Temperature is an important factor regulating microbial activity and shaping the soil microbial community. Little is known, however, on how temperature affects the most important groups of the soil microorganisms, the bacteria and the fungi, in situ. We have therefore measured the instantaneous total activity (respiration rate), bacterial activity (growth rate as thymidine incorporation rate) and fungal activity (growth rate as acetate-in-ergosterol incorporation rate) in soil at different temperatures (0–45 °C). Two soils were compared: one was an agricultural soil low in organic matter and with high pH, and the other was a forest humus soil with high organic matter content and low pH. Fungal and bacterial growth rates had optimum temperatures around 25–30 °C, while at higher temperatures lower values were found. This decrease was more drastic for fungi than for bacteria, resulting in an increase in the ratio of bacterial to fungal growth rate at higher temperatures. A tendency towards the opposite effect was observed at low temperatures, indicating that fungi were more adapted to low-temperature conditions than bacteria. The temperature dependence of all three activities was well modelled by the square root (Ratkowsky) model below the optimum temperature for fungal and bacterial growth. The respiration rate increased over almost the whole temperature range, showing the highest value at around 45 °C. Thus, at temperatures above 30 °C there was an uncoupling between the instantaneous respiration rate and bacterial and fungal activity. At these high temperatures, the respiration rate closely followed the Arrhenius temperature relationship.
Temperature, together with moisture content, is the most important environmental factor affecting microbial growth and activity in soils. To be able to understand fluctuations in microbial activity a reliable model for temperature dependency is therefore required. The importance of the temperature dependence of soil organisms has been further emphasized during recent years due to the global warming issue [2,3], since microorganisms are the main group producing CO2 during decomposition of organic material in soil.
The temperature dependence of soil microorganisms is usually studied by measuring the respiration rate (total activity), and numerous field and laboratory experiments have been performed on all types of soil habitats (see, e.g [2–4]). Less is known about the temperature dependence of different groups of soil microorganisms, such as bacteria and fungi. The temperature dependence of bacterial activity (growth rate) has, however, been determined using the thymidine and leucine incorporation methods to estimate the growth rate of the soil bacterial community in two soils, and these techniques were later used to study the temperature dependence of the bacterial community in heated peat and in soil incubated at different temperatures. The fungal activity (growth rate) can be estimated with the acetate-in-ergosterol incorporation technique, originally devised for aquatic habitats [8,9]. This technique was adapted to soil conditions by Bååth and used to determine the temperature dependence of the soil fungal community in one soil.
The instantaneous soil respiration rate often increases with temperature up to around 40 °C or more, even in soils from cold climates (e.g. [11–14]). This is not due to the growth of thermophilic organisms at higher temperatures, as the effect is seen even when short incubations times are used, which would not allow for substantial growth of the thermophilic community (e.g.). In contrast, soil bacterial and fungal growth rates in cold climates usually have optimum temperatures below 30 °C, with activity values decreasing at higher temperatures [7,10]. Pure cultures of bacterial or fungal isolates from temperate soils are usually found to have optimum growth temperatures below 30 °C [15,16]. One explanation of this discrepancy in optimum temperatures could, of course, be that different soils were studied using respiration techniques and the methods of determining fungal and bacterial activities. However, the uncoupling of respiration rate and microbial growth at higher temperatures can not be excluded, indicating the need for a direct comparison of these three different measures of activity.
It is often stated that fungi as a group are more adapted to low soil moisture conditions than bacteria [1,17], and would therefore be more important in dry soil. Less is known about the effect of temperature. Persson et al. found a difference in temperature dependence of respiration in a forest and an agricultural soil, in that a lower minimum temperature for respiration (tmin) was found in the former soil. One proposed explanation of this difference was a shift in the relative importance of fungi and bacteria as decomposers, i.e. that fungi are more important in the forest soil and are more active at low temperatures than bacteria [18–21]. However, no direct comparison has been made of the temperature dependence of soil fungal and bacterial communities.
The aim of the present study was to make such a comparison using two contrasting soil types from a temperate climate: one was an agricultural soil with high pH and low organic matter content and the other was a forest humus, with low pH and high organic matter content. First, we wanted to compare the activity (growth rate) of fungi and bacteria at different temperatures to determine whether one of the groups of organisms appears to be favoured at certain temperatures. Second, we wanted to ascertain whether there was an uncoupling of microbial growth and respiration rate at high temperatures. Third, we wanted to compare different ways of modelling the temperature relationship of total, bacterial and fungal activity.
2Materials and methods
We used two different soils originating from southern Sweden. One was an agricultural soil with a dry weight of 88% of the wet weight, a pH of 7.8 and an organic matter content of 5%. The other was a humus soil (the A01/A02 horizon) from a forest with mainly spruce, with a dry weight of 29% of the wet weight, a pH of 4.1 and an organic matter content of 82%. Both soils were collected in the spring of 2002, sieved (2.8 mm mesh size) and stored at 5 °C less than a month until used in the experiment. This storage time at low temperature has earlier been shown to have no effect on the bacterial community temperature relationship.
The total activity was estimated as the respiration rate. Samples consisting of 3 g of agricultural soil or 1 g of humus soil were transferred to 20 ml glass bottles. The samples were incubated at 0 °C (water with ice kept in a 4 °C cold room) and 4 °C for 120 h, at 10 °C for 72 h, at 14 °C for 49 h, at 18 °C and 25 °C for 24 h, at 30 °C for 8 h, at 35 °C for 7 h, at 40 °C for 6 h and at 45 °C for 5 h. Three replicates were incubated at each temperature. The CO2 evolution was measured with a gas chromatograph at the end of the incubation time.
Bacterial activity (growth rate) was estimated using the thymidine (TdR) incorporation technique on bacteria extracted from soil. The bacteria were extracted by transferring 1 g of soil to a glass flask and adding 40 ml of Milli Q water. The samples were shaken on a rotary shaker (200 rpm) for 15 min and then centrifuged at 1000g for 10 min. The supernatant with the extracted bacteria was then used. An aliquot (1.5 ml) of each sample was transferred to an Eppendorf tube and the tubes were distributed between the different temperature regimes. After 15 min, 5 μl methyl[3H]thymidine (926 GBq mmol−1, Amersham, UK) was added. Incubation was terminated by adding 75 μl cold 100% TCA. The incubation times were: 48 h at 0 °C, 24 h at 4 °C, 7 h at 10 and 14 °C, 4 h at 18 °C, 2 h at 25 °C, 1 h at 30 and 35 °C and 2 h at 40 and 45 °C. Three replicates (bacteria extracted from three different soil samples) were used. Removal of excess non-incorporated TdR and subsequent determination of incorporated radioactivity were carried out as described by Bååth et al..
The fungal activity (growth rate) was estimated using the 14C-acetate incorporation into ergosterol technique, modified for use in soil. One g of agricultural or 0.25 g of humus soil were transferred to small test-tubes with 1.5 ml distilled water, 0.05 ml 14C-acetate solution ([1,2-14C]acetic acid, sodium salt, 2.07 GBq/mmol, Amersham) and 0.45 ml 1 mM non-radioactive acetate. The tubes containing the soil slurry were incubated at 0 °C for 72 h, at 4 °C for 48 h, at 10 and 14 °C for 24 h, at 18 and 25 °C for 16 h, at 30 °C for 8 h, at 35 °C for 16 h and at 40 °C for 8 h. A temperature of 45 °C was not included in the fungal activity measurements, since it was assumed to be too high a temperature for fungal activity. Two replicates were used. One millilitre of 5% formalin was added to stop the incorporation of acetate. Zero time controls were made by adding formalin to samples before the labelled acetate to account for abiotic binding of acetate. Washing, extraction of ergosterol, measurement of ergosterol using HPLC, collecting of the ergosterol and the subsequent determination of incorporated radioactivity on a liquid scintillator were performed as described by Bååth.
For all measurements the incubation time was adjusted to be in the linear phase or incorporation at temperatures below optimum (determined in earlier experiments). Above optimum temperature we used the shortest incubation times compatible with detection of activity. The respiration rate was calculated as absolute values of CO2 evolution h−1 g−1 organic matter. For fungal and bacterial activities the measured entities (DPM acetate incorporation into ergosterol per g of soil for fungi or DPM TdR incorporated into bacteria extracted from soil) have no direct meaning, but only give relative values. The incorporation data for the TdR and acetate-in-ergosterol technique were therefore normalised, setting the mean incorporation rate at 25 °C to the value 1 for each soil.
The data were modelled using two common functions to describe temperature relationships. A square root relationship below optimum temperature has been shown to adequately model bacterial growth in pure culture: k1/2=b(t−tmin), where k is the “rate of the activity” (or growth rate in the case of bacteria and fungi) at temperature t (°C), tmin is the apparent minimum temperature for growth, and b is a slope parameter without any direct biological meaning (Ratkowsky et al. [23,24]). A plot of the square root of the activity against temperature will result in a linear relationship. tmin will in many environmental studies fall below the freezing point of water (e.g. [5,10,25]), which of course is physiologically meaningless. That is why tmin is denoted apparent minimum temperature for growth. However, tmin is useful as a comparison between different communities, in that lower values will indicate a better capacity to grow at low, permissible, temperatures.
The Arrhenius equation, originally used for enzyme kinetics, is one of many exponential functions used in modelling temperature relationships: k=A e−E/(R(t+ 273.15)), where A is a constant, R is the universal gas constant, and E is the activation energy. A plot of log activity against the inverse of the absolute temperature will result in a linear relationship if the activation energy is constant over the whole temperature interval. This function is very similar, over the temperature range used, to a simple exponential function, where a plot of log activity against temperature will result in a straight line if Q10 (the ratio of activity at two temperatures differing by 10 °C) is constant. To model an activation energy or Q10 value that varies with temperature, further variables have to be introduced [2–4]. The use of these models was, however, outside the scope of the present study.
The respiration rate increased with increasing temperature over the whole temperature range in the agricultural soil (Fig. 1(a)) and up to 40 °C in the humus soil (Fig. 1(b)). Thus, the maximum temperature for respiration (Table 1) was above these temperatures. The respiration rate at 45 °C was around 120 times higher than at 0 °C in the agricultural soil and 70 times higher in the humus soil.
Table 1. Minimum, optimum and maximum temperatures for total activity (respiration rate), bacterial (growth rate as thymidine incorporation rate) and fungal (growth rate as acetate-in-ergosterol incorporation rate) activity in an agricultural and a forest humus soil
The apparent minimum temperature (tmin) was calculated from the Ratkowsky equation, while the optimum and maximum temperatures were visually estimated from graphs.
The data showed good agreement with the square root (Ratkowsky) model up to 25 °C (Fig. 1(c), (d)), in that a linear relationship was found between temperature and the square root of the respiration rate (R2= 0.981 and 0.980, for the agricultural and humus soil, respectively). The respiration rate was about 20 times higher at 25 °C than at 0 °C. Above 25 °C, a steeper linear relationship between temperature and the square root of the respiration rate was observed for both soils. The use of the square root model up to 25 °C resulted in calculated apparent minimum temperatures (tmin) of around −6 °C for both soils (Table 1).
The respiration data were also plotted using the Arrhenius equation (Fig. 1(e), (f)). In both cases very good linear relationships were found over the whole temperature range (R2= 0.992 and 0.993 for the agricultural and humus soil, respectively, not including the lowest temperatures in the regression). However, in both cases the respiration rates at low temperatures were overestimated by this function.
3.2Bacterial growth rate
Optimum temperatures for thymidine incorporation rates of the bacterial community were between 25 and 30 °C for both soils (Table 1, Fig. 2(a), (b)). The bacterial activities at the optimum temperatures were around 14 and 9 times above that at 0 °C for the agricultural and humus soil, respectively. Above the optimum temperature the bacterial activity decreased, but some activity was observed even at 45 °C, indicating that the maximum temperature for growth of the bacterial community was above this temperature.
The bacterial activity was well described by the Ratkowsky function below the optimum temperature for growth in both soils (R2= 0.986 and 0.979 for the agricultural and humus soil, respectively; Fig. 2(c), (d)). The calculated apparent minimum temperature for bacterial growth (tmin) was −8.4 °C for the bacterial community from the agricultural soil and −12.1 °C for that from the humus soil.
Application of the linear Arrhenius equation using only data below optimum temperature for growth would result in overestimation of the bacterial activity at low temperatures (Fig. 2(e), (f)). However, no equations were fitted in the graphs, since the relationship appeared non-linear.
3.3Fungal growth rate
It was more difficult to measure the fungal activity with the acetate-in-ergosterol technique than performing measurements with the other two techniques due to a higher variation in data. The optimum temperatures for fungal growth were found to be between 25 and 30 °C for both soils (Table 1, Fig. 3(a), (b)). At this temperature, values of fungal activities were around 10 times higher than at 0 °C. Above this temperature the fungal activity decreased rapidly, resulting in a maximum temperature for growth of the fungal community of the forest humus soil at approx. 40 °C (Fig. 3(b), Table 1). In the agricultural soil high, but variable, values were found at this temperature (Fig. 3(a)) possible due to thermophilic fungi that started to grow during the incubation period. This data point was therefore not included in the evaluation.
The fungal activity below the optimum temperature for growth was well described by the Ratkowsky function in both soils (R2= 0.883 and 0.920 for the agricultural and humus soil, respectively; Fig. 3(c), (d)). The calculated apparent minimum temperature for fungal activity (tmin) was estimated to be −12.3 and −17.5 °C for the fungal community from the agricultural and humus soil, respectively.
Application of the linear Arrhenius equation using only data below optimum temperature for growth would result in overestimation of the fungal activity at low temperatures (Fig. 3(e), (f)). However, no equations were fitted in the graphs, since the relationship appeared non-linear, especially for the humus soil (Fig. 3(f)).
3.4Comparison of the temperature effect on bacterial and fungal growth rates
The activities at 25 °C were set to one, when comparing the bacterial and fungal growth rates at different temperatures (Fig. 4). Thus, a negative log ratio for the relative bacterial to fungal growth rate at a certain temperature indicates that bacterial growth was more negatively affected than fungi at this temperature, while a positive value indicates that fungal activity was more negatively affected. Both soils showed similar results. At 30 °C and below, the ratio of bacterial growth to fungal growth did not differ much from zero, although a tendency towards somewhat lower values was seen at the lowest temperatures, indicating that bacterial growth was slightly more negatively affected by low temperatures than fungi. At temperatures above 30 °C high ratios were found, showing that fungal growth was more negatively affected by high temperatures than bacterial activity. That the bacterial growth was more negatively affected by low temperatures compared to the fungal growth could also be deduced by comparing tmin values (Table 1). tmin for fungal growth was 3.9 and 5.4 °C lower than for bacterial growth in the agricultural and humus soil, respectively.
The temperature dependence of fungal and bacterial growth differed in that the former group was less inhibited by low temperatures and the latter less inhibited by higher temperatures (Fig. 4). This can also be seen by the lower apparent tmin for fungal growth than that for bacterial activity (Table 1). The advantage of fungi at low temperatures is in accordance with the finding that fungi dominated in high-altitude soils during winter and spring, when the soil was covered with snow, whereas bacteria appeared to dominate during summer under snow-free conditions [20,21,26]. The advantage of fungi at low temperatures may also explain the high amounts of fungal biomass found in forest soils during cold periods and in oligotrophic peat sites. In a comparison of fungal and bacterial growth on decomposing leaves in streams at different temperatures using similar techniques as in our study, fungi also appeared less negatively affected by low temperatures compared to bacteria (recalculated from Figs. 5 and 8 in).
One complicating factor is that the methodology used for determining the temperature dependence of growth of a microbial community can affect the results. Both leucine and thymidine incorporation can be used to estimate bacterial growth rates in soil. However, the use of leucine incorporation rate instead of thymidine incorporation rate resulted in higher values of the apparent tmin (a mean difference of 2.4 °C between thymidine and leucine incorporation) in two soils, resulting in the ratio of leucine to thymidine incorporation decreasing with decreasing temperature. This was also found in peat over a large temperature interval and in different aquatic habitats. The use of leucine instead of thymidine to determine the temperature dependency of the soil bacterial community will therefore result in a ratio of bacterial to fungal growth that is even lower at low temperatures than that shown in Fig. 4. Thus, the basic conclusion that fungal growth is less affected than bacterial activity at low temperatures is still valid, although caution must be exercised when stating the extent of this difference.
Another complicating factor is that fungal and bacterial growth rates were measured with different methods, the former reflecting membrane synthesis and the latter DNA synthesis. It is well known that temperature affects the membrane composition of microorganisms, e.g. the phospholipid fatty acid composition changes with temperature (e.g.). Ergosterol content might also be affected by temperature, although conflicting results exist in the literature. Both increasing and constant ergosterol content of fungi grown at higher temperatures have been reported [32–34]. However, at the moment there are no other available method for directly measuring fungal growth than the acetate-in-ergosterol technique. We have therefore chosen to present the comparison of fungal–bacterial growth at different temperatures in Fig. 4, although we are well aware that this relationship can be modified, when alternative methods to measure fungal growth rates in nature have been developed.
All three activity measurements followed the square root (Ratkowsky) function (a straight line of the square root of the activity vs. temperature) below the optimum temperature for growth of the fungal and bacterial communities (Figs. 1–3). This has earlier been shown to be the case for bacterial growth in soil and water, and for fungi in soil. The Ratkowsky model has also been found to adequately describe the temperature dependence of soil respiration [5,18,35] and the total activity under anaerobic conditions (denitrification,), and it has been used to model the decomposition of crop residues in soil [35,37,38]. Thus, the Ratkowsky function not only models the temperature dependency of bacterial and fungal growth in pure culture [23,24,39], but also the growth and activity of the whole microbial community. The apparent tmin values determined in the present study (Table 1) were similar to those found earlier for bacteria (−6.3 to −10.8 °C;), fungi (−11 °C;) and respiration (−6.0 °C;) in soil.
Using the Arrhenius equation or a straightforward Q10 relationship resulted in overestimation of activity at low temperatures (Figs. 1–3(e), (f)). This has been observed several times, both for pure cultures [23,24] and communities [5,25] and is due to Q10 not being constant but increasing at lower temperatures [2–4]. To adjust for the variation in Q10, models with more variables have been used [2–4,40,41]. This will result in temperature dependence being adequately modelled. However, the square root model, with an apparent tmin below 0 °C, will also result in an increase in Q10 with a decrease in temperature (Fig. 1 in). Using the equation proposed by Kirschbaum, based on several different studies of CO2 efflux from soil and litter, and recalculating it on a square root basis results in an almost perfect straight line relationship between respiration rate and temperature (R2= 0.998 with a value of apparent tmin of −4.2 °C). This does not mean that one should replace the equation used by Kirschbaum with the square root function, for example, in modelling large-scale effects of changing climate on the carbon balance of soils. The square root function will be very dependent on fitting the correct tmin value at low temperatures, while the equation with more variables used by Kirschbaum will give more flexibility in providing the best possible fit to the data. However, if the square root model provides an adequate description of the instantaneous growth rate and activity of soil microorganisms, any deviation from this relationship may be interpreted as an indication that temperature does not only have a direct effect on a particular activity. This can be exemplified by the “uncoupled” respiration above 30 °C, where little microbial growth occurred and where a breakpoint in the straight line between the square root of the respiration rate and temperature was found (Fig. 1(c), (d)). In other situations temperature might have complex effects, resulting in a temperature dependence that does not follow the square root model. This may be the case with soil methane efflux, where temperature not only directly affects the growth of methanogenic bacteria and the rate of methane production, but also significantly affects the pathway of carbon flow, that is, the production of precursor molecules for methane production, and the rate of methane consumption by the methanotrophic community [42,43]. This was also seen in a recent study on the temperature effects on N-transformations. N-mineralization closely followed the square root relationship (except at high soil moisture contents), while the denitrification rate deviated from this model being higher than expected at temperatures below zero. This anomaly was not due to a direct temperature effect, but on decreased oxygen diffusion through frozen soil, resulting in more anaerobic conditions facilitating denitrification.
The respiration rate above 30 °C appeared not to be coupled to microbial growth, since the former increased at higher temperatures, while the latter decreased (compare Fig. 1 with Figs. 2 and 3). This uncoupling could not be detected without the simultaneous measurement of respiration rate and microbial growth rates. It is likely that many earlier studies on instantaneous respiration at high temperatures [11–14] also included respiration not related to growth, although this would not be apparent without the complementary measurements of bacterial and fungal growth. For example, replotting the data of Anderson and Domsch according to the square root function revealed a similar situation to that found in the present study (Fig. 1(c), (d)), with a breakpoint around 30 °C. The reason for the uncoupling of soil respiration and microbial growth at higher temperatures is not known, but might be explained by observations on soil sterilized by irradiation. Soil respiration continues for weeks after irradiation even if the organisms are dead. This is because the enzymes are still functioning. Under such circumstances one would expect the CO2 efflux to become equivalent to a first-order enzymatic reaction, which will follow the Arrhenius function closely. This was also the case at temperatures above 30 °C (Fig. 1(e), (f)).
The uncoupling of the respiration rate from microbial growth at high temperatures has two implications. First, including respiration rates at temperatures above the optimum temperature for growth of the microbial community will obscure the fact that the respiration rate follows the square root model, and instead the Arrhenius or an exponential function with constant Q10 may appear to be a better choice. Second, it appears unlikely that the respiration rate at these high temperatures (above the optimum for growth of the soil microbial community) will allow us to compare the temperature dependence of microbial communities living in different thermal environments. Thus, including measurements of bacterial and fungal growth makes it possible to decide which temperatures to include for respiration measurements when studying microbial community adaptation to changing temperatures.
A partial uncoupling between respiration and microbial growth was also indicated by the different effects of temperature below optimum. Respiration increased around 20 times between 0 and 25 °C, while lower values were found for bacterial and fungal growth rates. Thus, it appears that the microorganisms released relatively more CO2 while growing at near optimum temperatures than at low temperatures.
We did not find any major differences in apparent tmin between the two soils studied for any of the activity measurements, being below freezing in all cases (Table 1). This contradicts the findings of Persson et al., that the respiration rate of an agricultural soil not only had a higher tmin than a forest soil, but also had a tmin above freezing point (+1.6 °C). However, they acknowledged that their respiration measurements of the agricultural soil at low temperatures were uncertain, which might have severely affected the calculated tmin value. It therefore seems likely that tmin in temperate and arctic soils is below 0 °C. However, one must bear in mind that tmin is an apparent value and can only be used to model activities under non-freezing conditions. For example, below freezing point the respiration rate will be affected by temperature in a different way from that above freezing, with Q10 values increasing abruptly [46,47].
It is important to keep the incubation time at high temperatures to a minimum in order to avoid the growth of a thermophilic community when studying the temperature dependency of the original community. This was easy with the respiration technique (5 h at 45 °C) and when measuring the bacterial activity (2 h at 45 °C), while longer times were needed for the fungal activity measurements to achieve values above the background. The growth of thermophilic fungi during incubation was probably the explanation of the anomalously high values of the rate of acetate-in-ergosterol incorporation at 40 °C in the agricultural soil (Fig. 3).
The main finding of the present study was that fungal and bacterial growth rates were affected differently by temperature. It is thus possible that changes in temperature due, for example, to climate changes, may alter the balance between these two main groups of soil microorganisms. However, one must bear in mind that the present study addressed instantaneous growth. Changes in temperature over a longer period of time may result in adaptation of the microbial community to the new conditions, probably through changes in species composition. This is a different situation from that studied here, and therefore the temperature dependence of the fungal and bacterial communities may be different.
This work was supported by a grant from the Swedish Research Council to E.B. and a grant from the Academy of Finland to J.P.