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

    1. Introduction

    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 [6] 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. [7].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: marie.pettersson@mbioekol.lu.se (M. Pettersson).

    FEMSEC 1517 2-6-03

    FEMS Microbiology Ecology 45 (2003) 13^21


  • 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 [10]. When the techniqueis used to measure community adaptation to temperature,the TdR incorporation of a bacterial community is mea-sured at dierent temperatures [11]. 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 [20].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 [21]. 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 [24] 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 [6].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

    2.1. Soils

    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.

    2.2. Experiments

    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

    FEMSEC 1517 2-6-03

    M. Pettersson,


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