Functional and structural responses of methanogenic microbial communities in Uruguayan soils to intermittent drainage

Soil Biology & Biochemistry 89 (2015) 238e247

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Functional and structural responses of methanogenic microbial communities in Uruguayan soils to intermittent drainage Yang Ji a, b, Ana Fernandez Scavino c, Melanie Klose b, Peter Claus b, Ralf Conrad b, * a Jiangsu Key Laboratory of Agricultural Meteorology, College of Applied Meteorology, Nanjing University of Information Science & Technology, Nanjing 210044, China b Max-Planck-Institute for Terrestrial Microbiology, Marburg 35043, Germany c Departamento de Biociencias, Facultad de Quimica, Universidad de la Republica, Montevideo 11800, Uruguay

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 May 2015 Received in revised form 9 July 2015 Accepted 20 July 2015 Available online 3 August 2015

Intermittent drainage is one of the most promising approaches to mitigate methane (CH4) emission from paddy fields. Irrigated rice fields in Uruguay are temporarily established on soils used as cattle pastures. We studied soil from the pasture-rice rotation (UR) as well as soil from a permanent cattle pasture (UT) hypothesizing that activity and structure of the bacterial and archaeal communities involved in production of CH4 change systematically with intermittent drainage. Methane production started after 7 days and 16 days of anoxic incubation in UR and UT soil, respectively. Then, production rates of CH4 were higher in UT than UR soil. Intermittent drainage significantly decreased the rates of CH4 production. Analysis of d13C indicated that CH4 was mainly produced from acetate both in UR (73e98%) and UT (51 e80%) soil. Intermittent drainage did not change the pathway of CH4 production. Quantitative PCR showed that methanogenic archaeal gene copy numbers (16S rRNA, mcrA) were much lower in UT than UR soil, but increased upon incubation under anoxic conditions. Terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the communities were clearly different between UR and UT soil. The bacterial community consisted of 9 phyla with relative abundance of >1% in both soils. Whereas the archaeal community in UR soil was dominated by Methanocellales and Methanosarcinaceae, that in UT soil was dominated by Crenarchaeota. Anoxic incubation affected the composition of the bacterial and archaeal communities in UT soil, but not so much in UR soil. In UT soil, the relative abundance of Clostridiales increased to 19%, and the archaeal community changed to dominance by Methanosarcinaceae and Methanobacteriales. Subsequent drainage and re-flooding, however, had comparatively little effect on the composition, although it decreased the rates of CH4 production in both soils. Difference in previous soil management and in the structures of the microbial communities apparently only affected their dynamics and functioning after the first flooding but not upon subsequent drainage and re-flooding. © 2015 Elsevier Ltd. All rights reserved.

Keywords: CH4 production Rice field soil Intermittent drainage Isotopic fractionation Microbial community Pyrosequencing

1. Introduction Methane (CH4) is after carbon dioxide (CO2) the second most important greenhouse gas. It is estimated that CH4 contributes around 18% to the overall global warming potential (Murray et al., 2001). Irrigated rice fields account for 51% of the global rice production area (Wassmann et al., 2000), and represent an important anthropogenic biological source of atmospheric CH4, contributing

* Corresponding author. Max-Planck-Institute for Terrestrial Microbiology, Karlvon-Frisch-Str. 10, 35043 Marburg, Germany. Tel.: þ49 6421 178801; fax: þ49 6421 178809. E-mail address: [email protected] (R. Conrad). http://dx.doi.org/10.1016/j.soilbio.2015.07.015 0038-0717/© 2015 Elsevier Ltd. All rights reserved.

about 33e40 Tg yr
1 (Ciais et al., 2013). Methane and CO2 are the end products of the degradation of organic matter under anaerobic conditions and are produced by a complex microbial community consisting of hydrolytic and fermentative bacteria and methanogenic archaea (Zinder, 1993; Conrad, 2007). Methane production occurs not only in flooded wetland soils, such as paddy rice fields, but occasionally also in aerated upland soils (Angel et al., 2011). Upland soils, even from desert areas, almost generally contain a methanogenic community, albeit at low abundance, which potentially can develop and display CH4 production when the soils are submerged (Angel et al., 2011, 2012a, 2012b). In all these soils there is sequential reduction of available inorganic electron acceptors and eventual production of CH4 (Peters and Conrad, 1996; Conrad,

Y. Ji et al. / Soil Biology & Biochemistry 89 (2015) 238e247

2002), although the structures (abundance and composition) of the methanogenic microbial communities are different. Intermittent drainage, an episode of drainage for several days in the middle of the rice season and drying-wetting alternation during the following period, is recognized as an important cultivation practice to mitigate CH4 emission in rice production (Yan et al., 2003, 2005). Moreover, this water management could reduce ineffective tillering, remove toxic products of anaerobic metabolism, and improve root activity (Ji et al., 2013). In rice field soils, the abundance and composition of the methanogenic archaeal community were found to be hardly affected by temporary drainage or oxygen exposure, while transcription of the mcrA gene, coding for a subunit of the methyl-CoM reductase, and expression of methanogenic activity were suppressed (Yuan et al., 2011; Ma et al., 2012). The methanogenic archaea depend on fermenting bacteria for provision of substrate, mostly acetate and H2. The composition of bacterial communities in irrigated rice field is rather complex (Asakawa and Kimura, 2008; Fernandez Scavino et al., 2013). The active part of the bacterial community was found to change with the time of flooding and to be different between the oxic and anoxic zones of irrigated rice field soil (Noll et al., 2005; Shrestha et al., 2007; Rui et al., 2009). Bacterial communities in upland soils are also rather complex, depending on soil type and soil conditions, pH in particular (Fierer and Jackson, 2006; Fernandez Scavino et al., 2013). Flooded rice fields are ecosystems that are regularly affected by drainage and desiccation. However, how the methanogenic bacterial and archaeal communities adapt to such conditions is poorly known. Rice fields in Uruguay are an interesting system for studying the influence of intermittent drainage, since they are only temporarily established on soils that normally are used as cattle pastures. Typically, four years of cattle pasture are alternated with two years of irrigated rice cultivation (Fernandez Scavino et al., 2013). The temporary establishment of flooded conditions helps to increase fertility of soil by mobilization of phosphorous, increases the production of grass plant biomass, and eventually results in larger production of beef (Deambrosi, 2007). The methanogenic microbial community in these soils was found to be rather stable over the rotational cycle between pasture and wetland rice cultivation (Fernandez Scavino et al., 2013). However, the behaviour of the microbial community after drying and rewetting has not been studied. We hypothesized that activity and structure of the bacterial and archaeal communities involved in production of CH4 would change systematically with a wetting-drying-wetting alternation and would be different between soil from a permanent pasture and from the pasture-rice rotation. 2. Materials and methods 2.1. Sampling Soil samples were taken at triplicate from different fields (32 S and 53 490 W) at 70 km of the Instituto Nacional de Inves
n Agropecuaria (INIA) at the city Treinta-y-Tres, Uruguay. tigacio The typical rotation is four consecutive years of cattle pasture followed by two consecutive years of flooded rice fields (Fernandez Scavino et al., 2013). This management has been going on for at least 15 years. The rice soil (UR; UR ¼ Uruguay Rice) was sampled (n ¼ 3) after drainage and before harvest during the second rice growing season in April 2011 from three different fields. At this time, the soils were no longer methanogenic and were in the course of drying. The pasture soil (UT; UT ¼ Uruguay Testigo, spanish for “Control”) was sampled (n ¼ 3) from three sites of a permanent cattle pasture not undergoing rotation. The pasture was on a site that was difficult to submerge so that rice was never grown. The 490

239

density of cows on pastures in Uruguay is generally rather low so cow feces did not accumulate. The two field types were found close to the station, and had the same soil type (planosol) with a texture of sand:silt:clay ¼ 37:40:23%. About 100 g soil was taken from the upper 10-cm soil layer. This was done by taking six cores from each of the three replicate locations. After air-drying, the soil from the six cores was mixed, thus giving a composite sample from each replicate plot. The dry soil samples were then shipped to Marburg, where the experiments were done, keeping the replicates separate. Storage of rice field soil under dry conditions has no detectable effect on the activity of the soil once submerged conditions are established (Mayer and Conrad, 1990). Chemical analyses (Table 1) were done as described before (Conrad and Klose, 2006). 2.2. Incubation experiments The incubation procedure was the following. Soil slurries were prepared in 26 ml pressure tubes using 5 g dry soil and 5 ml anoxic sterile water incubated at 25  C to mimic the flooding period for 2e4 weeks, and then the slurries were dried at 35  C as drainage period for one week. Finally the dry soil was rewetted and reincubated at 25  C to mimic the reflooding period for another 2e4 weeks. The tubes were closed with black rubber stoppers, flushed with N2, pressurized to 0.5 bar overpressure, and incubated until CH4 production was constant. Inhibition of aceticlastic methanogenesis was achieved by the addition of 3% methyl fluoride (Janssen and Frenzel, 1997). At regular time intervals, the headspace was analysed for CH4, CO2, H2, and for the d13C of CH4 and CO2 during the course of incubation and re-incubation. At the end of all incubations, the tubes were opened, and the liquid was analysed for pH, acetate and d13C of acetate The chemical analysis of gas and liquid samples was done as described before (Conrad et al., 2014). Briefly, CH4 and CO2 were analyzed by gas chromatography (GC), acetate by high-pressure liquid chromatography (HPLC) and the d13C by either GC combustion isotope ratio mass spectrometry (GC-C-IRMS) or HPLC-C-IRMS. The d13C of the methyl group of acetate was determined after offline pyrolysis. The d13C of organic matter was analyzed by the Centre for Stable Isotope Research and Analysis (KOSI) at the Uni€ ttingen using an elemental analyser coupled to an versity of Go IRMS. Values of d13C are given in permil relative to Vienna Pee Dee Belemnite standard. The fraction ðfH2 Þ of CH4 production by hydrogenotrophic methanogenesis was calculated by mass balance as described before (Conrad et al., 2010) using

.

d13 CCH4
mc
d13 CCH4
ma fH2 ¼ d13 CCH4
d13 CCH4
ma

(1)

with d13 CCH4 ¼ d13C of total CH4 produced, d13 CCH4
mc ¼ d13C of CH4 produced from hydrogenotrophic methanogenesis, which is equivalent to the CH4 produced in the presence of CH3F, and d13 CCH4
ma ¼ d13C of CH4 produced from acetotrophic methanogenesis. The d13 CCH4
ma was assumed to be equal to d13Cac-methyl if there is no fractionation during the reduction of acetate-methyl to CH4, or 10‰e20‰ lower than d13Cac-methyl if fractionation occurs. The d13C of total acetate was measured at the end of the incubation in the presence and absence of CH3F, that of acetate-methyl only in the presence of CH3F. 2.3. Molecular analyses DNA was extracted from soil using the Fast DNA Spin Kit (MP Biomedicals, Eschwege, Germany) for soil (Kolb et al., 2005). The extracted DNA was used for qPCR, T-RFLP and pyrosequencing. The abundance of bacterial and archaeal 16S rRNA gene copies and of

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Table 1 Chemical characteristics of the soils from rice field with pasture-rice rotation (UR) and from a permanent pasture (UT); mean ± SD, n ¼ 3.

UT UR

Sulfate (mM)

Nitrate (mM)

Fe (mmol/g)

Ctot (%)

Ntot (%)

d13Corg (‰)

0.148 ± 0.013 0.168 ± 0.011

0.001 ± 0.005 5.775 ± 0.521

16.07 ± 0.45 18.87 ± 1.81

1.62 ± 0.25 1.41 ± 0.10

0.13 ± 0.02 0.12 ± 0.01


17.17 ± 0.10
20.56 ± 0.06

methanogenic mcrA gene copies was determined by qPCR (Angel et al., 2012a, 2012b). The analysis of T-RFLP of 16S rRNA genes was done as described (Chin et al., 1999) using the primer combinations 109f/915r for Archaea (Grosskopf et al., 1998) with the reverse primer labelled with FAM (6-carboxyfluorescein) and 27f/ 907r for Bacteria (Lane, 1991; Weisburg et al., 1991) with the forward primer labelled with FAM. The 16S rRNA gene amplicons were digested with TaqI and MspI for Archaea and Bacteria, respectively, and the products were size-separated in an ABI 3130 DNA sequencer (Applied Biosystems, Darmstadt, Germany). For tagged pyrosequencing of bacterial and archaeal 16S rRNA gene fragments we used the primers F515 and R806 described by Bates et al. (2011). Each primer contained a unique 8-pb barcode. The PCR products from replicates of each soil were pooled and sequenced at the Max Planck Genome Centre in Cologne using a Roche 454 Genome Sequencer GS FLXþ. All analyses described in the current study were performed within version 1.22 of the mothur software package (http://www.mothur.org/) (Schloss et al., 2009). The pyrosequencing-based analysis resulted in recovery of 39,625 high quality sequences with a minimum read length of 200 bp across all samples. The raw sequences were sorted by primer sequence using appropriate commands. Those that did not match the primer sequences, were less than 200 bp, or contained any ambiguities were excluded from further analysis. For phylotype analyses, the remaining sequences were denoised (Schloss et al., 2011) and aligned against the SILVA bacterial and archaeal 16S rRNA gene database (Release 115) (Pruesse et al., 2007) in mothur. Sequence data were deposited under the study accession number SRP060560 for bacterial sequences in the NCBI Sequence Read Archive (SRA). 2.4. Statistical analyses Statistical analyses were done in R version 3.2.1 (R Development Core Team, 2011). Analysis of variance (ANOVA) and least significant differences (LSD) test were conducted with package agricolae version 1.2-1 (Mendiburu, 2015). Canonical correspondence analysis (CCA) of T-RFLP patterns of 16S rRNA genes were conducted with package vegan version 2.0-5 (Oksanen et al., 2015). T-RFLP data were normalized before CCA. CCA considered the effect of soil types (UT, UR) and incubation conditions (O, I, D, R) on the T-RFLP patterns. The trace values (given in the text) indicate the portion of the variance explained by the environmental variables (soil type; incubation). All levels of significance were defined at P  0.05. 3. Results 3.1. Rates, isotopic analysis, and path of methanogenesis The incubation experiments were done with soil samples from second-year rice fields of the pasture-rice rotation (UR) as well as from a permanent cattle pasture (UT). The main chemical characteristics of these soils are shown in Table 1. The contents of organic carbon, total nitrogen, total iron and sulfate were all very similar. Statistical comparison of data between UR and UT soils showed no significant difference. However, concentration of nitrate was one order of magnitude higher in the rice soil than the pasture soil (P < 0.001). The rice soil had a significantly lower d13C of organic

matter than the pasture soil (P < 0.001), which was probably influenced by the presence of C3 (rice) and C4 (various grasses) plants, respectively. Production of gaseous CH4 and CO2 together with the d13C values of CH4 and CO2 were measured during the two incubation courses both in the absence and the presence of methyl fluoride (CH3F), an inhibitor of aceticlastic methanogenesis. After the first incubation under submerged conditions the soil was dried for 7 days. Then, the same soil was rewetted and incubation under anoxic conditions was repeated. The time courses for both incubations of each soil are shown in Fig. S1. Methane production in the first incubation course started after a lag phase, which was about 7 days in UR and about 16 days in UT soil (Fig. S1). This lag was probably not due to inhibition of the methanogenic microbial community by the sulfate-reducing microbial community competing for the same substrates (Conrad, 2007; Conrad et al., 2011), since H2 partial pressures were sufficiently high to support methanogenesis (Fig. S1). Instead the population size of methanogens and/or expression of their activity may have been low during the lag phase (see below). Production rates of CH4 were higher in UT pasture soil than UR rice field soil (P < 0.05) (Fig. 1A). Addition of CH3F (3%) resulted in strong inhibition of CH4 production (Fig. 1A). The d13C values of the accumulating CH4 and CO2 were also measured during the course of the first incubation (Fig. S1). The d13C values of CO2 were constant, but those of CH4 changed with incubation time, especially in UT soil (Fig. S1). In order to compare the soils and incubation conditions, the averages of the d13C values of CH4 were calculated and are summarized in Fig. 1B. The average d13 CCH4 values were slightly lower in UT pasture soil (
52‰) than UR rice field soil (
48‰). They decreased strongly both in UT pasture soil (
74‰) and UR rice soil (
77‰) when CH3F was added to inhibit aceticlastic methanogenesis. The d13C of CH4 in the presence of CH3F is characteristic for hydrogenotrophically produced CH4. Addition of CH3F also strongly increased the concentration of acetate at the end of the incubation (Fig. 2A). The d13C values of total acetate were much lower in the presence than in the absence of CH3F (Fig. 2B). The acetate accumulated in the presence of CH3F was most probably due to microbial acetate formation, while that in the absence of CH3F was the residual acetate after microbial acetate consumption, most likely by aceticlastic methanogens. The d13C values of acetate-methyl ranged between
21‰ and
28‰ and were generally by about 10e20‰ lower than those of total acetate, both in the presence and absence of CH3F (Fig. 2C). In general, the concentration of acetate was higher in UT than UR soil, but the d13C values of total acetate and acetate-methyl were both similar between the two soils. These above data sets allowed the calculation of the percentage ðfH2 Þ of CH4 produced by hydrogenotrophic methanogenesis (the remainder being due to aceticlastic methanogenesis). The calculations were done for each time point showing that fH2 decreased with incubation time until a constant value was reached. For comparison, values of fH2 from all time points were averaged (variance given by SD) and shown in Fig. 1C. The calculations assumed either no fractionation during acetotrophic methanogenesis or a fractionation of
10‰, which is characteristic for the activity of Methanosaeta spp. (Penning et al., 2006) or of
20‰, which is characteristic for the activity of Methanosarcina spp.

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241

Fig. 2. (A) Concentrations of acetate and (B) d13C of acetate and (C) acetate-methyl accumulated until the end of incubation and re-incubation. The soils were from rice field with pasture-rice rotation (UR) and from a permanent pasture (UT); mean ± SD, n ¼ 3.

Fig. 1. (A) Rates, (B) d13C values, and (C) paths of potential CH4 production in soils from rice field with pasture-rice rotation (UR) and from a permanent pasture (UT) incubated and re-incubated under anoxic conditions in the absence and presence of CH3F; mean ± SD, n ¼ 3. Mean values of d13C of CH4 (B) were calculated from the replicate incubations for each date and then averaged for days 6e17 (UR) and 13e24 (UT). The paths of methanogenesis (C) were determined from mass balance of 13C in CH4 and acetate (Eq. (1)), assuming fractionation between acetate-methyl and CH4 being zero,
10‰ or
20‰.

(Krzycki et al., 1987). Irrespective of fractionation, values of fH2 were lower in UR than in UT soil (Fig. 1C). During the second incubation under re-wetted conditions CH4 production in the UR soil again started after a lag phase of about 7 days. However, in UT soil, the lag phase was about 6 days shorter than during the first incubation (Fig. S1). In both soils, partial pressures of H2 were lower during the second than the first incubation. Rates of CH4 production were about half compared to those before drying in both soils (P < 0.001), but were again higher in UT than in UR soil (Fig. 1A). Intermittent drainage resulted in the decrease of d13 CCH4 values (
85‰) in UR rice soil (Fig. 1B; P < 0.05), but had no effect in UT pasture soil (
74‰). However, d13C values of

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the methyl group of acetate increased after drying, which showed a stronger effect in UT than UR soil (Fig. 2C). Overall, these effects caused that average values of fH2 (and variance) were similar during the second and the first incubation (Fig. 1C). 3.2. Quantification (qPCR) of archaeal and bacterial gene copies Numbers of Bacteria were quantified by measuring the copy numbers of the bacterial 16S rRNA genes (Table 2). The measurements used the original soils before the anoxic incubation (O), at the end of the first incubation (I), after drying (D) and at the end of the re-incubation (R). The numbers of Bacteria were generally higher than those of Archaea. In UR rice soil, numbers were similar for all the treatments, except the first incubation, which resulted in decrease to less than half of the initial numbers. In UT pasture soil, however, numbers were more than doubled after drying and reincubation (Table 2). Numbers of Archaea decreased in UR soil after both incubation and re-incubation indicating death or degradation of archaeal DNA. Intermittent drainage, on the other hand, resulted in increase of the copy numbers by the same magnitude. In UT soil, by contrast, intermittent drainage did not change the copy numbers. However, the first incubation resulted in a large increase of the archaeal numbers by more than one order of magnitude (Table 2). The copy numbers of the mcrA gene, characteristic for the methanogens among the archaea, were also quantified (Table 2). The effects of intermittent drainage were similar for mcrA and archaeal 16S rRNA genes in UT soil. However, rewetting had a positive effect in UR soil, which was opposite with the results of archaeal and bacterial 16S rRNA genes, indicating partial recovery of the mcrA gene. Statistical comparison showed that the copy numbers of archaea and methanogens in original UR rice soil were significantly higher than original UT pasture soil by 1e2 orders of magnitude (P < 0.001), while no significant difference was found in the bacterial numbers. However, intermittent drainage weakened the differences between UR and UT soils, even resulting in higher copy numbers of the above three genes in the UT than the UR soil after rewetting.

Fig. 3. Canonical correspondence analysis of T-RFLP patterns recorded for (A) bacterial and (B) archaeal 16S rRNA genes in the soils from rice field with pasture-rice rotation (UR) and from a permanent pasture (UT) in original state (O), the end of incubation (I), dried state (D) and the end of re-incubation (R). Axis 1 and 2 of (A) explained 13% and 12% of the total bacterial variability, respectively. Axis 1 and 2 of (B) explained 30% and 21% of the total archaeal variability, respectively.

3.3. Community composition of bacteria The bacterial community composition was determined by analysis of terminal restriction fragment length polymorphism (TRFLP) of bacterial 16S rRNA genes. Canonical correspondence analysis (CCA) identified soil types (UT, UR; trace ¼ 0.10; P ¼ 0.002) and intermittent drainage (O, I, D, R; trace ¼ 0.28; P ¼ 0.001) to significantly affect the bacterial community explaining 10% and 28% of the variance, respectively (Fig. 3A). We made no attempt to assign the different T-RFs to phylogenetic groups of bacteria, but used the soil samples for pyrosequencing of 16S rRNA genes (see below).

Pyrotagsequencing was done to obtain information on the main bacterial phylotypes in the soils, rather than a statistically valid comparison of communities between the soils, which was basically achieved by T-RFLP analysis (see above). Therefore, the pyrosequencing in the different soils was not replicated. For each soil sample about 2200e15,700 bacterial high-quality 16S rRNA sequences were obtained, equivalent to about 960e3370 bacterial OTUs (Fig. S2). The community of Bacteria in the two soils contained >9 different phyla, from which 5 (i.e., Firmicutes, Proteobateria, Acidobacteria, Actinobacteria and Planctomycetes) made up

Table 2 Copy numbers of bacterial and archaeal 16S rRNA genes and mcrA genes in UR rice soil and UT pasture soil in original state (O), the end of incubation (I), dried state (D) and the end of re-incubation (R); mean ± SD, n ¼ 3. Bacteria gdw
1

Sample UR

UT

O I D R O I D R

(5.4 (1.4 (5.1 (4.1 (5.8 (8.1 (1.8 (1.7

± ± ± ± ± ± ± ±

0.6) 0.4) 2.2) 0.1) 2.1) 0.6) 0.6) 0.6)

       

Archaea gdw
1 8

10 108 108 108 108 108 109 109

(3.4 (6.4 (2.1 (5.7 (1.5 (2.4 (2.6 (1.5

± ± ± ± ± ± ± ±

0.3) 0.7) 0.1) 0.5) 0.1) 0.1) 0.1) 0.1)

       

mcrA gdw
1 8

10 107 108 107 107 108 108 108

(2.1 (6.5 (2.1 (5.2 (1.6 (3.8 (7.6 (1.0

± ± ± ± ± ± ± ±

0.6) 1.6) 0.6) 0.8) 0.1) 0.9) 4.1) 0.2)

       

107 106 107 107 106 107 107 108

Y. Ji et al. / Soil Biology & Biochemistry 89 (2015) 238e247

more than 10% (Fig. 4A). Compared to UT pasture soil, UR rice soil contained relatively more Chloroflexi, which comprised 17% and 11% after drying and after rewetting, respectively (Fig. 4A). In both soils, the relative abundance of Firmicutes increased upon incubation, drying and re-incubation, in the UT soil in particular, while Actinobacteria and Acidobacteria decreased. The Firmicutes were mainly

243

represented by the orders Clostridiales and Bacillales, which accounted for 13e58% and 19e61%, respectively (Fig. 4B). The first incubation under anoxic flooded conditions resulted in a dramatic increase of the relative abundance of Clostridiales in both soils, but the effect was much stronger in UT than UR soil (Fig. 4C). In UT soil the relative abundance of Clostridiales strongly increased from 1% in

Fig. 4. Pyrosequencing-based relative abundance of (A) bacterial phyla (relative to total Bacteria), (B) orders of Firmicutes (relative to total Firmicutes), and (C) Clostridiales and Bacillales (relative to total Bacteria) in the soils from rice field with pasture-rice rotation (UR) and from a permanent pasture (UT) in original state (O), the end of incubation (I), dried state (D) and the end of re-incubation (R). Phylogenetic groups accounting for <1% of all classified sequences are summarized in the artificial group ‘others’.

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the original state to 19% after incubation. In UR soil it increased from 2% to 5%. The following drying did not show an apparent effect on the relative abundance of Clostridiales in both soils. After reincubation, the relative abundance of Clostridiales decreased to 14% in UT soil, while it changed only marginally in UR soil. Note, however, that these changes were not verified by statistical tests. 3.4. Community composition of archaea Canonical correspondence analysis was also done with the TRFLP patterns of the archaeal communities (Fig. 3B), showing that soil types (UT, UR; trace ¼ 0.29, P ¼ 0.001) and intermittent drainage (O, I, D, R; trace ¼ 0.33, P ¼ 0.001) significantly affected the archaeal community explaining 29% and 33% of the variance, respectively. The UR soil was dominated by T-RFs of 186 bp (about 40% relative abundance) and 391 bp (about 50%). The UT soil, on the other hand, was dominated by the 186-bp T-RF (about 45%), which increased to up to 60%e80% relative abundance after incubation (I), drying (D) and re-incubation (R). In addition, also the 91-bp T-RF with about 9e31% relative abundance appeared (Fig. 5). According to our previous work (Fernandez Scavino et al., 2013), cloning and sequencing of archaeal 16S rRNA genes showed that the 186-bp TRF was representative for both Methanosarcinaceae and Crenarchaeota/Thaumarchaeota, while the 391-bp T-RF was representative for Methanocellales (Table S1). Pyrosequencing of 16S rRNA genes resulted only in 450 archaeal sequences. Nevertheless, the taxonomic affiliation of these sequences was largely consistent with the T-RFLP results (Fig. S3). In summary, the archaeal community composition was similar in all samples of UR soil (O, I, D, R) with Methanocellaceae and Methanosarcinaceae accounting for about 35e45% and 20e30%, respectively. By contrast, the archaeal community in UT soil shifted from an original dominance of Crenarchaeota (O) to a dominance of Methanosarcinaceae (40e50%) and Methanobacteriaceae (40e60%) (I, D, R). 4. Discussion Experiments mimicking intermittent drainage in two differently managed soils (UT and UR) showed a similar effect on the activities and pathways of CH4 production, and also affected the structures of

the soil microbial communities only little, once flooded anoxic conditions had been established. Although CCA analysis showed significant effects of intermittent drainage on the microbial community composition, the effects were only small and explained less than 35% of the total variance. The similarity in the response of the UT and UR soil to intermittent drainage was in contrast to their difference with respect to the abundance and composition of their original microbial communities. These differences mainly concerned the much lower abundance of methanogenic archaea in soil from the pasture (UT) than the pasture-rice rotation (UR), and the dominance of methanogenic Methanocellales and Methanosarcinaceae in UR soil and non-methanogenic Crenarchaeota (or Thaumarchaeota) in UT soil. These differences probably explained why CH4 production activity appeared after a longer lag phase in UT than in UR soil, confirming results obtained earlier (Fernandez Scavino et al., 2013). The differences in microbial community structure were probably the main reason that the first anoxic incubation under flooded conditions had a systematically different effect on activity and microbial community structure in the two soils. Anoxic incubation of UT soil resulted in a dramatic increase of the numbers of methanogenic mcrA genes, while those in UR soil were already at an elevated level. The increase of mcrA copies in UT soil was accompanied by a shift from non-methanogenic Crenarchaeota to Methanosarcinaceae and additional Methanobacteriales. Among the Bacteria, anoxic incubation resulted in the increase of the relative abundance of Clostridiales being much stronger in UT soil (from 1 to 19%) than in UR soil (from 2 to 5%). Hence, the microbial community in the hitherto non-methanogenic UT soil apparently changed by proliferation of methanogenic archaea and anaerobic clostridia, eventually allowing the formation of CH4. Once established, rates of CH4 production were even somewhat larger in UT than in UR soil. In addition, hydrogenotrophic methanogenesis apparently contributed more to CH4 in UT than in UR soil. In summary, both soils had developed methanogenic activity, which slightly differed in the rates and paths of CH4 production, achieved by a different composition of their archaeal and bacterial communities. However, once anoxic flooded conditions had been established, intermittent drainage had little effect on the structure and function of the microbial communities in both soils. Gene copy numbers stayed similar and were apparently not much affected by desiccation and re-flooding in both soils. The archaeal and bacterial

Fig. 5. T-RFLP patterns of archaeal 16S rRNA genes in the soils from rice field with pasture-rice rotation (UR) and from a permanent pasture (UT) in original state (O), the end of incubation (I), dried state (D) and the end of re-incubation (R); mean ± SD, n ¼ 3.

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compositions also stayed relatively constant in both soils. Intermittent drainage had also little effect on CH4 production. The contribution of hydrogenotrophic methanogenesis in both soils hardly changed after desiccation, being larger in UT (about 40%) than in UR (about 30%) soil. Also lag phases in the two soils were now similar, and in both soils potential rates of CH4 production became lower after desiccation. This decrease in activity may be the result of decrease in the availability of decomposable organic matter. Hence, intermittent drainage had comparatively little effect on structure and function of the methanogenic microbial communities in these two soils. The initial differences in microbial community structures apparently affected the microbial processes after the first initiation of methanogenic activity upon flooding, but not so much in the subsequent drainage re-flooding events. More generally, one may hypothesize that the history of soil management (here, permanent pasture versus pasture-rice rotation) shapes the structure of the soil microbial community and the eventual response upon flooding. Permanent pasture is an upland soil environment, which is mainly oxic and develops methanogenic activity only after proliferation of key members of the microbial methanogenic community. Indeed, similar events as in the pasture soil have been observed in other upland soils, including desert soil (Peters and Conrad, 1996; Angel et al., 2011). The major differences among upland soils may be the initial structure of the methanogenic community. Desert soils have been found to contain only few methanogenic archaea, either belonging to Methanocellales or Methanosarcinacea, which then proliferate to larger populations once the soil becomes anoxic upon flooding (Angel et al., 2011, 2012a). In the UT pasture soil, however, it were members of Methanobacteriales that proliferated besides Methanosarcinaceae. The presence of these different groups of methanogens probably determines the path of CH4 production, since only members of Methanosarcinaceae are able to utilize acetate in addition to H2/CO2 as methanogenic substrate. Biological desert soil crusts with only Methanocellales apparently produced CH4 hydrogenotrophically (Angel et al., 2011), whereas the UT soil (and other upland soils) (Angel et al., 2012a, 2012b) containing Methanosarcinales produced CH4 both aceticlastically and hydrogenotrophically. Among the Bacteria, Clostridiales seem to play a central role in the resuscitation cascade of dry soil, as their preferential proliferation has also been observed in desert soil crusts (Angel and Conrad, 2013) and dried lake sediments (Conrad et al., 2014) upon reflooding. The Clostridiales are endospore-forming bacteria that can well survive dry and oxic conditions, and members of this order are known to possess a large variety of hydrolytic enzymes and fermenting pathways. Thus, they can provide the crucial function in degrading organic matter to methanogenic substrates. Soils from the pasture-crop rotation (UR) have a more complicated history, as they systematically changed between upland (4 years) and flooded (2 years) condition. Thus, they are in between wetland rice fields that are annually flooded and pastures or fields with upland crops that are never flooded. The methanogenic archaeal communities in wetland rice fields have been found to change only moderately during season and upon water saving treatments (Krüger et al., 2005; Watanabe et al., 2007, 2013; Ahn et al., 2014). Bacterial communities change only early after flooding with stimulation of clostridial populations (Noll et al., 2005). The response to environmental conditions seems to be regulated mainly by changes in transcription and enzyme expression (Shrestha et al., 2009; Yuan et al., 2011; Ma et al., 2012). The methanogenic archaeal communities in wetland rice fields are quite diverse, typically containing members of Methanocellales, Methanosarcinaceae, Methanosaetaceae, Methanobacteriales and Methanomicrobiales (Lueders et al., 2001; Conrad, 2007). The situation in rotational fields is a bit more complicated, but again

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microbial community structures appear to be relatively stable and change little upon flooding and drainage (Watanabe et al., 2006; Lopes et al., 2014; Zhao et al., 2014; Breidenbach and Conrad, 2015). However, once rotational fields were converted to upland conditions for more than one year, methanogenic archaeal community structure was strongly affected and some members were lost (Liu et al., 2015). The path of methanogenesis in both wetland rice and rotational rice soils is usually characterized by a mixture of aceticlastic and hydrogenotrophic methanogenesis, usually at a ratio of 2:1 (Conrad, 1999; Yao and Conrad, 2000), similarly as observed here. In all these soils, intermittent drainage seems to have little effect on the structure and function of the methanogenic microbial communities, once they have established after the first flooding event. Here, this conclusion has been drawn from experiments with soils from upland and rotational fields. Literature data also indicate this conclusion from studies of wetland rice and various rice-crop rotations. Nevertheless, one should note there are still not thus many studies to allow a final conclusion that is fit for textbook. However, drying and re-flooding events seem to have effects on microbial structure and function in lake sediments that are (almost) permanently flooded and active in methanogenesis. Drainage and re-flooding of such environments happen rarely in nature, but then seem to result in decrease of abundance and change in composition of the bacterial and archaeal communities (Conrad et al., 2014). These authors showed that after re-flooding and re-establishment of methanogenic conditions, Clostridiales among the Bacteria and Methanocellales and Methanosarcinaceae among the Archaea have become relatively more abundant. The methanogenic activity has been stimulated, probably as recalcitrant organic matter became available by the drying and re-flooding events, and contribution of aceticlastic methanogenesis has increased. In conclusion, our results together with literature data indicate that the history of field management under upland or flooded conditions shapes the structure of the bacterial and archaeal communities, which eventually determine the function of methanogenic degradation of organic matter once the soils are flooded. Although the number of relevant experiments and observations is still scarce, data indicate that structure and function of the differently managed soils is relatively robust as long as rotation between upland and flooded conditions are sufficiently frequent, e.g. annual rotation. However, even a rotation of 4 years upland and 2 years flooded resulted in a rather stable microbial community sufficiently poised for methanogenic activity. More permanent situations, on the other hand, such as in desert soil or lake sediments, resulted in methanogenic communities with either very low abundance and simple composition or with high sensitivity to desiccation, respectively. We therefore hypothesize, that the key microbial populations in wetland or rotational rice, but also in desert soil, are €k, i.e. adapted to changing soil water and/or species that are euryo €k. O2 availability, whereas those in lake sediments are steno Acknowledgements The study was financially supported by the German Research Foundation (DFG) within the Collaborative Research Center 987. The authors acknowledge Alvaro Roel and Guillermina Cantou from INIA and Alex de Oliveira Chagas (Labrustar) and Hugo Firpo for their collaboration in the soil sampling and for providing agronomical information. YJ received a scholarship in the Joint Doctoral Promotion Program of the Max-Planck-Society and the Chinese Academy of Sciences, AFS a fellowship from the Max-PlanckSociety in 2012, and a postdoctoral scholarship from the China Scholarship Council (CSC) in 2015. We thank two anonymous reviewers for helpful comments.

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2015.07.015.

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