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Keywords:

  • Nitrate reduction;
  • Sulfate reduction;
  • Ferric iron reduction;
  • Rice roots;
  • 16S rRNA;
  • T-RFLP

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Leakage of O2 from roots of aquatic plants supports the oxidation of ammonia to nitrate and of sulfide to sulfate in the rhizosphere, so that these electron acceptors may become available to the root microbial communities and affect their activity. We studied the composition of the bacterial community active in anoxically incubated rice roots by analysis of terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequencing targeting bacterial 16S rRNA. The bacterial ribosomal abundance in unamended rice roots, which were naturally encrusted with ferric iron, were initially dominated (about 65% of total 16S rRNA) by Clostridium, Bacillus and Geobacter/Pelobacter, but after 5 d clostridia decreased and members of the CytophagaFlavobacteriumBacteroides (CFB) phylum increased (up to 30% of total 16S rRNA). Addition of nitrate or sulfate to the root incubations resulted in bacterial growth detected by fluorescent in situ hybridization (FISH). It also affected the steady state concentrations of H2, acetate, propionate and butyrate that were measured in the root incubations. Nitrate reducers were apparently involved in consumption of all of these compounds. Sulfate reducers, on the other hand, showed net production of acetate during utilization of propionate. Nitrate stimulated populations of Bacillus and Dechloromonas to become active, the latter temporarily increasing to 25% of total 16S rRNA, but suppressed the increase of CFB bacteria. Sulfate, on the other hand, stimulated Desulfosporosinus and Geobacter/Pelobacter, increasing to about 15% of total 16S rRNA, and suppressed CFB bacteria to become active. In conclusion, our study showed the potential effect of exogenous electron acceptors on the composition and activity of the bacterial community in rice root incubations, and identified the phylogenetic groups of the root microbial communities that respond to an increased availability of nitrate or sulfate.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Rice plants and rice fields have served as a model for wetland biogeochemistry and microbiology [1–3]. Aquatic plants live in anoxic soil habitats and thus have to ensure that their roots are supplied with O2. This creates a unique environment, in which the bulk soil is anoxic while the rhizosphere is partially oxic. The rice rhizosphere apparently sustains aerobic processes such as oxidation of ammonia, sulfur, iron and CH4 as well as anaerobic processes such as reduction of nitrate, sulfate, ferric iron and methanogenesis [1–4]. This is possible, since rice roots leak O2, preferentially at the tips and where lateral roots emerge [5]. The leakage of O2 supports the oxidation of ammonia to nitrate and of sulfide to sulfate [6] in the rhizosphere, so that these electron acceptors may become available to the root microbial communities and affect their activity. On the other hand, roots also leak organic substrates, which serve as substrates for fermentation and eventually CH4 production [7]. Organic substrates are alternatively supplied by degradation of root material, where roots from old nodes start to decay as soon as younger nodes emerge [8]. The organic substrates not only support methanogenesis, but alternatively allow reduction of sulfate or nitrate if these electron acceptors are available [6,9,10]. Some of these substrates may also be degraded by the aerobic root microbial communities, but this contribution is unknown. Nitrogen, mostly in form of urea or ammonium sulfate, and sulfate, as ammonium sulfate, are regularly supplied for fertilization of the crop [11]. Sulfate addition in form of gypsum is considered as a possible option for mitigation of CH4 emission [12,13]. Hence, rice roots can support complete nitrogen and sulfur cycles as long as organic substrates and O2 are supplied to the root microbial communities. Reduction of nitrate and sulfate may inhibit methanogenesis because of competition for common electron donors and accumulation of toxic intermediates [14–16].

The methanogenic archaeal communities on rice roots seem to be different from that in anoxic soil [17,18]. The archaeal community structure on the roots apparently changes when environmental conditions change. For example, the presence of high phosphate concentrations suppresses the proliferation of Methanosarcinaceae[19]. Recently, we showed that the activity of nitrate- and sulfate-reducing bacteria resulted in suppression of Methanosarcinaceae and Methanomicrobiaceae/Rice cluster I methanogens, respectively [20]. However, it is not known which bacterial populations are actively involved in the suppression of the methanogens.

The community structure of nitrate reducers on rice roots is unknown. The community structure of Gram-negative sulfate reducers has been characterized by using primers specific for their 16S rRNA genes, and was found to be similar to that in rice soil [21]. The most abundant Gram-negative sulfate reducers were within the radiation of the genera Desulfosarcina and Desulforhabdus. However, members of Desulfovibrio[21], Desulfotomaculum[22] and Desulfosporosinus[23] have also been detected on rice roots. The community structure of iron reducers on rice roots has not been studied comprehensively, but contains a novel iron-reducing Anaeromyxobacter[24].

In order to learn which bacteria might potentially be involved in reduction of nitrate, sulfate and ferric iron on rice roots, we used excised rice roots that were incubated in the presence and absence of external electron acceptors as a model system. In this model system, organic substrates were provided by the anaerobic decomposition of root material in the absence of O2. The abundance and activity of Bacteria were analyzed by targeting ribosomal RNA. The structure of the active Bacteria community was analyzed by T-RFLP after reverse transcription of 16S rRNA and sequence analysis of bacterial clone libraries.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Preparation of root samples and chemical analyses

Rice plants (Oryza sativa var. Roma, type japonica) were grown in the greenhouse using soil from rice fields at the Italian Rice Research Institute in Vercelli, Italy. Soil samples were taken in 1998 and 2000. A description of soil characteristics and the sampling site can be found in previous reports [25]. The preparation of soil, germination of rice seeds and growing conditions have been described before [26]. The rice plants were grown for approximately 80 d. The preparation of rice roots, the incubation conditions and chemical analyses have been described in detail by Scheid et al. [20]. Briefly, pieces (5 g) of roots were transferred into 120-ml glass bottles. After the addition of 5 g of CaCO3 (marble granules) for buffering and 53 ml of demineralized, autoclaved and anoxic water, the roots were incubated in triplicate at 25 °C under N2 atmosphere in the presence or absence of exogenous nitrate (8 mM NaNO3) or sulfate (5 mM Na2SO4). The amounts of nitrate and sulfate were equal in terms of electron equivalents required for complete reduction. They were reasonable with respect to usual N-fertilization practice (i.e., 100–200 kg N ha−1 or about 100 μmol cm−2) or sulfate content in not yet flooded rice field soil [27]. The incubation vessels were shaken vigorously to achieve equilibrium between the gas and the liquid phases before sampling. Methane, CO2 and H2 were analyzed in gas samples (0.25–0.5 ml); fatty acids, nitrate, nitrite, sulfate and iron(II) were analyzed in liquid samples (0.5 ml) as described by Scheid et al. [20]. In some experiments, roots were incubated in the presence of [2-14C]acetate. The temporal change of the radioactivity in the acetate, CO2 and CH4 fractions were followed by HPLC and GC equipped with a radiodetector [28].

2.2Molecular analyses

Microorganisms were detached from incubated rice roots as described by Weber et al. [29] and combined with cells from the water, which was used for incubation. Fluorescent in situ hybridization (FISH) was done as described by Scheid et al. [20] using general probes for bacteria (Eub338; [30]) and archaea (Arc915, Arc344; [31]).

The rRNA extraction method has been described previously [32] and was applied as described in detail by Scheid et al. [20]. Bacterial 16S rRNA was amplified from total RNA extracts using a one step RT-PCR system (Access QuickTM RT-PCR-System, Promega, Mannheim, Germany) with the primer combination Ba27f (5′ AGA GTT TGA TCC TGG CTC AG 3′) and Ba907r (5′ CCG TCA ATT C(AC)T TT(AG) AGT TT 3′) [33]. The reaction mixture (50 μl) of the one step RT-PCR system contained 1 μl of template RNA, 0.5 μM of each primer (MWG Biotech, Ebersberg, Germany), 25 μl of 2× reaction buffer and 1 μl (5 U) of AMV reverse transcriptase supplied by the manufacturer (Promega, Mannheim, Germany). The 2× reaction buffer included 3 mM MgSO4 (Final conc. 1.5 mM), Tfl DNA polymerase and the deoxynucleoside triphosphates. The reverse transcription reaction was carried out at 52 °C for 30–45 min, which was found to be more optimal in terms of specificity without compromising efficiency than the recommended maximum temperature of 48 °C. The reverse transcription reaction was followed by a denaturing step at 94 °C for 2 min to inactivate the AMV reverse transcriptase and to activate the Tfl DNA polymerase. The thermal profile of the PCR included 17 to 22 cycles of primer annealing at 52 °C for 0.45 s, primer extension at 68 °C for 1 min, and denaturing at 94 °C for 45 s. For the T-RFLP analyses the forward primer was labeled 5′ terminal with FAM (6-carboxyfluorescein).

T-RFLP analyses were performed as described by Chin et al. [34]. The principle of this method has been described by Liu et al. [35]. After PCR amplification with the specific primer set, the fluorescent-labeled PCR fragments were purified using Qiaquick columnsTM (Qiagen, Heidelberg, Germany). Aliquots were digested by Msp I for 3 h at 37 °C following the protocol of the manufacturer (Promega, Mannheim, Germany). Further analyses were performed on a DNA sequencer (373 DNA sequencer, PE Applied Biosystems, Weiterstadt, Germany) as described by Chin et al. [34]. Only peaks >50 bp and larger than a threshold of 100 (peak height, arbitrary units) were considered. The sum of all peak heights in each T-RFLP pattern was calculated as an indication of the total DNA quantity represented by each pattern. This DNA quantity was standardized between all patterns of the same experiment by proportionally reducing the height of each peak in the pattern with the larger amount of DNA. For this the proportion of the smallest DNA quantity and a larger DNA quantity was calculated and used as a correction factor as described by Dunbar et al. [36]. The relative abundance of a detected terminal fragment length (T-RF) was calculated as the respective signal height of this peak divided by the signal heights of all peaks of the electropherogram. The T-RF representing chloroplast rRNA gene were disregarded. Each analysis was performed in three parallels. Peaks representing >1% of the total in all three replicates and at all sampling times were analyzed individually. Peaks that only occasionally reached a high abundance (usually >2% of the total) were also analyzed individually. The other peaks were combined and designated as “other T-RF”.

16S rcDNA amplicons were cloned using the pGEM-T Vector System (Promega, Mannheim, Germany). Clone libraries were screened by T-RFLP for significant T-RFs according to the patterns from the incubations. The respective 16S rcDNA clones were sequenced with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Weiterstadt, Germany) and the primers Ba519r and Ba907r [33]. Sequences (fragment lengths between 415 and 874 bp) were assigned to phylogenetic groups with a Wu-Blast2 search (EMBL-nucleotide-sequence database; [37]; http://www.ebi.ac.uk/blast2/). Alignment, comparison and phylogenetic trees were accomplished with the ARB software package [38] and the database from June 2002. Additional sequences, which showed highest matches in the Blast search were obtained from the EMBL database and integrated in the phylogenetic analysis. Trees were calculated using neighbor joining and maximum-likelihood methods [38]. To omit highly variable regions within the 16S rRNA genes, filters for the Dechloromonas, the Desulfosporosinus or the δ-Proteobacteria were used. Escherichia coli, Serratia marcescens and Salmonella typhimurium were used as outgroup references.

The 16S rcDNA sequences were deposited at the EMBL database under the following Accession Nos.: AJ621940–AJ622014.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Reduction of electron acceptors

The experimental results concerning sequential reduction of nitrate, ferric iron and sulfate on rice roots have already been presented by Scheid et al. [20]. Briefly, addition of equivalent amounts of nitrate and sulfate to Fe(III)-encrusted roots resulted first in the reduction of nitrate (7.5 mM), which was depleted within 1 d with nitrite intermediately accumulating for another day. Sulfate (5 mM) was completely reduced between day 5 and 12. Roots were generally encrusted with ferric iron, so that there was no need to add exogenous Fe(III) (control). Accumulation of Fe(II) (3.5 mM) started from the beginning of the experiment and ended after about 12 d. At this time, vigorous CH4 production started [20]. The reduction pattern was similar whether or not 3.5 mM propionate was added as electron donor.

3.2Growth of microorganisms

The abundance of microorganisms was determined by microscopically counting total microorganisms using DAPI and Bacteria and Archaea using FISH with domain-specific probes. The microorganisms were counted during the phase of nitrate reduction (1 d) and sulfate reduction (8 d) both in the incubations with exogenous electron acceptors and in the unamended control. In the control, numbers of Bacteria only slightly increased with incubation time (up to 20 d). However, addition of nitrate or sulfate resulted in a tenfold increase of numbers of Bacteria after 1 and 8 d, respectively (Fig. 1). On the other hand, addition of nitrate resulted in a tenfold decrease of Archaea within 1 d. Sulfate addition did not have such an effect on the archaeal numbers. In the control (containing only Fe(III) incrustations), however, numbers of Archaea increased by more than tenfold after 20 d incubation, i.e., several days after sulfate and ferric iron had been depleted and vigorous CH4 production had started (Fig. 1).

image

Figure 1. Change of numbers of Bacteria and Archaea in rice root incubations in the absence and presence of nitrate and sulfate. Microbial numbers were counted under the microscope stained with DAPI (total counts) or using FISH targeting Bacteria (EUB) or Archaea. Mean ± SD; n= 3.

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3.3T-RFLP analysis of the bacterial community

The community structure of active Bacteria was determined by T-RFLP analysis targeting ribosomal RNA (16S rRNA). T-RFLP of 16S rRNA measures the relative abundance of the bacterial ribosomes having different terminal restriction fragments (T-RF). Thus it is a measure of both the relative abundance of different bacterial populations and of their ribosomal content, being indicative of activity. The T-RFLP patterns were compared between nitrate treatment, sulfate treatment and unamended control after different incubation times (Fig. 2). The incubation times were chosen according to the duration of the phases of nitrate reduction (0–1 d) and sulfate reduction (5–12 d). Sixteen different T-RF were identified.

image

Figure 2. Temporal change of the bacterial community structure in rice root incubations determined by T-RFLP targeting 16S rRNA; (a) unamended control, (b) plus sulfate, (c) plus nitrate; mean values of n= 3, SD see Fig. 6.

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In order to assign the different T-RF to bacterial taxa, clone libraries (based on rcDNA of 16S rRNA) were created from the control incubations after 1 d (DS6) and 5 d (DS7), as well as from the incubations with nitrate after 1 d (DS4) and with sulfate after 9 d (DS5). All T-RF could be assigned to one or several bacterial taxa, except the T-RF with 478, 535 and 529 bp length (Table 1).

Table 1.  Phylogenetic affiliation of cloned 16S rcDNA sequences and length of T-RFa
CloneT-RFNext relative (EMBL entry name)Similarity
  1. aLength of sequence: 415–874 bp; primers used for sequencing: 519r, 907r. Designation of clones: DS4 (24 h, + nitrate); DS5 (9 d, +sulfate); DS6 (24 h, control); DS7 (5 d, control).

  2. bCharacterization of acetogenic bacterium isolated from an anaerobic reactor (Fernandez and Tiedje, unpublished).

IRR-DS5–21138Bacillus sp. (YKJ-10 AF221061)0.96
IRR-DS5–9, 5–12, 5–30138Desulfosporosinus spp. (AF076248, AY007667)0.96–0.99
IRR-DS5–15138Desulfotomaculum aeronauticum (DA16S)0.94
IRR-DS5–26, 6–2152α-Proteobacterium (UBA387860)0.99,1.00
IRR-DS7–4, 7–16, 7–27, 4–6, 4–33152Bacillus spp. (UEA229195, AF221061, BA16SRRA)0.95–0.97
IRR-DS6–4163Pelobacter propionicus (AF400105)0.95
IRR-DS7–21163Geobacter pelophilus (U96918)0.92
IRR-DS5–3163Desulfovibrio sp. (USU012594)0.91
IRR-DS5–4, 5–17, 5–20, 5–32163Geobacter sp. (GSPY19190)0.96–0.97
IRR-DS4–26, 5–7, 5–11, 7–3, 7–17, 7–25201CFB-Phylum (BSA229237, BSA229236)0.93–0.99
IRR-DS5–13205Clostridium sp. (CSA229250)0.99
IRR-DS5–29206Anaerobacter polyendosporus (APO18189)0.94
IRR-DS5–24208Clostridium sp. (CSA229248)0.94
IRR-DS5–16, 6–9, 6–26, 7–8, 7–14, 7–18, 7–20271Clostridium spp. (UEA229230, UBA387879, UBA387878)0.96–0.98
IRR-DS7–2, 7–13, 6–22289Clostridium spp. (UEA229210, AF371590)0.92
IRR-DS4–3, 4–7, 4–8, 4–9, 4–21, 4–23433Dechloromonas sp. strain JJ (AY032611)0.97–0.99
IRR-DS4–1433Dechloromonas sp. (AF137507)0.94
IRR-DS4–4, 4–10433β-Proteobacteria (AB024608, AB017489)0.94,0.95
IRR-DS6–1, 6–8488Aquaspirillum sp. (UBA387862)1.00
IRR-DS4–19, 4–20, 4–22, 4–24, 7–6, 7–10492β-Proteobacteria (UBA295498, U54470, AF204250)0.93–0.95
IRR-DS7–9, 7–19508Geobacter pelophilus (U96918)0.99
IRR-DS6–23, 7–15, 7–28512Clostridium sp. (UEA229186, UEA229206)0.92–0.96
IRR-DS6–6, 6–7, 6–10, 6–20, 6–21, 6–29, 7–7518Clostridium scatologenes strain SL1 (CSCY18813)0.94–0.96
  Clostridium sp. T7 (AF281142)b0.96–0.98
IRR-DS6–19518Clostridium sp. (AF191250)0.96
IRR-DS6–11, 6–18, 6–24, 6–27521Clostridium quinii (CQDSM16SR)0.98–0.99
IRR-DS6–28, 7–1521Clostridium sp. (UBA387883, CSDSM16SR)0.98,0.99
IRR-DS6–5521Clostridium cylindrosporum (CCY18179)0.90

Initially, clostridia were the most dominant bacterial group (T-RF of 271, 289, 512, 521 bp). The different clostridia remained a prominent group in the unamended control, but members of CFB-phylum (201 bp) became the single most important group (Fig. 2(a)).

In the incubations with exogenous nitrate or sulfate, however, the bacterial community structure developed differently. In the nitrate treatments, members of the Dechloromonas group (433 bp) became abundant during days 1–5 and persisted until day 12. To a lesser extent, also the T-RF with 433 bp (β-Proteobacteria), 478 bp (not assigned) and 488 bp (Aquaspirillum sp.) increased in abundance during the same period (Fig. 2(c)). The dominance of Dechloromonas was intriguing. Five different clones closely related to the genus Dechloromonas were obtained from the nitrate treatment. The phylogenetic placement of these clone sequences showed that they fell within the phylogenetic radiation of known Dechloromonas strains (Fig. 3). Three almost identical clone sequences (IRR-DS4–3, -7, -21) had a sequence similarity of approximately 99% to Dechloromonas strain JJ [39]. The closest relative to clone sequences IRR-DS-4–9, -23 was Dechloromonas strain CL24 (sequence similarity of 98.3–99.2%).

image

Figure 3. Phylogenetic affiliation of cloned 16S rRNA gene sequences from rice roots related to Dechloromonas (dendrogram calculated using maximum-likelihood method). The scale bar indicates the estimated number of base changes per nucleotide sequence position.

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In the sulfate treatments, on the other hand, there was a relative increase in abundance of members of the groups of Desulfosporosinus/Desulfotomaculum (138 bp) and Pelobacter/Geobacter (163, 508 bp) (Fig. 2(b)). The closest relatives of Desulfosporosinus affiliated clone sequences were Desulfosporosinus strain PFB and strain S5 (Fig. 4). The sequence similarity among the clone sequences IRR-DS-5–9, -30 and strain PFB was between 96.8% and 97.2%. Clone sequence IRR-DS-5–12 branched together with the Desulfosporosinus strain S5 (sequence similarity: 98%) and strain T1 (sequence similarity: 97.6%).

image

Figure 4. Phylogenetic affiliation of cloned 16S rRNA gene sequences from rice roots related to Desulfosporosinus (dendrogram calculated using maximum-likelihood method). The scale bar indicates the estimated number of base changes per nucleotide sequence position.

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Within the Geobacter/Pelobacter group two clone sequences (IRR-DS-7–9, -19) fell with a sequence similarity value of approximately 99.8% close to Geobacter pelophilus (Fig. 5). Other clone sequences were closely related to Geobacter chapelleii (IRR-DS-5–17, -20: approximately 98%) or to uncultured bacteria (IRR-DS-5–4, -32, similarity to Banisveld landfill sequences: 94.6–98.1%; [40]).

image

Figure 5. Phylogenetic affiliation of cloned 16S rRNA gene sequences from rice roots related to Geobacter (dendrogram calculated using maximum-likelihood method). The scale bar indicates the estimated number of base changes per nucleotide sequence position.

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3.4Temporal change of activity and community structure

The change of prominent T-RF representing different groups of Bacteria was followed in the different incubations together with the change in substrate concentrations.

Hydrogen partial pressures were low (1.6 Pa) during the phase of nitrate reduction compared to about 100–170 Pa in the control (Fig. 6(a)). During the sulfate reduction phase, H2 partial pressures (1.4–3.6 Pa) were also lower than in the control (14–50 Pa). When sulfate was depleted the H2 partial pressures increased again to 14 Pa (Fig. 6(a)).

image

Figure 6. Temporal change of bacterial metabolites, and of the relative abundance of individual bacterial groups defined by T-RFLP analysis in rice root incubations in the absence (control) and the presence of nitrate and sulfate. (a) H2; (b) propionate; (c) butyrate; (d) acetate; (e) clostridia, T-RF=271, 512, 521 bp; (f) Dechloromonas, T-RF=433 bp; (g) Bacillus, T-RF=152 bp; (h) CFB phylum, T-RF=201 bp; (i) Desulfosporosinus, T-RF=138 bp; (j) Geobacter, T-RF=163 bp. Mean ± SD; n= 3. The data of H2 and acetate have already been shown [20].

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Concentrations of propionate and butyrate slightly increased with time in the control, but decreased during the reduction of nitrate and nitrite (Fig. 6(b) and (c)). The reduction of sulfate was accompanied by a decrease of propionate concentrations and, to a lesser extent, of butyrate concentrations. Acetate accumulated steadily with time in all experiments, but accumulation was lower during the nitrate reduction phase (Fig. 6(d)). After onset of sulfate reduction, i.e., 5 d of incubation, decomposition of propionate resulted in increased acetate accumulation. The addition of 2-14C-labeled acetate to incubated rice roots showed that during the periods of nitrate and sulfate reduction acetate was oxidized to CO2 (not shown).

The T-RF (271, 512, 521 bp) assigned to clostridia were the most abundant group at the beginning of incubation accounting for 56% of total bacterial ribosomes, but decreased with time in all incubations (Fig. 6(e)), although it never decreased below 10%.

Nitrate treatment resulted in a conspicuous increase of the T-RF (433 bp) assigned to Dechloromonas reaching a maximum of about 25% after 5 d incubation (Fig. 6(f)). The T-RF (152 bp) assigned to Bacillus sp. was relatively high compared to the control up to day 1 when nitrate became depleted (Fig. 6(g)). Interestingly, the abundance of the CFB-Phylum (201 bp) was relatively low in the nitrate treatment until day 12–17 (Fig. 6(h)).

Sulfate treatment, on the other hand, resulted in a short increased abundance of Desulfosporosinus on day 9 that was not seen in any of the other incubations (Fig. 6(i)). After day 9, the T-RF (163 bp) assigned to Geobacter became significantly more abundant than in the control (Fig. 6(j)). A similar pattern was seen with the T-RF of 508 bp length (not shown), which was also assigned to Geobacter. The CFB-Phylum, on the other hand, was less abundant in the sulfate treatment compared to the control during 9–17 d of incubation (Fig. 6(h)) with incubation time.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Our results showed that the presence of nitrate or sulfate increased the size and affected the composition of the bacterial community in rice root incubations. The size of the archaeal community, on the other hand, only increased in the control incubations after endogenous electron acceptors (i.e., ferric iron) were reduced.

We investigated the composition of the active bacterial communities by determining the relative abundance of group-specific rRNA using T-RFLP analysis, assuming that bacterial cells of active populations have a higher content of ribosomes [41,42]. The active bacterial community that was initially present on the roots mainly consisted of clostridia (about 60% of the total). The clostridia probably lived by fermentation and were responsible for the production of H2, butyrate, propionate and acetate, in particular, during the first days of incubation. However, within 5 days members of the CFB phylum replaced this initial community, then representing about 30% of the total bacterial community. In addition, members of the genera Bacillus and Geobacter/Pelobacter were represented by about 5% each. They may have contributed to the fermentation activity or to reduction of the iron oxide encrustation of the rice roots. The latter activity is quite likely for Geobacter[43]. Geobacter has previously been found on rice roots [32]. In the soil environment, Geobacter is able to respond to favorable conditions. For example, the Geobacteraceae family was found to make a strong contribution to the bacterial community in an iron-reducing plume of a landfill-leachate polluted aquifer [40].

The bacterial community in the root incubations exhibited a completely different development, when exogenous electron acceptors (nitrate or sulfate) were added. The added nitrate was rapidly reduced via nitrite. Nevertheless, it resulted in drastic community change, notably by first increasing the relative abundance of Bacillus and then, even more pronounced, that of Dechloromonas. Similarly to Dechloromonas, albeit to much less extent, T-RF representing β-proteobacteria (492 bp) and Aquaspirillum (488 bp) were also increased. The activity of these bacterial groups is not quite clear, but they were possibly involved in causing the relatively low H2 partial pressures, the propionate consumption and the delayed production of acetate and butyrate observed during the first days of incubation. The temporal increase of Bacillus is in agreement with the nitrate reduction activity of this genus [44]. Members of β-proteobacteria and Aquaspirillum are also able to reduce nitrate [45]. Dechloromonas sp. reduces chlorate and perchlorate, but also nitrate and nitrite. The closely related Ferribacterium limneticum is able to reduce ferric iron [39,46]. Dechloromonas strain JJ, which is closely related to 3 clones obtained from roots amended with nitrate, is able to oxidize simple organic acids with nitrate [39]; other strains can utilize H2 as electron donor [47]. It is intriguing to see that Dechloromonas can become a dominant population in the rice root microbial community when nitrate is supplied.

On the other hand, nitrate amendment decreased the relative abundance of clostridia and Geobacter/Pelobacter and retarded the development of CFB bacteria in the root preparations. Possibly, these bacterial groups were sensitive to nitrite or other toxic productions of denitrification, as recently found for methanogens, Methanosarcina in particular, which were also suppressed by these compounds [48,16,20].

Sulfate amendment also changed the bacterial community during the period when sulfate was reduced, i.e., between days 5 and 12. Most pronounced was a temporal increase of the abundance of Gram-positive Desulfosporosinus and a gradual increase of Geobacter/Pelobacter, whereas the further increase of CFB bacteria stopped after onset of sulfate reduction. The detected Desulfosporosinus were phylogenetically related to Desulfosporosinus meridiei isolates from groundwater polluted with hydrocarbons [49,50]. This group was not detected by isolation of sulfate-reducing bacteria with different carbon sources, that revealed Gram-positive organisms related to Desulfotomaculum but not Desulfosporosinus[51]. Just recently, however, Desulfosporosinus has been detected on rice roots from Japanese rice fields [52]. Desulfosporosinus likely contributed to the consumption of H2 and the previously observed suppression of methanogens by sulfate [20]. However, it remains unknown which sulfate reducers were responsible for the consumption of propionate and butyrate that was observed in our root incubations. Propionate-consuming Gram-negative sulfate reducers have previously been found on rice roots [21,51]. Therefore, it is likely that 16S rRNA of these groups was present on rice roots, but the contribution to total 16S rRNA was apparently marginal and did not increase upon sulfate amendment.

The reason why the rRNA content of Geobacter/Pelobacter increased upon sulfate amendment is also not clear, but may be due to the increased production of acetate. Acetate production was stimulated in the presence of sulfate, possibly because consumption of propionate was enhanced. Members of the Geobacteraceae are known for their ability to use acetate as electron donor [43,53]. Iron reduction was going on simultaneously with sulfate reduction until about day 12 of the incubation, and acetate-utilizing Geobacter possibly contributed to this process. Finally, sulfate amendment resulted in suppression of CFB bacteria, as did nitrate. We assume that both nitrate reducers and sulfate reducers competed with members of the CFB phylum for common electron donors, e.g., fermentable organic substrates.

In conclusion, our study demonstrates the potential effect of exogenous electron donors on the composition and activity of the bacterial community in rice root incubations. Although our results were obtained by using a model system, we expect that similar effects are likely occurring at the root of intact rice plants. Our study identified the phylogenetic groups of the root microbial community that responded to an increased availability of nitrate or sulfate. In addition it generally showed how different microbial groups potentially interact with each other by turning over electron donors and acceptors, thus affecting the living conditions. Similar interactions may not only occur on rice roots but also in other environments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This study was part of the Sonderforschungsbereich 395 of the Deutsche Forschungsgemeinschaft “Interaction, adaptation and catalytic capacity of terrestrial microorganisms”.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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