ORIGINAL PAPER

Soil microbial recolonisation after a firein a Mediterranean forest

Gema Bárcenas-Moreno & Fuensanta García-Orenes &

Jorge Mataix-Solera & Jorge Mataix-Beneyto &

Erland Bååth

Received: 14 April 2010 /Revised: 8 December 2010 /Accepted: 21 December 2010 /Published online: 7 January 2011# Springer-Verlag 2011

Abstract The capacity of different microbial groups torecolonise soil after a fire event will be decisive indetermining the microbial community after the fire.Microbial recovery after a wildfire that occurred in Sierrala Grana (Alicante province, southeast Spain) was trackedfor 32 months after the fire. Colony forming units (CFUs)of different microbial groups, microbial biomass, soilrespiration, bacterial growth (leucine incorporation) andchanges in the microbial community structure (phospholipidfatty acid (PLFA) analysis) were determined directly after thefire and four times during the recovery period. Direct effects

were reflected by low values of most microbiologicalvariables measured immediately after the fire. Microbialbiomass increased during the first year after the fire but wasbelow the unburned reference site 32 months after the fire.Bacterial activity and soil respiration showed the highestvalues immediately after the fire, but decreased to valuessimilar to that of the unburned reference site or even lower(respiration) 32 months after the fire. Colony forming units ofbacterial groups estimated by the plate count method peaked8 months after the fire, but then decreased, showing valuessimilar to the unburned reference site at the end of the study,with the exception of spore formers, which were 20 timeshigher than the reference site 32 months after the fire. FungalCFUs were more sensitive to the fire and recovered moreslowly than bacteria. Fungi recovering less rapidly thanbacteria were also indicated by the PLFA pattern, with PLFAsindicative of fungi being less common after the fire. Therecovery of microbial biomass and activity was mirroredby the initially very high levels of dissolved organiccarbon being consumed and decreasing within 8 monthsafter the fire. The wildfire event had thus resulted in adecrease in microbial biomass, with a more bacteria-dominated microbial community.

Keywords Forest fire . Soil . Microbial community .

Microbial recolonisation . Fungi . Bacteria . Actinomycetes .

Spore formers

Introduction

Forest wildfires are one of the most critical threats toMediterranean forest ecosystems in the southeast of Spaindue to shortage of forested areas and human pressures onforest use. Mediterranean forests and wildfire, however,

G. Bárcenas-Moreno (*)MEDSoil Research Group, Department of Crystallography,Mineralogy and Agricultural Chemistry, Seville University,Chemistry Building, C/Prof. García González,41012 Seville s/n, Spaine-mail: gbarcenas@us.es

G. Bárcenas-Moreno : F. García-Orenes : J. Mataix-Solera :J. Mataix-BeneytoGEA—Grupo de Edafología Ambiental–Environmental SoilScience Group, Department of Agrochemistry and Environment,Miguel Hernández University,Avda. de la Universidad s/n,03202 Elche, Alicante, Spain

F. García-Orenese-mail: fuensanta.garcia@umh.es

J. Mataix-Solerae-mail: jorge.mataix@umh.es

J. Mataix-Beneytoe-mail: j.matix@umh.es

E. BååthMicrobial Ecology, Department of Biology, Ecology Building,Lund University,SE-223 62 Lund, Swedene-mail: erland.baath@mbioekol.lu.se

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have always evolved together, mainly because the climaticcharacteristics of Mediterranean areas favour wildfires(long drought periods and frequent temperatures above30°C). Nevertheless, at the end of the twentieth century, theaverage annual number of forest fires throughout theMediterranean basin was close to 50,000, i.e. twice asmany as during the 1970s. The annual cumulated burnedarea in the Mediterranean countries was estimated to beapproximately 600,000 ha, and only in Spain, it increasedfrom 50,000 ha in the 1970s to 208,000 ha in the 1990s(Alexandrian et al. 1999). Thus, changes in the intensityand timing of fires have, mainly through human influence,induced a critical situation where the few surviving forestedareas face a serious risk of being destroyed in the event of awildfire.

High-intensity fire can partially sterilise the surface soil,and the capacity to be competitive in recolonising the soilcan be decisive in determining the microbial communityafter a fire (Pietikäinen and Fritze 1995). Some groups ofmicroorganisms have been considered more sensitive toheat than others, for example fungi compared to bacteria(Bollen 1969; Dunn et al. 1985). It is also often reportedthat after heating bacteria will recover more rapidly and tohigher levels than fungi (Bollen 1969; Vázquez et al. 1993;Bååth et al. 1995; Badía and Martí 2003; Guerrero et al.2005; Mabuhay et al. 2006; Ponder et al. 2009). Previouswork in the Mediterranean area has shown that bacteriarecover faster than fungi in short-term laboratory experi-ments with heated soil (Guerrero et al. 2005; Bárcenas-Moreno and Bååth 2009), possibly due to a high pHfavouring bacterial growth (Rousk et al. 2009) andincreasing concentration of dissolved organic C due to theheating treatments inducing rapid growth.

Depending on fire intensity and severity, the soilecosystem could be transformed into a different environ-ment for soil microorganisms after the fire. Fire-inducedchanges in the quantity and quality of soil organic matter(SOM) are known as a major factor controlling ecosystemresponse after the fire (Widden and Parkinson 1975;Almendros et al. 1990; Vázquez et al. 1993; Pietikäinenand Fritze 1995; Díaz-Raviña et al. 1996; Prieto-Fernándezet al. 1998; Guerrero et al. 2005). Depending on the type ofvegetation, Romanyà et al. (2001) recorded maximumtemperatures at ground level between 285°C and 525°C inprescribed fires of Mediterranean grasslands and shrub-lands. A general decrease in organic C has been reported attemperatures above 200–250°C (Giovannini et al. 1988).The combustion of SOM not only leads to losses of soilC but also to altered proportions of SOM fractions(Fernández et al. 1997; Martín et al. 2009) and even theformation of toxic substances affecting the soil micro-organisms (Almendros et al. 1990; Díaz-Raviña et al.1996). The damage caused by fire to the vegetation can

also influence microbial recovery, either directly due toplant cover decrease or as a consequence of changes inplant species composition (Hart et al. 2005). The sum ofthe factors described above and other fire-induced changessuch as pH or nutrient availability will determine thecomposition of the microbial community during recoloni-sation after a fire. However, field studies over a longerperiod of time following the recovery of microbialbiomass, activity and community structure after a fire areless common (but see, e.g. Boerner et al. 2005, 2006),especially in the Mediterranean area, although Fioretto etal. (2005) studied the development of different microbialactivities in soil after fires of different intensities.

The main objective of this study was to determinemicrobial recolonisation processes in a field situation bymonitoring microbial biomass, activity and communitystructure. The study was conducted during 32 months aftera wildfire in a pine-oak forest in Spain. We especiallydirected the study towards how the wildfire affected thebalance of fungi and bacteria, as well as microbialsuccession, during the 32 months after the fire. This is thefirst time that several aspects on the balance between fungiand bacteria, including classic plate counts, biomass-basedand more sensitive growth-based methods, are studied in afield situation over a long period of time.

Material and methods

Study area

Soil was collected in a forest located in Torremanzanas (inthe province of Alicante, southeast Spain) at 38°36′ N, 0°25′ W and 750 m above sea level. A 178-ha area wasaffected by a wildfire in August 2005. Mediterraneansclerophyllous vegetation characterises this area. Pinushalepensis (Aleppo pine) is the dominant tree species,accompanied by Quercus coccifera (kermes oak) as themain woody shrub, sharing space with a diverse shrub cover(dominated by Rosmarinus officinalis, Ulex palviflorus,Juniperus oxicedrus, Cistus albidus and Arbutus unedo).The mean annual temperature is 15°C, and the mean annualprecipitation is 385 mm with a typical Mediterraneanprecipitation regime. The fire affected area was sampled ina northerly direction with a medium slope (around 20º). Thesoil is a typic Xerorthent on marly limestone as the parentalmaterial with a loam texture according to the USDA soiltaxonomy classification.

The burned area was sampled immediately and 8, 12, 24and 32 months after the fire. Vegetation recovery was notdirectly measured but visual examination allowed for somequalitative information. Thus, immediately after the fire, thewhole vegetation was destroyed and only charred stems

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remained. After 8 months, numerous bryophytes colonisedsoil and pine seedling (3–5 cm height) and kermes oak (5–15 cm) started to sprout. Some grasses also appeared inplaces. The first year after the fire the most abundantspecies were pine (20–15 cm height) and kermes oak (30–50 cm). At that time, C. albidus also appeared frequentlyand other species present in Mediterranean forest areasfrom this location were found like Asparagus sp., A. unedoand Rubia peregrina. During the second year, pine andkermes oak continued to grow, with increased grass growthat the fourth sampling. Finally, 32 months after the wildfire,the pines were between 1.20 and 1.50 m in height. Theplant cover was quite dense, although plant cover anddiversity in the burned area were still below the selectedreference area. After 32 months, the developed plantcommunity allowed for the selection of an unburnedreference forest situated near the fire area with similarvegetation, orientation and slope as the burned area. Theselection of such a reference stand was not possible beforedue to the wildfire destroying the vegetation. Soil parametersof this reference area (Table 1) were used to evaluate thefire’s effect 32 months after the fire. Since seasonal variationin the unburned area was not measured, we can only use it asa reference to evaluate the recovery process 32 months afterthe fire. Although we should take into account that seasonalcharacteristics in this area promote good water availabilityand temperature conditions which will favour microbialproliferation during spring compared with other seasons,Bastida et al. (2008) found no significant differences inmicrobial biomass and respiration in similar areas in thesoutheast of Spain.

Soil sampling

Soil samples were randomly collected from the upper5 cm after first removing the litter. The soil samples

were under the influence of pine or shrubs, taking threesub-samples around each plant stem to form onecomposite sample, repeating the process through theselected area until ten composite samples per samplingoccasion and site were obtained. Soil samples weresieved (2 mm mesh), thoroughly mixed and divided intotwo samples: fresh samples stored at 4°C to measurecolony forming units (CFUs) and air-dried samples forchemical properties, respiration, microbial biomass andbacterial growth. Since our study site has a semiaridclimate, we consider air-dried samples more representa-tive than freezing, since some of the measurement hadto be delayed for more than a month due to logisticreasons (analyses at a laboratory with different loca-tions). These samples were rewetted before microbialmeasurements. Soil water content was measured in bothfresh and air-dried samples to express the results pergram of dry soil.

Physical and chemical analyses

The particle size distribution was measured using aBouyoucos hydrometer (Bouyoucos 1951). The water-holding capacity was determined according to Foster(1995). The soil water content was estimated by drying at105°C overnight, and the soil pH and electrical conduc-tivity (EC) were measured in deionised water (1:2.5 and1:5 w/w, respectively) at 20°C. The soil organic C (SOC)was analysed using rapid dichromate oxidation of organicC (Walkley and Black 1934), and the dissolved organic C(DOC) was measured in non-fumigated soil samples, asreported for microbial biomass C (see below). The soilnitrogen (Nk) was estimated by Kjeldahl digestion (Bremmerand Mulvaney 1982), and the available phosphorus (P) wasextracted and measured using the Burriel–Hernando method(Díez 1982).

Table 1 Physical and chemical variables (mean of n=10±SE) measured in the unburned reference soil and in burned samples immediately (0)and 8, 12, 24 and 32 months after the fire

0 months 8 months 12 months 24 months 32 months Reference

Corg (g C kg−1 dry soil) 43.4±2.4a 33.7±2.9ab 33.2±1.7b 30.5±2.1b 31.7±2.7b,* 71.6±4.1

DOC (g C kg−1 dry soil) 1.7±0.1a 0.38±0.05b 0.38±0.04b 0.27±0.02b 0.35±0.03b 0.42±0.03

Nk (g N kg−1 dry soil) 2.4±0.15 2.0±0.20 2.3±0.21 2.1±0.13 2.2±0.18* 4.7±0.30

Soil C/N ratio 18.4±0.55 17.5±1.03 15.7±1.53 14.4±0.33 14.6±1.31 15.5±0.51

P (10−3 g N kg−1 dry soil) 22.6±2.8a 13.7±1.9b 13.1±1.6bc 6.0±0.5c 9.5±1.2bc 16.8±5.9

pH 7.9±0.03c 8.1±0.04abc 8.0±0.1bc 8.2±0.027a 8.2±0.03ab,* 8.0±0.04

Electric conductivity (μS cm–1) 874±47a 377±26b 438±35b 352±11b 366±29b 437±20.3

% Soil water content 3.3±0.26c 14.4±1.28a 6.2±0.32b 5.4±0.39bc 5.7±0.65bc,* 11.3±1.47

Different letters in the same row indicate significant differences over time in burnt soils (Tukey’s post hoc test, P<0.05)

Corg organic C, DOC dissolved organic C, Nk Kjeldahl nitrogen, P available phosphorous

*Indicates significant differences between unburned reference soil and burned samples 32 months after the fire (t test, P<0.05)

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Colony forming units

We estimated the number of aerobic bacteria, sporeformers, filamentous fungi and actinomycetes using theplate count method following Zuberer (1994). Tryptic soyagar was used to isolate both total aerobic bacteria andspore formers. For the latter, samples were subjected to10 min at 80°C in a water bath before inoculation.Actinomycetes were counted on actinomycete isolationagar (Difco TM) supplemented with 5 g l−1 of glycerol(Panreac), 10 mg l−1 of nalidixic acid (Panreac) and50 mg l−1 of cycloheximide (Panreac). Filamentous fungiwere grown on rose bengal chloramphenicol (0.1 g l−1)agar. Different dilutions were used in triplicate. Theincubation temperature was 25°C for all groups, butdifferent incubation times were used: 3 days for aerobicbacteria and spore formers and 4 days for actinomycetesand fungi.

Microbial biomass

The microbial biomass carbon (Cmic) was determined usingfumigation-extraction (Vance et al. 1987). Two 5-g samplesfrom each replicate were fumigated under CHCl3 atmo-sphere in the dark for 24 h, while two control samples werenot fumigated but also kept in the dark for 24 h. Organic Cwas extracted with 20 ml of 0.5 M K2SO4. The amount ofDOC was then measured colorimetrically at 590 nm aftersulphuric acid–potassium dichromate digestion (Sims andHaby 1971). The microbial biomass C was calculated usingthe following equation:

Cmic ¼ 2:66 DOCF � DOCNF½ �

where F and NF denote fumigated and non-fumigatedsamples, respectively.

Microbial activity

Basal respiration was measured in a closed system with amultisensor respirometer (Mycro-Oxymax, Columbus, OH,USA). Both CO2 and O2 were monitored for 4 days at 25°Cafter rewetting of air-dried samples at 60% of water-holdingcapacity. Respiratory quotient (RQ) was calculated bydividing moles of CO2 evolved by moles of O2 consumedand was used as an index of potential changes in the sourceof substrate used for respiration and/or the pathway ofrespiration (Lipp and Andersen 2003).

Bacterial growth was estimated using the leucine (Leu)incorporation technique, which provides a relative estimateof bacterial growth (Bååth 1994a; Bååth et al. 2001).Briefly, air-dried samples were rewetted 4 days before the

measurements. This is enough time to recover the bacterialactivity (Iovieno and Bååth 2008). Then, 2 g of soil wasmixed with 20 ml of distilled water and shaken atmaximum intensity for 3 min on a vortex shaker. Sampleswere subjected to a low-speed centrifugation (1,000×g for10 min), after which 1.5 ml of the bacterial suspension(the supernatant) was placed in 2-ml microcentrifuga-tion vials. L-[4, 5-3H]-Leucine (2 μl, 155 Ci mmol−1,Amersham, UK) and nonlabelled Leu were added,resulting in 275 nM Leu in the bacterial suspension.The bacteria were killed after 2 h by adding 75 μl of100% trichloroacetic acid. Centrifugation and washingwere performed as described by Bååth et al. (2001).Scintillation cocktail (Ultima Gold, PerkinElmer) wasadded, and the radioactivity was measured using a liquidscintillation counter. The amount of leucine incorporatedinto the extracted bacteria (picomoles incorporated perhour per gram of soil) was used as a measure of bacterialgrowth.

Composition of microbial community

The composition and relative bacterial and fungal biomassof the microbial community were estimated using phos-pholipid fatty acid (PLFA) analysis mainly according toFrostegård et al. (1993). Briefly, soil was extracted in a onephase mixture of chloroform/methanol/citrate buffer(1:2:0.8). Extracted lipids were fractionated on pre-packedsilica columns (Bond Elut®, Varian Inc., Palo Alto, CA,USA), followed by mild alkaline methanolyses. ThePLFAs used to calculate the bacterial biomass indexwere as follows: i15:0, a15:0, i16:0, 16:1ω9, 16:1ω7t,i17:0, a17:0, cy17:0, 17:0, 18:1ω7 and cy19:0. Thefungal biomass index was based on the amount of18:2ω6,9 (Frostegård and Bååth 1996). The PLFA patternwas only determined on four of the composite samples foreach sampling time.

Statistical analysis

Normal distribution and variance homogeneity weretested for all samples. Colony forming unit (CFU) datawere necessary to transform using log10(x+1). Burnedand unburned areas were compared using t test 32 monthsafter the fire. Changes in different variables over timeafter the fire were analysed using one-way ANOVA andTukey’s test. The SPSS 16.0 statistical program wasused.

The changes in PLFA composition were analysed byprincipal component analysis (PCA) using mole percentafter standardising to unit variance. PCA analysis was madeby MVSP 3.0. Differences were considered statisticallysignificant at P<0.05.

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Results

Chemical analyses

SOC and Kjeldahl N (Nk) were negatively affected by thefire. Immediately after the fire, SOC were 43 g C kg−1 andNk 2.4 g N kg−1 (about 50% to 60% of the reference), but inthe following samplings, SOC decreased even more(Table 1) while Nk remained at the same level for 32 monthsafter the fire (Table 1). Dissolved organic C levels in theburned area were significantly higher immediately after thefire (1.7 g C kg−1) than during the remaining samplings.Dissolved organic C values then decreased to levels similarto the unburned reference (Table 1). This level was thenmaintained during the remaining study period. Burnedsamples showed a higher C/N ratio immediately after thefire than the remaining samplings, since the ratio decreasedto levels similar to the unburned reference site after the firstsampling (Table 1). Available P followed a similar patternas DOC, showing higher values immediately after the firethan in the following samplings (Table 1). Immediatelyafter the fire, the pH in burned samples was not differentfrom that of the unburned reference area, around pH 8.However, 8 months after the fire, the pH became slightlyhigher in the burned area and was then kept at this level.Similar to DOC or P content, EC was significantly higherimmediately after the fire than in the following samplings(Table 1).

Microbial diversity and microbial biomass

Microbial CFUs showed the lowest values immediatelyafter the fire (Fig. 1): 8.4×103 CFU g−1 for spore formers(10% of the unburned reference site, Fig. 1b), 9×104 CFU g−1 for actinomycetes (2.1% of the reference,Fig. 1c) and 6.2×104 for aerobic bacteria (1.8% of thereference, Fig. 1a), down to less than 1% of the referencefor fungi (5.8×102 CFU g−1, Fig. 1d). Eight months afterthe fire, a significant increase was found for all microbialgroups. Spore formers reached 1.6×107 CFU g−1 (50 timesthe reference, Fig. 1b), followed by aerobic bacteria (4.7×107 CFU g−1, Fig. 2a) and actinomycetes (2.9×107 CFU g−1, Fig. 1c) with about 8 to 15 times highervalues than the reference. Filamentous fungi increased onlyto values 1.5 to 3 times higher than the reference (1.9×106 CFU g−1, Fig. 2d). Thus, the fungal/bacterial ratiobased on CFUs increased from around 0.01 to 0.07 after32 months, but were still significantly lower than in thereference site (Fig. 1e).

During the second and third years after the fire, allmicrobial CFUs started to decrease. Bacterial counts wereabove the reference site values 32 months after the fire,while CFUs of filamentous fungi decreased to levels similar

to those in the reference. Bacterial spore formers showedvalues more than 20 times higher than the reference siteafter 32 months.

Microbial biomass C (Cmic), estimated by fumigation-extraction, showed the sterilisation effect immediately afterthe fire, in that most of the samples were below detectionlevels, with only a few replicates having low amounts ofsurviving biomass (Fig. 2a). Microbial biomass C of burnedsamples showed a marked increase after 8 months, to about380 μg C g−1 (35% of Cmic found in the reference soil).This level was then maintained during the rest of the study,remaining significantly below the reference 32 months afterthe fire.

Similar to the CFU measurements, fungal and bacterialPLFAs immediately after the fire showed varying sensitivitiesto fire, with a higher relative amount of bacterial PLFAsremaining compared with fungal PLFA. The concentration ofPLFAs indicative of both groups then increased slightly overtime, but they were below that of the reference site 32 monthsafter the fire (Fig. 2b).

PCA of the PLFA pattern indicated that it was affectednot only directly by the fire but also during the recolonisa-tion period after the fire (Fig. 3a). PC1, accounting for42.8% of the variation, separated reference samples fromsamples immediately after the fire and especially 8 to24 months after. Burned soil samples 32 months after thefire, shown to the left in the graph, are similar to thereference samples. This indicates that the microbial com-munity was recovering. However, PC2, accounting for12.2% of the variation, mainly differentiated between thereference and the 32-month samples. Thus, PC1 indicatedthe short-term effects of fire on the PLFA pattern, whilePC2 indicated long-term effects.

The relative abundance of individual PLFAs wasreflected in the loading plot (Fig. 3b). The unburnedreference samples were characterised by relatively highamounts of some PLFAs related to fungi and gram-negativebacteria (18:2ω6,9, 16:1ω5 and 18:1ω7 shown to the leftalong PC1). On the other hand, burned samples weremainly characterised by relatively high amounts ofbranched PLFAs such as iso- and ante-iso-PLFAs as wellas 10Me types (shown to the right along PC1). Althoughburned samples had partially recovered 32 months after thefire, some PLFAs were more common in the burnedsamples than in the unburned reference. This included16:1ω7c, 17:1ω8, 18:1ω9, cy17:0 and 10Me17:0 (negativevalues along PC2).

Similar effects of the fire that were found for relativeconcentrations of PLFAs (mole percent) were also found forabsolute abundance of PLFAs (nanomole per gram drysoil). An immediate decrease, followed by a steadyincrease, was found, for example, for the PLFAs a15:0and cy17:0 during recolonisation after the fire (Fig. 3c),

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while the fungal-related PLFA 18:2ω6,9 started to increaseonly 24 months after the fire (Fig. 2b). Since the totalamount of PLFA, indicating total biomass, never recoveredto values found in the unburned reference site, the absolute

concentration of all PLFAs did not exceeded that of thereference 32 months after the fire. Also the fungal/bacterialratio based on PLFAs was lower than in the reference sitethroughout the study (Fig. 3d).

Fig. 1 Microbial abundance estimated by plate count during a 32-month period following a wildfire: total aerobic bacteria (a), sporeformers (b), actinomycetes (c), filamentous fungi (d) and fungi-to-bacteria (F:B) ratio (e). Mean values ± SE (n=10). Ref. denotes anunburned reference soil, sampled at the same time as the sampling

32 months after the wildfire. *Indicates significant differencesbetween unburned reference soil and burned samples 32 months afterthe fire (t test, P<0.05). Different letters indicate significant differ-ences over time in burned soils (Tukey’s post hoc test, P<0.05)

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Microbial activity

Total microbial activity, determined by respiration rate, andLeu incorporation, estimating bacterial growth, were theonly microbial variables that showed higher values imme-diately after the fire than in the remaining samplings andunburned reference site (Fig. 4a, b). However, one shouldkeep in mind that activity was not measured directly afterthe fire, but 4 days after rewetting, and thus had time torecover.

Leu incorporation shortly after the fire was 280 pmolLeu h−1 g−1 (around 3 times higher than the unburnedreference site). High values were still found 8 months afterthe fire, but then bacterial growth started to decrease,eventually reaching 100 pmol Leu h−1 g−1, levels similar tothe unburned reference at the end of the study (Fig. 4a).

Soil basal respiration shortly after the fire was almost 2times the reference (Fig. 4b). Then, respiration ratedecreased to around 0.2 μg CO2 h−1 g−1 8 months afterthe fire and then remained at this level (approximately 50%of the reference values), until the end of the study.Oxygen consumption and CO2 evolution were closelycorrelated (r=0.992). Thus, the respiratory quotient variedlittle in burned samples throughout, but immediately afterthe fire significant higher values were found than in theremaining samplings (Fig. 4c).

Discussion

The present study took advantage of a real wildfire. Thus,in order to sample directly after the fire, it was not time tochoose a control site. Furthermore, due to the fire destroy-ing the vegetation, we could not select a proper referencesite until vegetation had started to regrow. Therefore, thisstudy was designed as a longitudinal one. To avoidconfounding seasonal effects, samples were taken in thesame month in the early autumn (0, 12 and 24 months afterthe fire) or in spring (8 and 32 months after the fire). After32 months, regrowth of the vegetation allowed us toinclude a reference stand with similar vegetation, slopeand orientation. Especially the latter has been shown to beof importance for the biochemical and microbiologicalproperties of soils in this area, while both biomass andrespiration did not vary significantly between samples takenin spring and autumn (Bastida et al. 2008). Thus, althoughwe cannot exclude confounding seasonal effects on themicrobiological variables, they appear to be much lesspronounced in this area than the large effects seen directlyafter the fire (decreasing to less than 10% of the referencesite in most cases) and found later during recovery.

Fire effects on nutrient availability

Fire impact on total organic matter is usually reflected inthe diminution of total SOC content and even in thealteration of different fractions of organic matter. In ourstudy, both SOC and Nk decreased directly after the fire,shown by lower values than obtained in the unburnedreference area. This indicates the level of severity reachedduring the fire, since the combustion of organic matter andsubsequent losses of organic C and N appear at temper-atures of 220–460°C (Giovannini et al. 1990; DeBano et al.1998). On the contrary, an increase in the availability of Pand DOC immediately after the fire was found, similar tothe levels reported in other studies (Pietikäinen and Fritze1995; Díaz-Raviña et al. 1992; Choromanska and DeLuca2002; Prokushkin and Tokareva 2007). This increase inavailable nutrients apparently promoted good conditions for

Fig. 2 Microbial biomass as estimated by fumigation-extraction (a)and PLFA abundance of bacterial (filled symbols) and fungal (opensymbols) (b) of indicator PLFAs during 32 months after a wildfire.Ref. denotes an unburned reference soil, sampled at the same time asthe sampling 32 months after the wildfire. Mean values ± SE (n=10).*Indicates significant differences between unburned reference soil andburned samples 32 months after the fire (t test, P<0.05). Differentletters indicate significant differences over time in burned soils(Tukey’s post hoc test, P<0.05)

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short-term increases in microbial activity, both bacterialgrowth and respiration (Fig. 4) (Fernández et al. 1997;Prieto-Fernández et al. 1998; Andersson et al. 2004;Guerrero et al. 2005; Bárcenas-Moreno and Bååth 2009).Over the longer term (i.e., until the end of the study), DOCdecreased, and the levels of organic C and N remained low,emphasising the long period of time necessary for the build-up of soil organic matter, which is essential for therestoration of soil microbial biomass (see below).

Direct effects of fire on soil microorganisms

Partial sterilisation of the soil was manifested by an initiallydrastic decrease in CFUs (Fig. 1) and Cmic, but only apartial decrease in PLFAs (Fig. 2b), since the latterappeared less affected by fire than the other methods. Theproblem in estimating the direct damage produced by

heating using PLFA measurements has previously beenobserved by Bárcenas-Moreno and Bååth (2009). Only thehighest temperatures applied in their study (400–500°C)destroyed almost all PLFAs, while Cmic and bacterial andfungal growth had already decreased to virtually zero at200°C. They suggested that a decrease in PLFAs was dueboth to death of the organisms and direct destruction ofsome PLFAs and to a subsequent degradation of PLFAsfrom dead organisms that was not immediately destroyedby heating. Any subsequent degradation of PLFAs would,however, be confounded by an increase in PLFAs resultingfrom the emerging new community, which was growing onthe released available carbon. Even if almost the wholemicrobial community were killed by a heating event, withactivity decreasing to zero, one would not necessarily findzero levels of PLFAs. Thus, the immediate effect of thewildfire clearly seen in an altered PLFA pattern (Fig. 3) was

Fig. 3 Principal component analysis of the PLFA pattern in theunburned reference (Ref) and burned soil samples (indicated by thecircles) measured immediately (0) and 8, 12, 24 and 32 months after awildfire. Scores from the different samplings (a), loadings of theindividual PLFAs (b), abundance of PLFAs cy17:0 and a15:0 during32 months after a wildfire (c) and fungi-to-bacteria (F:B) ratio (d).

Ref. is an unburned reference soil, sampled at the same time as thesampling 32 months after the wildfire. Mean values ± SE (n=4).*Indicates significant differences between unburned reference soil andburned samples 32 months after the fire (t test, P<0.05). Different letters(bold for a15:0 and normal for cy17:0 in C) indicate significantdifferences over time in burned soils (Tukey’s post hoc test, P<0.05)

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most likely underestimating the fire effect on the livingmicrobial biomass.

Guerrero et al. (2005) reported that bacterial CFUs werenot below detectable levels until the soil was heated to 500°Cor above, while fungal CFUs were below detectable levels at300°C or above. Different sensitivities of different soilmicrobial groups were also highlighted in our study by CFUsand PLFA, indicating more fire resistance of bacteria,especially for those able to form spores, while fungi weremore sensitive. Higher fungal sensitivity to heating than forbacteria has previously been reported (Bollen 1969; Dunn etal. 1985) and should be taken into account in evaluatingrecovery after a fire, since this could influence the inoculumsize of different organism groups present for recolonisation.

Unlike microbial abundance measurements, microbialactivity, both total respiration and bacterial growth, hadhigh values immediately after the fire (Fig. 4) if comparedwith the remaining sampling times and the unburnedreference site. However, this was due to logistic reasonsdelaying the measurements. Both respiration and bacterialgrowth were measured on dried samples that had beenpreincubated for 4 days after rewetting. With the largeamount of DOC released by the fire, this period will belong enough for substantial growth and respiration to occur.In a laboratory study using similar soil (Bárcenas-Morenoand Bååth 2009), a heating temperature that initiallyresulted in no significant bacterial growth, already after2 days of incubation showed bacterial growth that was upto 9 times that of the control. For respiration, this effect waseven more rapid, with the highest respiration rates recordedwithin 24 h after the heating treatment. Thus, it is verylikely that the immediate effect of the wildfire in the presentstudy would have been very low microbial activity directlyafter heating. Nevertheless, the high activity levels by a fewsurviving microorganisms in our incubated and rewettedsamples a few days after the fire suggested that in the fieldsituation during the first months after the fire, the increasein DOC initially stimulated rapidly growing bacteria.

Long-term microbial recolonisation after the fire

After the initial burst in microbial activity, the DOCconcentration decreased to levels close to or below thelevels found in the unburned reference site, with aconcomitant decrease in respiration. The growth of themicroorganisms was transformed into new biomass, withincreasing Cmic 8 months after the fire. Nevertheless,microbial biomass measured 32 months after the fire wassignificantly lower than in the unburned area, most likelybecause the soil organic C, which determines microbialbiomass (Wardle 1992), also remained at low levels.

Although CFUs will not indicate absolute biomass ofdifferent microbial groups, they can be used to suggest

Fig. 4 Bacterial growth (Leu incorporation) (a), respiration rate (b)and respiratory quotient (O2/CO2) (c) during 32 months after awildfire. Ref. denotes an unburned reference soil, sampled at the sametime as the sampling 32 months after the wildfire. Mean values ± SE(n=10). *Indicates significant differences between unburned referencesoil and burned samples 32 months after the fire (t test, P<0.05).Different letters indicate significant differences over time in burnedsoils (Tukey’s post hoc test, P<0.05)

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relative changes over time. CFUs of all microorganismgroups peaked 8 months after the fire and then decreased.Although the same pattern was found throughout allmicrobial groups, the peak values were smallest for fungi(see discussion below) and largest for spore formers whencompared with the reference site. Not only did the sporeformers survive the direct heating event better than theother groups studied, but they probably also had an initialadvantage in using the released DOC. After the initial rapidgrowth, they also have the potential to survive for longperiods as spores in the soil. Thus, the wildfire was clearlyevident in the increased levels of spore formers even32 months after the fire (Fig. 1b), as previously reported inother fire studies (Vázquez et al. 1993; Yeager et al. 2005).Judging from the slope of the decrease in spore formers, anenhanced number would still be present for many moreyears.

Several authors have reported that bacteria recover fasterand to higher levels after a heating event than fungi. Todetermine the varying levels of recovery, these researchershave used measurements of CFUs (Bollen 1969; Vázquezet al. 1993; Badía and Martí 2003; Guerrero et al. 2005;Mabuhay et al. 2006), Biolog® utilisation patterns (Giaiand Boerner 2007), the PLFA composition (Bååth et al.1995; Ponder et al. 2009) or fungal and bacterial growthestimations (Bárcenas-Moreno and Bååth 2009). Thedifferential effect on bacterial and fungal CFUs (Fig. 1)and the more rapid recovery of many bacterial PLFAs, likecy17:0 and a15:0 (Fig. 3), indicate that bacteria werefavoured over fungi after a fire also in our study. Bacteriadominating after a fire have been attributed to high pH,which favours bacterial growth (see, e.g. Rousk et al.2009). In our study, pH did not change substantially afterthe fire during the entire study; nevertheless, the originalsoil pH was high (around 8), presumably favouring bacteriaalso during recolonisation. Better rates of survival ofbacteria compared to fungi could also have resulted in alarger initial inoculum of bacterial cells. Once the bacteriahad recolonised the soil substrate, they could be antagonistictoward fungi, delaying the recovery of fungi even further, assuggested by Bárcenas-Moreno and Bååth (2009).Thebacterial/fungal ratio after the fire could also be affectedby changes in organic matter (Vázquez et al. 1993). Boththe presence of “pyromorphic” compounds (Widden andParkinson 1975; Almendros et al. 1990; Vázquez et al.1993; Pietikäinen and Fritze 1995; Guerrero et al. 2005)and the destruction of some fraction of organic matter likecellulose (Acea and Carballas 1996; Fernández et al. 1997;Prieto-Fernández et al. 1998) have been reported tocondition lower fungal recovery after the fire.

Similar to the number of aerobic bacterial CFUs, thebacterial growth peaked after 8 months and then decreasedslowly, attaining levels similar to the unburned reference

site after 32 months (Fig. 4). Bacterial CFUs behavingsimilar to bacterial growth have been found previously(Bååth 1994b; Söderberg et al. 2002, 2004; Bååth et al.2005). The correlation between bacterial growth and CFUscontrasts with the lack of good correlation betweenbacterial growth and respiration (Fig. 4). In our study, thelatter peaked directly after the fire event and then decreaseddramatically after 8 months, showing lower levels than theunburned reference site 32 months after the fire. Bacterialgrowth, on the other hand, peaked after 8 months andshowed high values even 12 months after the fire, and thendecreased more progressively than respiration. This dis-crepancy is a common phenomenon, however, as lack ofcorrelation between respiration and bacterial growth hasbeen found frequently after the addition of fresh substrate(Rousk and Bååth 2007; Meidute et al. 2008).

The close correlation between DOC and respirationindicates a key role of the former in determining respira-tion. We suggest that, 8 months after the fire, the mostenergy-rich compounds (DOC) are exhausted. The decreasein RQ after the first sampling could also indicate a changein the source of substrate used in respiration during ourstudy, from easily decomposable to more recalcitrantcompounds (Lipp and Andersen 2003).

Similar to a short-term laboratory study (Bárcenas-Moreno and Bååth 2009), the PLFAs that increased themost after the initial decrease due to heating were 16:1ω7c,cy17:0 and a15:0. The two former PLFAs are usuallythought to reflect gram-negative bacteria, and increasingconcentrations are often found in connection with increasedbacterial growth, e.g. in the rhizosphere (Olsson et al. 1996;Söderberg et al. 2004). However, PLFAs indicative ofgram-positive bacteria, such as a15:0, also increased overtime after the fire, presumably reflecting growth of gram-positive spore-forming bacteria. The ratio of the PLFAscy17:0/16:1ω7c has been used to indicate nutrient stress,since it is known to increase in the stationary phase duringgrowth of bacteria in pure culture (Härtig et al. 2005).Although this ratio increased with time after the fire, whichmight be interpreted as an increased nutrient limitation forthe bacteria, this might be a simplistic interpretation.Changes in the composition of the bacterial communityover time, with bacteria having different contents of thesePLFAs, could also explain the changes in the ratio ofcy17:0/16:1ω7c after a heating event.

Conclusion

The effect of a wildfire can be categorised into differentphases of the recovery process. First is the initial partialsterilisation of soil microorganisms and the rapid, but short,increase in the availability of nutrients, which then

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promotes the proliferation of fast-growing bacteria. Then,when available substrate decreases, microbial activitydecreases as well, although the build-up of microbialbiomass continues. We hypothesise that the progressiverecovery of vegetation will further normalise the microbialparameters, except that the abundance values will remainbelow an unburned area until total restoration of thevegetation.

Acknowledgements This work was supported by a grant from the“Plan Nacional de Formación de Profesorado Universitario” fellowshipfrom the SpanishMinistry of Education to G.B.-M. and from the SwedishResearch Council (project no. 2009-4503) to E.B.

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