single cells of psychrophilic microorganisms is critical. Intracellular substrates, proteins, nucleic acids, polysaccharides, and other cellular constituents are most likely metabolized and fuel endogenous cell respiration due to their proximity to cell organelles and enzymes. The utilization of added extracellular substrates may be harder to access as compared to intracellular constituents, due to their low mobility in the frozen soil. The decrease in free water with decreasing temperatures may explain why the ratio 14 C-to-total-C progressively declined with cooling from w35% at the freezing point to w5% at K33 8C (Fig. 5). 4.4. The stimulatory and inhibitory effects of added substrates Glucose and yeast extract stimulated soil respiration, while acetate inhibited respiration. Acetate is one of the key intermediates of the cellular metabolic system. However, in acidic unfrozen soils, the acetate amendment always caused

The larger rate of respiration below 0 8C of tundra soils compared to boreal/forest soils clearly indicate the adaptive nature of the cold respiration, indicating that CO 2 production below 0 8C is caused, at least partly, by a specialized ecological group of microorganisms adapted to extreme Arctic conditions and life at sub-zero temperatures. Similar indications have been mentioned by Flanagan and Scarborough (1974); Flanagan (1981). Similarly, deeper, colder soils do relatively better at low temperatures and warmer (summer) upper soil layers do relatively better at warmer temperatures. This is consistent with emergent ecological theory on acclimation and adaptation to cold conditions in Arctic/boreal organisms, including animals and higher plants (Mooney and Billings, 1961; Tjoelker et al., 1999), as well as microorganisms (Lloyd and Taylor, 1994; Panikov, 1997). 4.6. Environmental significance Our goal was to help provide a biological basis for the significant, recently discovered winter CO2 emission from tundra ecosystems to the atmosphere (Zimov et al., 1993, 1996; Oechel et al., 1997; Fahnestock et al., 1999). Since the cold-season soil respiration is mainly due to microbial activity (with some possible contribution from plant roots) these results can be used to help model future winter CO2 emissions under a warmer climate. Intriguing opportunities exist to identify those obligate psychrophilic microbes that are responsible for metabolic activity in deeply frozen soil. Also, elucidation of the particular biochemical and

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

physiological attributes that provide exceptionally high resistance to cold and drought stress is highly desirable.

Acknowledgements This research was supported by the National Science Foundation: Microbial Observatories and Microbial Interactions and Processes, Arctic Systems Science, LandAtmosphere-Ice Interactions Program and Division of Environmental Biology, and Joint Program on Terrestrial Ecology and Global Change; the US Department of Energy: Office of Health and Environmental Research; and by the EU 4th Framework Programme (CONGAS). We thank Steve Hastings, Rommel Zulueta, Glen Kinoshita, Gus Lindquist, Craig Tweedie, and Patrick Webber for soil sampling in Barrow, as well as Larry Hinzman and Vladimir Romanovsky for discussion and input, especially on the unfrozen water content of sub-zero soils.

References Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 2002. Molecular Biology of the Cell, fourth ed. Garland Science Publishing, New York. Amann, R.I., Ludwig, W., Schleifer, K.-H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews 59, 143–169. Brown, J., Miller, P.L., Tieszen, L.L., Bunnell, F., 1980. An Arctic Ecosystem: The Coastal Tundra at Barrow, Alaska. Hutchinson & Ross, Stroudsburg. Coyne, P.I., Kelley, J.J., 1971. Release of carbon dioxide from frozen soil to the arctic atmosphere. Nature 234, 407–408. Elberling, B., Brandt, K.K., 2003. Uncoupling of microbial CO2 production and release in frozen soil and its implications for field studies of arctic C cycling. Soil Biology & Biochemistry 35, 263–272. Fahnestock, J.T., Jones, M.H., Brooks, P.D., Walker, D.A., Welker, J.M., 1998. Winter and early spring CO2 flux from tundra communities of northern Alaska. Journal of Geophysical Research 102 (D22), 29925– 29931. Fahnestock, J.T., Jones, M.H., Welker, J.M., 1999. Wintertime CO2 efflux from arctic soils: implications for annual carbon budgets. Global Biogeochemical Cycles 13, 775–780. Finegold, L., 1996. Molecular and biophysical aspects of adaptation of life to temperatures below the freezing point. Advances in Space Research 18, 87–95. Flanagan, P.W., 1981. Fungal taxa, physiological groups and biomass. In: Wicklow, D.T., Carroll, G.C. (Eds.), The Fungal Community. Marcel Dekker, Inc., New York, pp. 569–592. Flanagan, P.W., Bunnell, F.L., 1980. Microflora activities and decomposition. In: Brown, J., Miller, P.C, Tieszen, L.L., Bunnell, F.L. (Eds.), An Arctic Ecosystem: The Coastal Tundra at Barrow, Alaska. Dowden, Hutchinson & Ross, Stroudsburg, pp. 291–335. Flanagan, P.W., Scarborough, W., 1974. Physiological groups of decomposer fungi in tundra plant remains. In: Holding, A.J., Heal, O.W., MacLean, S.F., Flanagan, P.W. (Eds.), Soil Organisms and Decomposition in Tundra. International Biological Programme Tundra Biome Steering Committee, Stockholm, pp. 159–181. Flanagan, P.W., Veum, A.K., 1974. Relationships between respiration, weight loss, temperature, and moisture in organic residues in tundra, Ibid, pp.. In: Holding, A.J., Heal, O.W., MacLean, S.F., Flanagan, P.W.

793

(Eds.), Soil Organisms and Decomposition in Tundra. International Biological Programme Tundra Biome Steering, Stockholm, pp. 249– 278. Geiges, O., 1996. Microbial processes in frozen food. Advances in Space Research 18, 109–118. Hinkel, K.M., Outcult, S.I., 1994. Identification of heat-transfer processes during cooling, freezing, and thaw in central Alaska. Permafrost and Periglacial Processes 5, 217–235. Jones, M.H., Fahnestock, J.T., Welker, J.M., 1999. Early and late winter CO2 efflux from arctic tundra in the Kuparuk river watershed Alaska. Arctic, Antarctic, and Alpine Research 31, 187–190. Kappen, L.B., Schroeter, B., Scheidegger, C., Sommerkorn, M., Hestmark, G., 1996. Cold resistance and metabolic activity of lichens below 0 8C. Advances in Space Research 18, 119–128. Klute, A. (Ed.), 1986. Methods of Soil Analysis. Part 1. Physical and Mineralogical Methods. Soil Science Society of America, Madison. Kushner, D., 1981. Extreme environments: are there any limits to life?. In: Ponnamperuna, C. (Ed.), Comets and the Origin of Life. Reidel, New York, pp. 241–248. Larkin, J.M., Stokes, J.L., 1968. Growth of psychrophilic microorganisms at subzero temperatures. Canadian Journal of Microbiology 14, 97–101. Lloyd, J.L., Taylor, J.A., 1994. On the temperature dependence of soil respiration. Functional Ecology 8, 315–323. Malmer, N., 1962. Studies on mire vegetation in the archaean area of southwestern Go¨taland (South Sweden). Opera Botanica 7, 1–322. Marion, G.M., Grant, S.A., 1997. Physical Chemistry of Geochemical Solutions at Subzero Temperatures. US Army Cold Regions Research and Engineering Laboratory, Hanover, CRREL Special Report 97–10. Mazur, P., 1980. Limits to life at low temperatures and at reduced water contents and water activities. Origins of Life 10, 137–159. Mikan, C.J., Schimel, J.P., Doyle, A.P., 2002. Temperature controls of microbial respiration in arctic tundra soils above and below freezing. Soil Biology & Biochemistry 34, 1785–1795. Monger, H.C., Wilding, L.P., 2002. Inorganic carbon: composition and formation. In: Lal, R. (Ed.), Encyclopedia of Soil Science. Marcel Dekker, New York, pp. 701–705. Mooney, H.A., Billings, W.D., 1961. Comparative physiological ecology of arctic and alpine populations of Oxyria digyna. Ecological Monographs 31, 1–29. Oechel, W.C., Vourlitis, G., Hastings, S.J., 1997. Cold season CO2emission from arctic soils. Global Biogeochemical Cycles 11, 163–172. Panikov, N.S., 1995. Microbial Growth Kinetics. Chapman & Hall, London. Panikov, N.S., 1997. A kinetic approach to microbial ecology in Arctic and boreal ecosystems in relation to global change. In: Oechel, W., Callagan, T., Gilmanov, T., Holsten, J.I., Maxwell, B., Molau, U., Sveinbjornsson, B. (Eds.), Global Change and Arctic Terrestrial Ecosystems. Ecological Studies 124. Springer, New York, pp. 171–189. Panikov, N.S., 1999. Fluxes of CO2 and CH4 in high latitude wetlands: measuring, modeling, and predicting response to climate change. Polar Research 18, 237–244. Panikov, N.S., Dedysh, S.N., 2000. Cold season CH4 and CO2 emission from boreal peat bogs (West Siberia): winter fluxes and thaw activation dynamics. Global Biogeochemical Cycles 14, 1071–1080. Panikov, N.S., Nizovceva, D.V., Semenov, A.M., Sizova, M.V., 1997. Effects of mineral compounds on the respiratory activity of microbial community in ombrotrophic peat bog. Mikrobiologiya 66 (2), 165–171. Price, B.P., 2000. A habitat for psychrophiles in deep antarctic ice. Proceedings of the National Academy of Sciences 97, 1247–1251. Rivkina, E.M., Friedmann, E.I., McKay, C.P., Gilichinsky, D.A., 2000. Metabolic activity of permafrost bacteria below the freezing point. Applied & Environmental Microbiology 66, 3230–3233. Romanovsky, V.E., Ostercamp, T.E., 2000. Effects of unfrozen water on heat and mass transport processes in the active layer and permafrost. Permafrost & Periglacial Processes 11, 219–239.

794

N.S. Panikov et al. / Soil Biology & Biochemistry 38 (2006) 785–794

Romanovsky, V.E., Osterkamp, T.E., 1995. Interannual variations of the thermal regime of the active layer and near-surface permafrost in Northern Alaska. Permafrost & Periglacial Processes 6, 313–335. Romanovsky, V.E., Sergueev, D.O., Osterkamp, T.E., 2003. Temporal variations in the active layer and near-surface permafrost temperatures at the long-term observatories in Northern Alaska. In: Phillips, M., Springman, S., Arenson, L.U. (Eds.), Permafrost. Swets & Zeitlinger, Lisse, The Netherlands, pp. 989–994. Russell, N.J., 1990. Cold adaptation of microorganisms. Philosophical Transactions of the Royal Society 326, 595–611. Schimel, J.S., Bilbrough, C.B., Welker, J.M., 2004. The effect of changing snow cover on year-round soil nitrogen dynamics in arctic tundra ecosystems. Soil Biology & Biochemistry 36, 217–227. Schroeter, B., Green, T.G.A., Kappen, L., Seppelt, R.A., 1994. Carbon dioxide exchange at subzero temperatures: field measurements on Umbilicaria aprina in Antarctica. Cryptogramic Botany 4, 233–241. Sommerfeld, R.A., Mosier, A.R., Musselman, R.C., 1993. CO2, CH4, and N2O flux through a wyoming snowpack and implications for global budgets. Nature 361, 140–142.

Straka, R.P., Stokes, J.L., 1960. Psychrophilic bacteria from Antarctica. Journal of Bacteriology 80, 622–625. Tjoelker, M.G., Oleksyn, J., Reich, P.B., 1999. Acclimation of respiration to temperature and CO2 in seedlings of boreal tree species in relation to plant size and relative growth rate. Global Change Biology 49, 679–691. Welker, J.M., Fahnestock, J.T., Jones, M.H., 2000. Annual CO2 flux from dry and moist arctic tundra: field responses to increases in summer temperature and winter snow depth. Climatic Change 44, 139– 150. Zimov, S.A., Zimova, G.M., Davidov, S.P., Davidova, A.I., Voropaev, Y.V., Voropaeva, Z.V., Prosiannikov, S.F., Prosiannikova, O.V., Semiletova, I.V., Semiletov, I.P., 1993. Winter biotic activity and production of CO2 in Siberian soils: a factor in the greenhouse effect. Journal of Geophysical Research 98, 5017–5023. Zimov, S.A., Davidov, S.P., Voropaev, Y.V., Prosiannikov, S.F., Semiletov, I.P., Chapin, M.C., Chapin, F.S., 1996. Siberian CO2 efflux in winter as a CO2 source and cause of seasonality in atmospheric CO2. Climatic Change 33, 111–120.