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Soil Biology & Biochemistry 50 (2012) 22e25

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lbio

Short communication

Heat stress and methane-oxidizing bacteria: Effects on activity and populationdynamics

Adrian Ho, Peter Frenzel*

Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, Germany

a r t i c l e i n f o

Article history:Received 17 November 2011Received in revised form16 February 2012Accepted 24 February 2012Available online 7 March 2012

Keywords:Methane-oxidizing bacteriapmoAHeat stressResiliencePopulation dynamics

* Corresponding author. Tel.: þ49 6421 178 820; faE-mail addresses: frenzel@mpi-marburg.mpg.de,

(P. Frenzel).

0038-0717/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.soilbio.2012.02.023

a b s t r a c t

We studied the effects of an acute temperature increase (heat stress) on methane oxidation andmethanotroph community structure in a laboratory-scale experiment with paddy soil. Methane oxida-tion was resilient, recovering already six days after heat stress, and later on even reached higher valuesthan in the control. It was consistently shown by qPCR and by terminal restriction length polymorphism(T-RFLP) that type II methanotrophs increased over time. While this was a general trend, the initialincrease of type II was much more pronounced after heat stress at 45 �C. Type I methanotrophs wereinversely correlated to type II and temperature. Overall, heat stress is a potential factor shifting thecommunity towards a dominance of type II methanotrophs.

� 2012 Elsevier Ltd. All rights reserved.

Rice fields are anthropogenic methane sources contributingaround 10% to the atmospheric methane burden (Conrad, 2009).Methane emission from paddy fields would even be higher if notfor the role methanotrophs play at oxiceanoxic interfaces (Conradand Frenzel, 2002).With no or a sparse leaf canopy during early ricegrowing season, the soilefloodwater interface is subject to strongtemperature fluctuations (dial temperature range 25e37 �C),affecting methane emission (Neue et al., 1997) and makingtemperature a potential control factor for methanotrophs thrivingat this interface. While microorganisms may deal very well withgradually changing temperature, an acute increase may affectcommunity composition and/or have adverse effects on func-tioning. Methane oxidation in paddy soils functions well at 25 �C(Eller and Frenzel, 2001), but decreased drastically when incubatedat 40e45 �C (Mohanty et al., 2007). A detailed analysis of howcommunity structure is affected by temperature is lacking so far,but would help solving the enigma of how so many methanotrophsmay coexist in one particular soil (Lüke et al., 2010; Lüke andFrenzel, 2011). Hence, we designed an experiment aiming atdetermining both the effects of an acute temperature increase (heatstress) on methane oxidation, and on methanotrophic communitystructure.

x: þ49 6421 178 809.frenzel@staff.uni-marburg.de

All rights reserved.

Traditional taxonomy has divided methanotrophs into type I(Gammaproteobacteria) and type II (Alphaproteobacteria) with type Ibranching into type Ia and Ib. Type I and II methanotrophs aredistinguished based on their physiology, biochemistry andmorphology (Trotsenko and Murrell, 2008; Semrau et al., 2010).While Type Ia methanotrophs (e.g. Methylomonas, Methylobacter,and Methylomicrobium) are generally mesophilic or psychrophilic,type Ib contains thermotolerant and thermophilic taxa belonging togenera Methylococcus and Methylocaldum (Bodrossy et al., 1997;Bowman, 2000), and appears to be the dominant active methano-trophs in paddy soils (Ho et al., 2011a). Verrucomicrobial meth-anotrophs were discovered recently (Op den Camp et al., 2009), buthave so far not been found outside extreme environments.

Focussing on the soilefloodwater interface, we used a paddy soilfrom Vercelli (Italy). Soil microcosms (Ho et al., 2011b) were pre-incubated at 25 �C under 10% methane in air in the dark for 9days. Controls, continuously incubated at 25 �C, were compared tomicrocosms exposed to 37 �C and 45 �C, respectively, for up to 42 h.Afterwards, these microcosms were returned to 25 �C for furtherincubation up to 69 days. Soil sampling and methane fluxmeasurement were performed in triplicates at 18 and 42 h, and at 6and 69 days after heat stress. Water loss was compensated byadding autoclaved de-ionizedwater. After sampling, soil was storedat �80 �C till further analyses. Community structure and pop-ulation dynamics were analyzed by pmoA-based group-specificquantitative PCR (qPCR) analyses, and by terminal restriction

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A. Ho, P. Frenzel / Soil Biology & Biochemistry 50 (2012) 22e25 23

fragment length polymorphism (T-RFLP). The pmoA gene encodesa subunit of the particulate methane monooxygenase, a keyenzyme for methane oxidation found in virtually all knownmethanotrophs.

Resilience of methanotrophic activity to heat stress: Methaneuptake rates were determined by linear regression for individualmicrocosms incubated in flux chambers (Ho et al., 2011b). Methanewas measured by GC-FID (SRI-9300A; SRI Instruments, Torrance,CA, USA). Methane uptake was not compromised by exposure to37 �C (Fig. 1). Indeed, this temperature was only slightly above theoptimum temperature range (25e35 �C) for methane oxidationfound previously in a sample taken from the same field (Mohantyet al., 2007). Following heat stress at 45 �C, methane uptake waslower than in the control, but recovered after six days reachingeventually values twice as high as in the control.

Response of the methanotrophic community to heat stress: ThreeqPCR assays: TYPEII, MBAC, and MCOC (Kolb et al., 2003; Ho et al.,2011b) targeting the pmoA gene of type II, Ia, and Ibmethanotrophs,respectively, and a qPCR assay targeting the 16S rRNA gene(Stubner, 2002) were run three times per microcosm and timepoint (Fig. 2). The coverage of the assays TYPEII, MBAC, andMCOC is83%, 93%, and 91%, respectively, considering 279,164, and 136 clonesequences retrieved from the same soil (Ho et al., 2011b). In general,we observed an increase of type II methanotrophswith time in bothcontrol and heat stressed microcosms, indicating that this kind ofsuccession may be characteristic for the experimental setup. Uponexposure to 45 �C, type II methanotrophs increased drasticallyalready after six days. This was consistently shown in a constrainedanalysis (RDA, Fig. 3), and in a non-metric ordination (NMDS; notshown). However, after 69 days, type II abundance was similar as inthe control, indicating that the one-off heat stress accelerated typeII dominance, but did not leave a lasting imprint on the methano-trophic community. Considering total microbial abundance, theEUBAC assay gave relatively constant numbers, and did not reflectthe lower methane uptake rates soon after heat stress. Interest-ingly, heat stress did not induce growth of type Ib methanotrophsin spite of thermotolerant taxa clustering within this group(Bodrossy et al., 1997). Besides the cultured representatives of typeIb, Rice Paddy Cluster-1 (RPC-1) is predominant in paddy soils(Lüke et al., 2010).

Based on methane uptake rates and qPCR analysis, the apparentcell specific activity was calculated showing an increase fromimmediately after treatment (day 6) till day 69. In microcosmsexposed to 37 �C and 45 �C, oxidation rate ranged from 7.3 to

Fig. 1. Response of methane uptake rates to heat stress (mean� sd). Three replicatesper treatment and time point, except for 45 �C after 18 h and 69 days (n¼ 2). Anasterisk indicates significant differences between control and 45 �C-treatment (t-test;p< 0.05). No significant differences were found between control and 37 �C-treatment.

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Fig. 2. qPCR analysis showing the effect of heat stress. Copy numbers for sub-populations of methanotrophs (TYPEII, MBAC, MCOC; Kolb et al., 2003) and theeubacterial 16s rRNA gene (EUBAC; Stubner, 2002) in (a) control, and (b) 37 �C- and (c)45 �C-treated microcosms. Each time point represents the mean of nine replicates fromthree microcosms (�sd). The lower detection limit of the assays was 104 targetmolecules g soil�1.

Fig. 3. Biplot of a redundancy analysis (RDA) with treatment and time as constraintsdisplaying three replicate quantifications per day, microcosm, and treatment. Ordi-nation was done on log10 transformed data with vegan (Oksanen et al., 2011) in R (RDevelopment Core Team, 2011). Only samples taken after pre-incubation and at days6 and 69 after heat stress were considered. RDA explains 55.4% of total inertia; theordination is highly significant with p� 0.005. Yellow, light and dark brown denotecontrol, and heat stress at 37 �C and 45 �C, respectively. Triangles indicate the scores ofsubgroups measured by the respective qPCR assays: TYPEII, MBAC and MCOC.Constraints are labelled in blue. Time is given in black numbers at the centre of thepolygons (days 0 and 69) and the ‘spider plot’ (day 6). Data points form a coherentcluster at day 0, but split up in well separated clusters upon heat stress (light bluepolygons, day 6; points connected by grey lines to their common centroid). Finally, thedifferent treatments form again a joint cluster at day 69. (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version ofthis article.)

A. Ho, P. Frenzel / Soil Biology & Biochemistry 50 (2012) 22e2524

39.8 fmol CH4 h�1 cell�1, and from 6.6 to 15.6 fmol CH4 h�1 cell�1,respectively. The apparent cell specific activity for the controlremained relatively constant with 2.6e7.1 fmol CH4 h�1 cell�1. Thisdifference corresponded most probably to the activity of type IImethanotrophs that increased by number, while type I remainednearly constant (MBAC) or even decreased (MCOC; t-test, p< 0.05;Fig. 2). The die-off of heat sensitive organisms may have providednutrients and even surfaces to grow on for the survivors, but thedifferent behaviour shows clearly that the three subgroups of

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Fig. 4. T-RFLP analysis of control and heat-treated microcosms with three replicates per treand the pmoA2 of some type II MOB by T-RF 277. AOB are represented by a T-RF indicative fofragment named RA21 is characteristic for a cluster of sequences in an intermediate positionwere assigned considering 500 clones retrieved from the same soil (Lüke et al., 2010).

methanotrophs have divergent adaptations. As methanotrophs areable to form exospores or cysts (Whittenbury et al., 1970a), a largepart of the population, in particular type II, is assumed to forma microbial seed bank (Eller et al., 2005). We suggest that heatstress and the subsequent return to ambient temperature havetriggered the transformation from dormant to active state, even-tually resulting in growth and higher methane uptake. Indeed, typeII exospores and cysts are heat resistant, and at least exposure totemperature >70 �C seems to induce their germination(Whittenbury et al., 1970b).

Additional information on the population dynamics wasprovided by T-RFLP (Fig. 4). Analysis was carried out in triplicatesper time point as described before (Lüke et al., 2010). The threedominant T-RFs were indicative for type II (244 bp) and type I (74and 221 bp). In addition, a T-RF indicative for pmoA2 was detected(277 bp), but represented only a small fraction (<10%). Fragmentsaffiliated to amoA were of minor importance. T-RFs were assignedconsidering 500 clone sequences retrieved from the same soil(Lüke et al., 2010). Relative abundances of type II (rSpearman¼ 0.59,p< 0.001, n¼ 45) and pmoA2 (rSpearman¼ 0.38, p< 0.001)increased with time, while the sum of fragments indicative fortype I was inversely related to type II (rSpearman¼�0.99,p< 0.001). In addition, we used T-RFLP profiles to test for effectsof heat stress on community structure. We tested distancematrices generated from T-RFLP profiles measured 6 days afterheat stress (n¼ 9). Using BrayeCurtis distances, the followingtests as implemented in VEGAN (Oksanen et al., 2011) were per-formed: ANOSIM (analysis of similarity; Clarke, 1993), ADONIS(permutational multivariate analysis of variance, Anderson,2001), and MRPP (multi response permutation procedure,Oksanen et al., 2011). All tests indicated significant differences incommunity structure (p< 0.05). Hence, T-RFLP confirmed qPCRanalysis in all aspects.

Overall, methane oxidation was resilient compensating quicklyfor a transient drop-down after heat stress. At the communitylevel, type II methanotrophs increased with time and becamedominant. However, based on the apparent cell specific activityand statistical analyses, type II growth was transiently stimulatedupon brief exposure to 45 �C. Hence, heat stress is a potentialfactor inducing a community shift towards a dominance of type IImethanotrophs. One may speculate that this will become evenmore important with repeated exposure to heat stress. Previousstudies suggest that type I methanotrophs favour lower temper-atures, generally not exceeding 15 �C while type II prefer highertemperature (Mohanty et al., 2007). Therefore, cold stress mayrender a different response.

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atment and time point as a colour-coded heatmap. Type II is represented by T-RF 244,r the homologous gene of the ammonium monooxygenase subunit a (amoA), while thebetween pmoA and amoA. The other fragments belong to type I methanotrophs. T-RFs

A. Ho, P. Frenzel / Soil Biology & Biochemistry 50 (2012) 22e25 25

Acknowledgements

This study was financially supported by the International MaxPlanck Research School for Environmental, Cellular and MolecularMicrobiology by a grant to AH. This work, as part of the EuropeanScience Foundation EUROCORES Programme, is a contribution toEuroEEFG e MECOMECON.

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