Temperature-dependent changes in the soil bacterial community in limed and unlimed soil
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Temperature-dependent changes in the soil bacterial community inlimed and unlimed soil
Marie Pettersson , Erland BaathDepartment of Microbial Ecology, Ecology Building, Lund University, SE-223 62 Lund, Sweden
Received 5 February 2003; received in revised form 21 March 2003; accepted 21 March 2003
First published online 23 April 2003
A humus soil with a pH(H2O) of 4.9 was limed to a pH of 7.5 and was incubated together with samples from unlimed and field limed(pH 6.1) soils at 5, 20 and 30C for up to 80 days. The changes in the phospholipid fatty acid (PLFA) pattern were most rapid for thebacterial community of the soil incubated at 30C, while no changes were found in the soil incubated at 5C. The response of thecommunity activity to temperature was measured using the thymidine incorporation method on bacteria extracted from the soil. Thebacterial community in soil incubated at 30C became more adapted to high temperature than that in soil samples incubated at 5C.When soil samples incubated at 30C and 20C were returned to 5C for 35 days, only small changes in the PLFA pattern were found. Nosignificant shift in community temperature adaptation was found. Thus, higher temperatures (with higher turnover) led to higher rates ofchange in both the PLFA pattern and the activity response to temperature, compared with lower temperatures. No effect of liming as away of increasing substrate availability and turnover on the rate of change was observed. Changes in the PLFA pattern appeared soonerthan changes in the activity response to temperature, indicating that changes in the PLFA pattern were mainly due to phenotypicacclimation and not to species replacement.7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: Temperature response; Phenotypic plasticity ; Phospholipid fatty acid analysis ; Thymidine incorporation
Temperature is one of the most important environmen-tal factors regulating the activity and determining thecomposition of the bacterial community. Bacterial com-munities inhabiting environments with dierent tempera-ture regimes therefore have dierent temperature relation-ships with cardinal points (minimum, optimum andmaximum) related to the environmental conditions [1^5].If the temperature is changed, a selection pressure will
be applied, altering the bacterial community. Bacterialspecies more adapted to the new conditions will growfaster, while those not well adapted will be outcompeted.Initially, this will be seen as a change in the temperaturerelationship of the bacterial community with cardinalpoints more suitable to the new conditions. With time,the bacterial community structure will change.
The eect of temperature actually consists of two parts.First, the greater the temperature shift, the greater magni-tude of the selection pressure, and thus the increase incommunity adaptation. However, temperature may alsoaect the rate of change of the community. Petterssonand Baath  found that the rate of community adapta-tion to pH was faster at 30C than at 5C. They explainedthis as an increased turnover of bacteria, where a higherturnover would enable the more rapid replacement of lessadapted species by more adapted ones. Similar results re-garding the eect of temperature on the rate of commu-nity adaptation to heavy metals were found by Diaz-Ra-vina et al. .Another way of inuencing the rate of community
adaptation by increasing the turnover of the communityis to add substrate (carbon) to the soil. This would avoidthe two confounding eects of temperature describedabove. One way of increasing substrate availability is byliming an acid soil, since the increase in pH releases or-ganic matter in forms more available for microorganisms[8,9], thereby increasing the activity and growth (and turn-over) of the microbial community.
0168-6496 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.doi :10.1016/S0168-6496(03)00106-5
* Corresponding author. Tel. : +46 (46) 222 3760;Fax: +46 (46) 222 4158.E-mail address: email@example.com (M. Pettersson).
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To measure the adaptation of the bacterial communityto dierent temperatures we used a technique based ondirect measurement of the bacterial activity, the thymidine(TdR) incorporation technique. The basic technique forsoil has been described by Baath . When the techniqueis used to measure community adaptation to temperature,the TdR incorporation of a bacterial community is mea-sured at dierent temperatures . Since TdR incorpora-tion measures instant activity, dormant and inactive cellswill have no inuence. The TdR incorporation techniquehas previously been used to measure the inuence of tem-perature change on the activity of the bacterial communityin soil [12,13] and to measure temperature as a regulatingfactor in aquatic systems [14^17].The phospholipid fatty acid (PLFA) pattern has previ-
ously been used to study changes in the soil bacterial com-munity. By studying PLFAs one can determine changes inthe community without determining the exact species com-position, since a change in the PLFA pattern is supposedto indicate an altered community. The technique has beenused to measure community dynamics during temperaturechanges in compost and heated peat [18,19] and microbialcommunity development to elevated atmospheric temper-ature in a model terrestrial ecosystem .The eect of temperature on bacterial communities has
also been studied using molecular approaches. For exam-ple, the soil bacterial community structure measured usingDGGE was altered after a geothermal heating event in theYellowstone National Park . T-RFLP has been used tomeasure how dierent temperature stress aected thestructure of the methanogenic archaeal community ofrice eld soil [22,23].In a study of bacteria in peat  it was shown that the
change in the community temperature relationship was, asexpected, dependent on the extent of the environmentaltemperature change. However, since they studied self-heated peat, the temperature range was quite extreme,with temperatures rising to those well outside the temper-ature range of the initial community. The objective of ourwork was therefore to study the eect of temperature onthe soil bacterial community using a more relevant tem-perature range.In the present study we have compared the eect of
temperature changes on the PLFA pattern and the tem-perature response based on activity measurements. Theeects of both increasing and decreasing temperaturewere studied. Several hypotheses were tested in this paper.First, the change in bacterial PLFA pattern and commu-nity tolerance to a new temperature would be most rapidwhen the turnover of the community was highest. Theturnover was aected by both temperature and substrate(liming). Regarding the latter treatment we hypothesizedthat the shift in PLFA pattern and community tolerancewould be faster for limed soil than for unlimed soil, sincethe activity and turnover are higher in the former soil .Secondly, we hypothesized that changes in the PLFA pat-
tern and community tolerance with temperature would befaster when changing the soil temperature from 5 to 30C,than from 30 to 5C, since the turnover of the soil bacte-rial community is lower at low temperatures. Further-more, we wanted to compare how rapid changes in themicrobial community due to temperature were detectedbased on activity measurements and PLFA compositionto try and elucidate to what extent the changes in thePLFA pattern were due to changes in species compositionor to phenotypic changes.
2. Materials and methods
Two humus soils from an experimental liming area ofconiferous trees (mainly spruce) in Hasslov (5624PN,130PE), southern Sweden, with an approximate yearlysoil temperature ranging between frozen and 20C wereused. One of the soils was unlimed and had a pH(H2O)of 4.9. The other had been limed in the eld 15 years agowith 8750 kg ha31 dolomite and had a pH(H2O) of 6.1.The soils were collected at the end of August (soil temper-ature approximately 10C), sieved the same day (2.8 mmmesh size) and used immediately.
Samples of the unlimed soil were limed with 2.2 gCaCO3 per 100 g soil. The pH(H2O) was 7.5 in this ex-perimentally limed soil. The following abbreviations willbe used for the dierent soil samples : UL=unlimed soil,FL=eld limed soil and EL= experimentally limed soil.Subscripts indicate soil incubation temperature, e.g. EL30indicates experimentally limed soil that was incubated at30C. When two temperatures are given, the rst indicatesthe initial incubation temperature, while the second indi-cates the nal incubation temperature. For example,EL30; 5 indicates an experimentally limed soil rst incuba-ted at 30C and then at 5C.We performed two dierent types of temperature experi-
ments. The increased temperature (IT) experiment wasperformed twice. In the initial (rst) experiment, two ELsamples were incubated at 5C, 20C and 30C, and twoUL and two FL samples were incubated at 5C. The ULsamples were used as a low-pH control, giving the baselineof the measurements, while the FL samples were used as ahigh-pH control with an already high-pH-tolerant commu-nity in order to be able to dierentiate between pH- andtemperature-related eects. In the main (second) IT ex-periment the same treatment as above was applied, butthe UL and FL soils were also incubated at 30C.In the other type of experiment we decreased the tem-
perature. The decreased temperature (DT) experiment wasalso performed twice (Fig. 1). In the rst DT experiment
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we took advantage of the main IT experiment. After 83days of the main IT experiment the samples incubated at30C were incubated at 5C and followed for another 34days. In the second DT experiment replicate samples fromthe EL (limed on day 0), UL and FL soils were incubatedat 5, 20 and 30C. After 36 days, half of the samples wereincubated at 5C, while the other half remained at theoriginal temperature for a further 35 days.
2.3. PLFA analysis
Samples of 0.5 g were taken and stored at 318C. Anal-yses were performed on samples taken at eight occasionsat intervals up to 50 days in the initial experiment and at11 occasions up to 82 days in the main IT experiment.Samples were taken at six occasions up to 34 days in therst and up to 35 days in the second DT experiment.Lipids were extracted and PLFAs quantied by a proce-dure described previously .
2.4. Bacterial community temperature response based onactivity measurements
Bacterial activity was measured on bacteria extractedfrom soil. TdR incorporation was measured according toBaath  in the initial experiment. In the main IT andrst and second DT experiments the modied method ofBaath et al.  was used. Briey, 1 g of soil was put intoa glass ask and 40 ml of Milli Q water was added. Thesamples were shaken on a rotary shaker (200 rpm) for 15min and then centrifuged at 1000Ug for 10 min. Thesupernatant with the extracted bacteria was then used.1.5 ml of each sample was put in an Eppendorf tubeand 15 Wl methyl[3H]TdR (926 GBq mmol31, Amersham)diluted 1:3 was added. Isotope dilution  was not takeninto account. Incubation was terminated after 1 h at 30and 35C, 2 h at 25C, 4 h at 17C and 24 h at 5C byadding 75 Wl cold 100% TCA. Non-incorporated TdR wasremoved with repeated centrifugation washings(13 000Ug) with 1.5 ml of cold 5% TCA and 1.5 ml cold80% ethanol. Then 0.2 ml of NaOH was added and thesamples were incubated at 90C for 60 min. Finally 1 ml
of scintillation cocktail (Ultima Gold, Canberra-Packard)was added before scintillation counting.After 44 days of the initial IT experiment the temper-
ature response of the bacterial community was measured.Bacterial suspensions, extracted from the EL30, EL20 andEL5 soils, were incubated at 5, 20 and 30C, using threereplicates of each at each incubation temperature. Similartemperature response tests were performed after 35 and 69days in the main IT experiment using a range of temper-atures (5^35C). The TdR incorporation rates were nor-malized to that found at 25C in order to facilitate com-parisons. In the rst and second DT experiments thetemperature tolerance was measured at intervals up to35 days after the change to the lower temperature. TdRincorporation was only measured at two temperatures(5 and 35C) and the log of the ratio of these values (TdRincorporation at 35C/TdR incorporation at 5C) wasused as an index of the community response to temper-ature. A high value of this ratio indicates a communitybetter adapted to high temperature.
Concentrations of the individual PLFAs (expressed asmol%) were subjected to principal component analysis(PCA) after scaling to unit variance. Thirty-one bacterialPLFAs in the initial and 33 in the main IT experimentwere identied and used to compare the eects of thedierent forms of treatment. Thirty-two bacterial PLFAsin the rst and 33 in the second DT experiment were usedto compare the dierent kinds of treatment. For thePLFA analysis n=1 and for the TdR analysis n=3, exceptfor the initial experiment (n=2). In the second DT experi-ment a two-way analysis of variance (ANOVA) with timeas one factor and treatment (UL, EL and FL) as the otherwas made for the scores of the rst principal component.The response of the bacterial community activity to tem-perature for the measurements on days 35 and 69 wasanalyzed with a paired t-test where similar treatments(UL, EL and FL) were compared.
3.1. PLFA pattern
A shift in the PLFA pattern with incubation tem-perature was evident when the dierent treatments werecompared using PCA (Fig. 2). The low-temperature treat-ment (5C incubation) resulted in low values for the rstcomponent in both IT experiments, while increasing tem-perature (20 and 30C) gave higher values along the com-ponent. The second principal component mainly dieren-tiated between soils with dierent pH, with low values forlow-pH treatment. The rst principal component ac-counted for 35.0% of the variation in the initial IT experi-
UL EL FL
5 oC 20 oC 30 oC 5 oC 20 oC 30 oC 5 oC 20 oC 30 oC
5 oC* 20 oC 5 oC 30 oC 5 oC* 5 oC* 20 oC 5 oC 30 oC 5 oC* 5 oC* 20 oC 5 oC 30 oC 5 oC*
Fig. 1. Depiction of the two DT experiments for the unlimed soil (UL),experimentally limed soil (EL) and eld limed soil (FL). The rst rowof temperatures (A) indicate the initial incubation temperature for 82days in the rst, and 35 days in the second DT experiment, before theactual experiments started. The second row of temperatures (B) are thetemperatures used in the DT experiments. An * indicates the incubationtemperatures in the rst DT experiment. In...