Editor: Christoph Tebbe
Effect of drying and rewetting on bacterial growth rates in soil
Article first published online: 10 JUN 2008
© 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Ecology
Volume 65, Issue 3, pages 400–407, September 2008
How to Cite
Iovieno, P. and Bååth, E. (2008), Effect of drying and rewetting on bacterial growth rates in soil. FEMS Microbiology Ecology, 65: 400–407. doi: 10.1111/j.1574-6941.2008.00524.x
- Issue published online: 7 AUG 2008
- Article first published online: 10 JUN 2008
- Received 9 January 2008; revised 25 April 2008; accepted 28 April 2008.First published online 10 June 2008.
- soil moisture;
- bacterial growth;
- thymidine incorporation;
- leucine incorporation;
The effect of soil moisture on bacterial growth was investigated, and the effects of rewetting were compared with glucose addition because both treatments increase substrate availability. Bacterial growth was estimated as thymidine and leucine incorporation, and was compared with respiration. Low growth rates were found in air-dried soil, increasing rapidly to high stable values in moist soils. Respiration and bacterial growth at different soil moisture contents were correlated. Rewetting air-dried soil resulted in a linear increase in bacterial growth with time, reaching the levels in moist soil (10 times higher) after about 7 h. Respiration rates increased within 1 h to a level >10 times higher than that in moist soil. After the initial flush, there was a gradual decrease in respiration rate, while bacterial growth increased to levels twice that of moist soil 24 h after rewetting, and decreased to levels similar to those in moist soil after 2 days. Adding glucose resulted in no positive effect on bacterial growth during the first 9 h, despite resulting in more than five times higher respiration. This indicated that the initial increase in bacterial growth after rewetting was not due to increased substrate availability.
Moisture conditions, temperature and substrate availability are the major environmental factors controlling microbial activity in soil. The effects of these factors on different aspects of microbial activity, such as decomposition, C mineralization and N transformations, are therefore well known. Not only are moisture conditions per se important but also drying–rewetting cycles will affect microbial processes, and thus carbon and nitrogen gaseous emissions (Burger et al., 2005). Drying–rewetting events will be especially important in arid and semiarid environments, but most surface soils will experience seasonal fluctuations in moisture content. This is not only a soil problem, but will, for example, also affect microbial activity in river sediments, because during summer in more arid areas, rivers will often dry out completely (Amalfitano et al., 2008). The effect of soil moisture on activity and process rates in soil has therefore been extensively studied. Less is known, however, on the direct effect of soil moisture on microbial growth in soil, and respiration rate is often used as a substitute for growth rate (e.g. Bloem et al., 1992). However, we cannot always use soil respiration rates to infer microbial growth rates. For example, Pietikäinen et al. (2005) indicated that optimum growth rates in temperate soils were obtained at around 25–30 °C, but the soil respiration increased with temperature up to 45 °C, indicating at least a short-term uncoupling of microbial growth and respiration at higher temperatures.
It is well known that microbial activity is very low in air-dried soil (O'Connell, 1990; Franzluebbers, 1999; Schimel et al., 1999; Borken et al., 2003; Hicks et al., 2003) and a positive correlation between microbial activity, as reflected by respiration rate, and water potential or water content is generally found. The previous history of moisture conditions will also be of importance. Rewetting a dry soil will result in a flush of respiration (Birch, 1958; Kieft et al., 1987; Van Gestel et al., 1993; Clein & Schimel, 1994; Fierer & Schimel, 2002; Miller et al., 2005), often increasing within an hour (Rudaz et al., 1991; Rey et al., 2005) to levels more than five times higher than soil kept constantly moist. This flush usually persists for up to 10 days after rewetting (Franzluebbers et al., 2000; Fierer & Schimel, 2003; Pesaro et al., 2004). Two explanations of this flush have been proposed. One is improved access to nonbiomass soil organic matter (physically protected organic matter) that becomes accessible for microbial degradation due to structural changes in the soil after drying–rewetting. The second is that the pulse of new substrate has a microorganism origin. The latter could involve the dying and disintegration of cells due to the rapid changes in osmotic potential during drying–rewetting or the release of osmo-regulatory substances during the rewetting event, allowing the cells to adjust to the drastic change in water potential. Recent studies indicate that both these mechanisms could account for changes in carbon cycling (Fierer & Schimel, 2003), although on different timescales.
To our knowledge, no direct measurements of the effect of soil water content on microbial growth rates have been made. Using an indirect method (frequency of dividing–divided cells, FDDC), it was estimated that bacterial growth rates increased by up to 2.3 times after rewetting compared with the rate in continuously moist soil, but that this increase was temporary (Bloem et al., 1992). No differences in FDDC were found between wet and dry soils, despite a considerable difference in respiration rate, indicating possible uncoupling between bacterial growth and respiration. However, the short-term event directly after rewetting was not studied. Furthermore, as noted by the authors, FDDC is known to overestimate growth rates in environments containing particles, such as sediments and soils. Others have studied microbial biomass before and after rewetting events using plate counts (e.g. Bloem et al., 1992), total bacterial counts (e.g. Bloem et al., 1992; Saetre & Stark, 2005), total biomass measurements (Pesaro et al., 2004; Saetre & Stark, 2005) or ATP (De Nobili et al., 2006) as measures of microbial growth. These studies all showed increased biomass a few days after rewetting, but due to the low precision in bacterial counts or the need for long measurement periods, e.g. using substrate-induced respiration (SIR) measurements for biomass estimation, they have provided little information about short-term changes in growth rates.
The aim of the present investigation therefore was to study the effect of soil moisture and rewetting on bacterial growth in soil using techniques that more directly estimate growth-related processes, such as thymidine and leucine incorporation into bacterial macromolecules. The specific questions addressed in this study were: How is bacterial growth related to respiration rate at different soil moisture levels? How does bacterial growth respond in the short term (hours) and long term (days) to a rewetting event and does this response correlate to respiration? Finally, we compared two treatments that are known to almost instantaneously increase microbial activity (respiration rate) due to increased substrate availability – rewetting and glucose addition (using concentrations applied in the SIR method) – and their effect on bacterial growth and respiration. If the rewetting effect is only due to increased substrate availability, we would expect a similar development of bacterial growth rates with these two treatments.
Materials and methods
Respiration and bacterial growth at different soil moisture contents
A grassland sandy loam soil [top 5 cm, pH 6.5, 4% organic matter content, sieved (2 mm)] was used. Water-holding capacity was 60% water (dry weight basis). Fourteen subsamples (50 g) were air-dried for different times in order to obtain a water content gradient; one of them was air-dried for 3 days until it reached a constant weight (2.9% water). The subsample with the highest water content (42%) was stored under its original field-moisture conditions (constantly moist soil).
Soil respiration was then measured. One gram soil was placed in a 20-mL glass vial, flushed with air for 30 s and sealed. Carbon dioxide was determined after 2 days at 20 °C using a gas chromatograph.
Bacterial growth was estimated using thymidine or leucine incorporation. Thymidine and leucine incorporation was determined using the homogenization–centrifugation method (Bååth, 1992, 1994) with the modifications introduced by Bååth et al. (2001). One gram of soil and 20 mL of distilled water were placed in 50-mL centrifuge tubes, shaken for 15 min on a rotary shaker at 300 r.p.m. and then centrifuged at 1000 g for 10 min. Two replicate 1.5-mL samples of each supernatant (bacterial suspension) were incubated with 5 μL methyl-[3H]-thymidine (37 MBq mL−1, 925 GBq mmol−1, Amersham; 130 nM final concentration) and 5 μL l-[U-14C]-leucine (1.85 MBq mL−1, 11.3 GBq mmol−1, Amersham; 520 nM final concentration) in microcentrifuge tubes for 2 h at 20 °C. The incubation was stopped and the macromolecules were precipitated by adding 75 μL cold 100% trichloroacetic acid (TCA). Washing and preparation for scintillation counting was according to Bååth et al. (2001).
Leucine incorporation using a slurry technique after rewetting
Air-dried grassland soil (3 days' air drying) was rewetted to moisture conditions giving maximum bacterial growth rates (30% water). Leucine incorporation was then measured in the dry soil, and every 30 min in the rewetted soil starting 15 min after rewetting, according to the soil slurry technique described by Bååth (1990) with some modifications. Fifty milligrams of soil, 1 mL distilled water and 20 μL l-[U-14C]-leucine (3.1 μM final concentration) were added to microcentrifuge tubes and incubated for 15 min at 20 °C using four replicates. Incubation was terminated by adding 1 mL 5% formalin. After centrifugation and removal of the supernatant, the soil was extracted with 1.5 mL 0.3 M NaOH, 25 mM EDTA and 0.1% sodium dodecyl sulfate at 60 °C for 20 h. After centrifugation, the supernatant was transferred to new microcentrifuge tubes, cooled, acidified with 0.39 mL 1 M HCl and 0.105 mL 100% TCA and kept on ice for 30 min to precipitate macromolecules. The precipitate was washed by centrifugation with 1.5 mL 5% TCA and then with 1.5 mL 80% ethanol. The precipitate was then resuspended in 0.2 mL 1 M NaOH and treated at 90 °C for 1 h, before scintillation cocktail was added (Ultima Gold, Perkin Elmer) and scintillation counting was performed.
Bacterial growth and respiration after rewetting and glucose addition
Air-dried grassland soil (3 days' air-drying) was rewetted to the same water content as soil kept constantly moist (30% water). This moist soil was also amended with 2 mg glucose-C and 0.1 mg NH4NO3-N with 2 mg talcum (to avoid clumping of the substrate) per gram of wet soil. This resulted in four different treatments, air-dried, rewetted, constantly moist and constantly moist with glucose addition.
The experiment was repeated two times to try and shorten the period required to obtain the bacterial suspension before measuring thymidine and leucine incorporation as much as possible. One gram of soil was added to 50-mL centrifuge tubes and 20 mL distilled water was added. The tubes were immediately vortexed at full speed for 3 min, followed by low-speed centrifugation at 1000 g for 10 min. A 1.5-mL sample was directly pipetted into a microcentrifuge tube, together with 5 μL methyl-[3H]-thymidine and 5 μL l-[U-14C]-leucine, and incubated for 1 h. The samples were then treated as above. The time elapsed from adding water to the soil sample for extraction of the bacteria to the addition of the radioactive substrate was thus only 15 min. In the first experiment, measurements were made on two replicates every hour for 7 h, thymidine and leucine being added to the first sample 15 min after rewetting or adding glucose to moist soil. Samples were also taken 24, 30 and 48 h after rewetting. In the second experiment, the effect of rewetting was studied over 4 days. Two different sets of soil samples were rewetted at different times (in the morning and in the afternoon using two replicates for each set). By sampling them at the same time, samples could be taken at fairly regular times after rewetting. Samples were taken two to three times per day for 4 days.
Respiration following rewetting and the effect of glucose addition were also studied in a separate experiment. Respiration was measured at 20 °C in a respirometer (Respicond IV, Nordgren Innovations, Sweden; Nordgren, 1988), using 20-g portions of soil. The respirometer was used instead of GC to allow for continuous measurements of respiration over several days. The air-dried soil had 5.5% water (four replicates) and the moist soil had 25.8% water (four replicates). It was decided to use a moist soil with a water content that was as low as possible, but that had a high bacterial activity (see the results of the soil moisture experiments with the same soil), to avoid anaerobic conditions when adding glucose in the long-term respiration experiment. Rewetting was started by adding water to the air-dried soil (six replicates), and the glucose treatment of moist soil was conducted by adding 2 mg glucose-C and 0.1 mg NH4NO3-N with 2 mg talcum per gram of wet soil (six replicates). The CO2 respired was recorded once every hour.
Effect of soil moisture content on respiration and bacterial growth rates
When comparing the effect of soil water content on bacterial growth determined using the leucine or the thymidine incorporation techniques, similar results were obtained (Fig. 1a and b). The driest soil (air-dried to a constant weight) had the lowest growth rate, which increased >10 times in wetter soil, showing a broad plateau with maximum values over a large range of water contents. Only in the driest soil did the ratio of leucine/thymidine incorporation change, being slightly higher than that in wetter soils (Fig. 1c). Soil respiration rate also increased, as expected, with increasing moisture content (Fig. 1d), but with a less evident plateau at higher soil moisture contents compared with thymidine and leucine incorporation (c.f. Fig. 1a and b with 1d). Thus, the soil respiration rate achieved maximum values at a higher soil water content than bacterial growth rates.
Effect of rewetting on leucine incorporation using a slurry technique
To minimize the time with added water to dry soil before adding the radioactive substrate, a slurry technique was used, where the substrate was added at the same time as water and the incubation times were made as short as possible (15 min). When the air-dried soil (lowest moisture content in Fig. 1) was rewetted, a linear increase in bacterial growth rate was obtained with time (Fig. 2). Already 15 min after rewetting the bacterial growth rates were threefold higher than in the air-dried soil. After 5–6 h, the increase in bacterial growth rate showed up to 10 times higher values than in the air-dried soil.
Comparison of rewetting and glucose addition on bacterial activity and respiration
The gradual increase in bacterial growth rate after rewetting was also seen when the homogenization–centrifugation technique was used instead of the soil-slurry technique to study the effect of rewetting. Bacterial growth increased linearly over 7 h to levels similar to those in the constantly moist soil (Fig. 3a, data for moist soil set to one). After 24 and 30 h, the growth rates in the rewetted soil were almost twice those in the moist soil, but this difference disappeared after 48 h. Bacterial growth was constantly lower in the moist soil with added glucose at the beginning of the experiment (83% of the wet soil, mean over the first 7 h), but bacterial growth then increased to levels 6–10 times higher than the moist soil (leucine incorporation after 24–48 h, Fig. 3a). Thymidine and leucine incorporation gave similar results (R=0.98 for the whole data set). However, calculating the ratio of leucine to thymidine incorporation (leucine/thymidine) revealed some differences (Fig. 3b). While this ratio decreased after rewetting (compared with that in constantly moist soil, which was set to 1), indicating that thymidine incorporation reacted more to the rewetting event than leucine incorporation, the opposite was found for glucose addition. The leucine/thymidine ratio increased for samples taken 24–48 h after addition of glucose (when bacterial growth increased significantly), showing that leucine incorporation rates increased more than thymidine incorporation rates in the growth phase after glucose addition.
Increased bacterial growth, compared with the constantly moist control soil, was also found when longer time periods after rewetting were studied, with maximum after around 24 h, decreasing to values similar to the moist soil after around 2 days (Fig. 4a). Thus, despite the initial low bacterial growth rates in the rewetted soil, integrating bacterial growth over the whole study period indicated that the rewetting event (over 4 days) increased bacterial growth by 25–30% compared with the moist control soil (integrating the data in Figs 3a and 4a). Although thymidine and leucine incorporation rates were well correlated (R=0.94 for the whole data set), the effect of rewetting was more evident for thymidine incorporation, resulting in lower leucine/thymidine incorporation ratios in recently rewetted soil than in the moist control soil (Fig. 4b). When the bacterial growth rates had returned to values found in the moist soil (after around 60 h; Fig. 4a), the leucine/thymidine incorporation ratio had reverted to the same value as in the moist soil.
The soil respiration rate was measured with the same soil and similar set up as for bacterial growth measurements over 13 days (Fig. 5, only first 100 h shown). Air-dried soil showed very low respiration rates throughout the experiment, with the constantly moist soil exhibiting consistently higher values. Adding glucose to the moist soil resulted in an increase in respiration (more than five times) within an hour, followed by stable values. After around 10 h, respiration increased again to a peak value after around 40 h. Rewetting the air-dried soil immediately resulted in high respiration rates (>10 times higher than the wet control soil), which then decreased exponentially over time eventually becoming similar to the wet soil. Higher respiration rates were thus found in the rewetted soil than in the moist soil treated with glucose during the first 2 h.
The main finding was that although rewetting caused an immediate increase in respiration rate, bacterial growth only recovered gradually in a linear fashion (c.f. Fig. 5 with Figs 2 and 3a). The increase in bacterial growth during the first 5–7 h could be explained by increased growth of a small number of surviving bacteria, growing on a substrate becoming more available after rewetting. This is, however, an unlikely explanation. First, such a growth increase would not result in a linear increase in growth, but an exponential increase. Second, the addition of glucose also increased the concentration of easily available substrate, but did not result in an increased growth rate during this time frame (Fig. 3a). Instead, we suggest that the increase in bacterial growth after rewetting was largely due to dormant bacteria becoming active after rewetting. This was also suggested by Saetre & Stark (2005). The activation may be a probabilistic event, similar to colony formation (Ishikuri & Hattori, 1985).
After the initial recovery in the rewetted soil, resulting in growth rates similar to those in the constantly moist soil, bacterial growth increased even further, to levels twice as high as in the moist soil after 24 h (Figs 3a and 4a). This is a time frame similar to that of the increase in bacterial growth after adding glucose, suggesting that this increase was due to growth on substrate made available by rewetting, and not due to further activation of dormant cells. It is unlikely that this increase was mainly due to growth on substances released from soil organic matter, because these substances are probably not available to a great extent. It is more likely that they have a biological origin, as discussed by Fierer & Schimel (2003). They also suggested that there could be two alternative sources of substances of biological origin: those released from organisms that have died as a result of the drying–rewetting treatment, or those resulting from cytoplasmic solutes rapidly released from the microorganisms due to the sudden change in osmotic potential upon rewetting. Although their data were not conclusive, they opted for the second explanation. Our data are compatible with either explanation, although one piece of evidence supports the latter explanation. Leucine incorportion rates increased slightly more than thymidine incorporation rates during the increased growth phase (>10 h) after glucose addition as indicated by an increase in the leucine/thymidine incorporation ratio (Fig. 3b). This has been observed previously after glucose addition (Aldén et al., 2001; Vinten et al., 2002) and after adding easily available plant material (Rousk & Bååth, 2007). However, rewetting resulted in a lower leucine/thymidine incorporation ratio, showing that thymidine incorporation responded more than leucine incorporation to those substances released by drying–rewetting, even when bacterial growth was higher than that in the control. This could be due to a large proportion of the substances released from the drying–rewetting treatment were amino acids, substances that would dilute the added radioactive-labeled leucine, resulting in apparently lower leucine-incorporation rates due to isotope dilution. Amino acids are the primary osmo-regulatory solutes in bacteria (Wood et al., 2001), and dilution stress of bacteria has been found to result in the release of large amounts of amino acids (Halverson et al., 2000). Adding a protein to soil also results in a lower increase in leucine incorporation compared with thymidine incorporation, probably due to similar dilution of the added labeled leucine with amino acids from the hydrolyzed protein (Meidute et al., 2008). Another explanation could of course be that different communities responded to these two treatments. It has, for example, frequently been reported that not all bacteria incorporate thymidine with the same efficiency (Christensen, 1993; Robarts & Zohary, 1993), and a shift in the community could thus result in altered leucine/thymidine incorporation ratios.
The long-term effect of rewetting on bacterial growth after the 24-h peak level was a revert to levels found in the constantly moist soil after around 2 days (Fig. 4). Most of the recovery in ATP content after rewetting recently dried soils was also observed after 2 days (De Nobili et al., 2006) and this is the same time frame in which Saetre & Stark (2005) found an increase in bacterial counts in a study on rewetting. A comparison of our data with those of Saetre & Stark (2005) demonstrates the much better sensitivity of measuring bacterial growth using the thymidine or the leucine incorporation technique than relying on bacterial counts. The bacterial biomass (calculated from microscopic counts) only increased from 310 to 460 μg C g−1 soil in air-dried and rewetted soil, while we found an increase of a factor of up to 20 for leucine incorporation between air-dried soil and rewetted soil after 24 h. This is probably due to a large part of the counted bacteria being in a dormant state in air-dried soil, bacteria that are alive, i.e. counted in the microscope, but not active, i.e. not incorporating thymidine or leucine.
The methods used to estimate bacterial growth involved adding a radioactive-labeled substrate to a water solution: either to a slurry or to a suspension of bacteria extracted from soil. This will of course affect the results. Bacterial growth in the air-dried samples was not measured under air-dried conditions, but during 15 min in a slurry (Fig. 2) or during 1 h in a water suspension (after 15 min to extract the bacteria) (Figs 3 and 4). This would, by necessity, overestimate the bacterial growth rate in dry soil. This is probably the reason why air-dried soil showed relatively lower respiration rates (<20 times lower than wet soils; Fig. 1d) than bacterial growth rates (about 10 times lower; Fig. 1a and b). However, the low bacterial growth rates in dry soil and the linear increase over a 5–7-h period could be detected using the present methodology despite this shortcoming. Low bacterial production was also found in benthic bacteria subjected to experimental drying using leucine incorporation (Amalfitano et al., 2008).
The glucose treatment was included to allow comparison with a treatment that increased substrate availability. The initial decrease (of about 17%, Fig. 3a) could be due to the high glucose concentration, resulting in a lower osmotic potential, decreasing bacterial activity. A lower osmotic potential due to salt addition has been found to decrease soil bacterial growth rates instantaneously (Bååth et al., 2001). The stable growth rates during up to around 8 h after glucose addition reflect the stable respiration values measured during this time, and are in accordance with a lag phase before growth starts to increase. This is also the time frame during which hardly any increase in dsDNA was found by Marstorp & Witter (1999), indicating no growth increase.
It is often assumed that the respiration rate can be used as an approximation of microbial growth in soil, that is, one assumes constant substrate efficiency and no wasteful metabolism. Thus, Bloem et al. (1992) compared bacterial production rates calculated using O2 consumption rates and FDDC after rewetting of soils. A correlation was indeed found between bacterial growth and respiration rates in soils with different soil moisture contents (Fig. 1). Our results, however, clearly showed that during rewetting there is no correlation between bacterial growth and respiration rate. Instead, there appeared to be wasteful respiration during the first hours, similar to the effect of adding glucose to the soil. Similar short-term decoupling of growth and respiration has also been found at high temperatures (Pietikäinen et al., 2005). Neither was there any correlation between respiration and bacterial growth up to 100 h after rewetting (c.f. Figs 4 and 5). This illustrates two important points. First, that respiration rate often provides a poor estimate of microbial growth rates in soil, and that there are several occasions when the apparent substrate-utilization efficiency varies considerably in soil. Second, the estimation of respiration and bacterial growth using the thymidine or the leucine incorporation method can be a suitable way of identifying situations in which wasteful metabolism occurs.
The long-term (4 days) discrepancy between bacterial growth and respiration rates could of course be explained by fungal growth and respiration. Fungal growth cannot, however, explain the short-term discrepancy. It is unlikely that fungi will be able to react within 1 h after rewetting to induce the high initial respirations, if bacteria will take 7 h to fully recover their activity. Thus, respiration by fungi could not explain the uncoupling between respiration and bacterial growth during the first few hours after rewetting. In the future, however, it will be important to simultaneously measure both bacterial and fungal growth to be able to fully understand the relationship between microbial growth and respiration rates in soil.
This study was supported by grants from the Swedish Research Council to E.B. We thank Dr A. Nordgren for measuring the rate of respiration in the rewetting study.
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