Anoxic Soil Research Articles

Vertical distribution of the methanotrophic community after drainage of rice field soil

Anoxic soils, such as flooded rice fields, are major sources of the greenhouse gas CH4 while oxic upland soils are major sinks of atmospheric CH4. Nevertheless, CH4 is also consumed in rice fields where up to 90% of the produced CH4 is oxidized in a narrow oxic zone around the rice roots and in the soil surface layer before it escapes into the atmosphere. After 1 day drainage of rice field soil, CH4 oxidation was detected in the top 2-mm soil layers, but after 8 days drainage the zone of CH4 oxidation extended to 8 mm depth. Simultaneously, the potential for CH4 production decreased, but some production was still detectable after 8 days drainage throughout the soil profile. The vertical distribution of the methanotrophic community was also monitored after 1 and 8 days drainage using denaturing gradient gel electrophoresis after PCR amplification with primer sets targeting two regions on the 16S rRNA gene that are relatively specific for methylotrophic α- and γ-Proteobacteria, and targeting two functional genes encoding subunits of key enzymes in all methanotrophs, i.e. the genes for the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Drainage stimulated the methanotrophic community. Eight days after drainage, new methanotrophic populations appeared and a distinct methanotrophic community developed. The population structure of type I and II methanotrophs was differently affected by drainage. Type II methanotrophs (α-Proteobacteria) were present throughout the soil core directly after drainage (1 day), and the community composition remained largely unchanged with depth. Only two new type II populations appeared after 8 days of drainage. Drainage had a more pronounced impact on the type I methanotrophic community (γ-Proteobacteria). Type I populations were not or only weakly detected 1 day after drainage. However, after 8 days of drainage, a large diversity of type I methanotrophs were detected, altough they were not evenly distributed throughout the soil core but dominated at different depths. A distinct type I community structure had developed within each soil section between 0 and 20 mm soil depth, indicating the widening of suitable habitats for methanotrophs in the rice field soil within 1 week of drainage.

Microbiology of flooded rice paddies.

Flooded rice paddies are one of the major biogenic sources of atmospheric methane. Apart from this contribution to the 'greenhouse' effect, rice paddy soil represents a suitable model system to study fundamental aspects of microbial ecology, such as diversity, structure, and dynamics of microbial communities as well as structure-function relationships between microbial groups. Flooded rice paddy soil can be considered as a system with three compartments (oxic surface soil, anoxic bulk soil, and rhizosphere) characterized by different physio-chemical conditions. After flooding, oxygen is rapidly depleted in the bulk soil. Anaerobic microorganisms, such as fermentative bacteria and methanogenic archaea, predominate within the microbial community, and thus methane is the final product of anaerobic degradation of organic matter. In the surface soil and the rhizosphere well-defined microscale chemical gradients can be measured. The oxygen profile seems to govern gradients of other electron acceptors (e.g., nitrate, iron(III), and sulfate) and reduced compounds (e.g., ammonium, iron(II), and sulfide). These gradients provide information about the activity and spatial distribution of functional groups of microorganisms. This review presents the current knowledge about the highly complex microbiology of flooded rice paddies. In Section 2 we describe the predominant microbial groups and their function with particular regard to bacterial populations utilizing polysaccharides and simple sugars, and to the methanogenic archaea. Section 3 describes the spatial and temporal development of microscale chemical gradients measured in experimentally defined model systems, including gradients of oxygen and dissolved and solid-phase iron(III) and iron(II). In Section 4, the results of measurements of microscale gradients of oxygen, pH, nitrate-nitrite, and methane in natural rice fields and natural rice soil cores taken to the laboratory will be presented. Finally, perspectives of future research are discussed (Section 5).

Molecular Retrieval of Large 16S rRNA Gene Fragments from an Italian Rice Paddy Soil Affiliated with the Class Actinobacteria

We designed a PCR assay specific for the 16S rRNA genes of members of the class Actinobacteria, and created a clone library using the amplification product of total community DNA extracted from anoxic Italian rice field soil. Eighteen out of 27 randomly sequenced clones were affiliated with Actinobacteria, i.e. Frankineae, Corynebacterineae, Micrococcineae, the bacterium candidatus "Microthrix parvicella" and a novel taxonomically undefined cluster.

Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil.

Temperature is an important factor controlling CH(4) production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37 degrees C or in a temperature gradient block covering the same temperature range at intervals of 1 degrees C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH(4) production rates increased with temperature, with an apparent activation energy of 61 kJ mol(-1). Steady-state partial pressures of the methanogenic precursor H(2) also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H(2) plus CO(2)-dependent methanogenesis was kept at -20 to -25 kJ mol of CH(4)(-1) over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 microM. Simultaneously, the relative contribution of H(2) as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to (14)CH(4), so that the carbon and electron flow to CH(4) was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.

Effect of temperature on reduction of iron and production of carbon dioxide and methane in anoxic wetland rice soils

Wetland rice soils from Italy (Pavia) and the Philippines (Bugallon, Luisiana, Maligaya) were incubated under anoxic conditions at 31 different temperatures ranging from 4.7 °C to 49.5 °C. Production of CO2 was most intensive at the beginning of the incubation (0–4 days) and was predominantly coupled to the reduction of free Fe(III). The optimum temperature for these processes was between 32 °C and 41 °C. After 9–16 days, CO2 production rates had decreased and the available Fe(III) had been completely reduced at the optimum temperatures. However, Fe(III) was still available at temperatures below and above the optimum. Maximum CH4 production rates were observed after 4–16 days (except in soil from Maligaya) with temperature optima between 32 °C and 41 °C, similar to those for CO2 production and Fe reduction. Since ongoing Fe reduction is known to suppress CH4 production, the temperature range of optimum CH4 production was restricted to those temperatures at which Fe(III) had already been depleted. Nevertheless, the temperature characteristics of both CO2 and CH4 production often exhibited two temperature optima at some time during the incubation, suggesting a complex pattern of adaptation of the methanogenic microbial community to temperature. When available Fe(III) was completely depleted by anoxic pre-incubation at 30 °C, CH4 was produced at a constant rate (steady state conditions) which increased with increasing temperature. Steady state CH4 production reached a first maximum at about 40 °C, but increased further up to at least 50 °C, suggesting the presence of thermophilic microorganisms whose activity was apparently masked when Fe had not been completely reduced. The apparent activation energy of CH4 production at steady state ranged between 48 kJ mol–1 and 65 kJ mol–1.

Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization

We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel ‘non-extreme’ environmental Archaea in the pelagic water of a boreal forest lake.

Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization(1).

We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel 'non-extreme' environmental Archaea in the pelagic water of a boreal forest lake.

Ecophysiology of Wetland Plant Roots: A Modelling Comparison of Aeration in Relation to Species Distribution

This study examined the potential for inter-specific differences in root aeration to determine wetland plant distribution in nature. We compared aeration in species that differ in the type of sediment and depth of water they colonize. Differences in root anatomy, structure and physiology were applied to aeration models that predicted the maximum possible aerobic lengths and development of anoxic zones in primary adventitious roots. Differences in anatomy and metabolism that provided higher axial fluxes of oxygen allowed deeper root growth in species that favour more reducing sediments and deeper water. Modelling identified factors that affected growth in anoxic soils through their effects on aeration. These included lateral root formation, which occurred at the expense of extension of the primary root because of the additional respiratory demand they imposed, reducing oxygen fluxes to the tip and stele, and the development of stelar anoxia. However, changes in sediment oxygen demand had little detectable effect on aeration in the primary roots due to their low wall permeability and high surface impedance, but appeared to reduce internal oxygen availability by accelerating loss from laterals. The development of pressurized convective gas flow in shoots and rhizomes was also found to be important in assisting root aeration, as it maintained higher basal oxygen concentrations at the rhizome–root junctions in species growing into deep water.

Electron balance during steady‐state production of CH4 and CO2 in anoxic rice soil

SummaryIn methanogenic rice‐field soil, organic matter (SOM) is anaerobically fermented to acetate, CO2 and H2 which then serve as substrates for methanogenesis. The whole process is a disproportionation reaction in which part of the SOM is oxidized to CO2 and part is reduced to CH4. We were interested in the electron balance during this process. The rates VCO2 and VCH4 at which CO2 and CH4 are produced by anaerobic degradation of SOM, and the fraction RH2 of the CH4 produced by reduction of CO2 with H2 (as opposed to acetotrophic methanogenesis), depend on (i) the presence or absence of inorganic oxidants and (ii) the electron balance ΔZ. Under pseudo steady‐state conditions, where inorganic oxidants are exhausted and the rate of SOM degradation is small compared with the size of the pool, VCO2, VCH4 and RH2 are constrained by ΔZ. Conversely, ΔZ may be determined from VCO2, VCH4 and RH2, all of which may be independently measured. We measured VCO2 (0.149–0.308 μmol g−1 day−1), VCH4 (0.169–0.466 μmol g−1 day−1) and RH2 (0.19–0.35) in eight soils and obtained values of ΔZ ranging from −0.918 to 0.035 μmol g−1 day−1. The majority (six) were negative, indicating a decrease in the oxidation state of the SOM carbon on degradation. This could be caused by humic acids acting as an electron acceptor and allowing more of the SOM to be oxidized to CO2 rather than reduced to CH4. Direct measurement of SOM carbon oxidation state Z produced values around zero (−0.1 ± 0.1), but is too insensitive to reveal changes of the magnitude of ΔZ.

Electron balance during steady-state production of CH4 and CO2 in anoxic rice soil

Electron balance during steady-state production of CH4 and CO2 in anoxic rice soil

Selective chemical extraction and grainsize normalisation for environmental assessment of anoxic sediments: validation of an integrated procedure

An integrated selective extraction and size normalisation procedure for use in metal partitioning and diagenetic studies of anoxic sediments and soils is presented. Data obtained by this procedure can readily be combined with other primary data (e.g. sulfur concentrations, carbonate concentrations, cation exchange capacity, etc.) and derived parameters (e.g. degrees of pyritisation and sulfidisation) that enhance interpretation of the behaviour of trace metals in anoxic sediments. Achieving size normalisation during extraction, allows direct comparison of sediments from dissimilar sedimentary environments, and simplifies assessment of the processes that determine whether a sediment is a source of or a sink for trace metals. Aspects of a study of trace metals in sediments from the Brisbane River estuary, Australia, are used to illustrate applications of the integrated procedure.

Nitrification and denitrification in the rhizosphere of rice: the detection of processes by a new multi-channel electrode

N turnover in flooded rice soils is characterized by a tight coupling between nitrification and denitrification. Nitrification is restricted to the millimetre-thin oxic surface layer while denitrification occurs in the adjacent anoxic soil. However, in planted rice soil O2 released from the rice roots may also support nitrification within the otherwise anoxic bulk soil. To locate root-associated nitrification and denitrification we constructed a new multi-channel microelectrode that measures NH4+, NO2–, and NO3– at the same point. Unfertilized, unplanted rice microcosms developed an oxic-anoxic interface with nitrification taking place above and denitrification below ca. 1 mm depth. In unfertilized microcosms with rice plants, NH4+, NO2– and NO3– could not be detected in the rhizosphere. Assimilation by the rice roots reduced the available N to a level where nitrification and denitrification virtually could not occur. However, a few hours after injecting (NH4)2HPO4 or urea, a high nitrification activity could be detected in the surface layer of planted microcosms and in a depth of 20–30 mm in the rooted soil. O2 concentrations of up to 150 μM were measured at the same depth, indicating O2 release from the rice roots. Nitrification occurred at a distance of 0–2 mm from the surface around individual roots, and denitrification occurred at a distance of 1.5–5.0 mm. Addition of urea to the floodwater of planted rice microcosms stimulated nitrification. Transpiration of the rice plants caused percolation of water resulting in a mass flow of NH4+ towards the roots, thus supporting nitrification.

Archaeal population dynamics during sequential reduction processes in rice field soil.

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe(3+), and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e., Methanosarcinaceae, Methanosaetaceae, Methanomicrobiaceae, and Methanobacteriaceae) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983-4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the Methanosaetaceae and Methanobacteriaceae. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific TaqI restriction sites. Sequence analysis showed a high degree of conservation of TaqI restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp), Methanosaetaceae (280 bp), and Methanosarcinaceae/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g., Methanomicrobiaceae and rice cluster III) and Methanosaetaceae did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as Methanobacteriaceae decreased over time. Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.

Effect of soil aggregate size on methanogenesis and archaeal community structure in anoxic rice field soil

In anoxically incubated slurries of Italian rice field soil, CH 4 production is initiated after a lag phase during which ferric iron and sulfate are reduced. The production of CH 4 was affected by the size of soil aggregates used for the preparation of the soil slurry. Rates of CH 4 production were lowest with small aggregates (<50 and 50–100 μm), were highest with aggregates of 200–2000 μm size and were intermediate with aggregates of 2000–15 000 μm size. The different amounts of CH 4 accumulated were positively correlated to the concentrations of acetate, propionate and caproate that transiently accumulated in the slurries prepared from different aggregate sizes and also to the organic carbon content. The addition of organic debris that was collected from large-size aggregates to the aggregate size fractions <200 and <50 μm resulted in an increase of CH 4 production to amounts that were comparable to those measured in unamended aggregates of 200–2000 μm size, indicating that CH 4 production in the different aggregate size fractions was limited by substrate. The distribution of archaeal small-subunit rRNA genes in the different soil aggregate fractions was analyzed by terminal restriction fragment length polymorphism which allowed seven different archaeal ribotypes to be distinguished. Ribotype-182 (consisting of members of the Methanosarcinaceae and rice cluster VI), ribotype-389 (rice cluster I and II) and ribotype-820 (undigested DNA, rice cluster IV and members of the Methanosarcinaceae) accounted for >20, >30 and >10% of the total, respectively. The other ribotypes accounted for <10% of the total. The relative quantity of the individual ribotypes changed only slightly with incubation time and was almost the same among the different soil aggregate fractions. Ribotype-389, for example, slightly decreased with time, whereas ribotype-182 slightly increased. At the end of incubation, the relative quantity of ribotype-182 seemed to be slightly higher in soil fractions with larger than with smaller aggregates, whereas it was the opposite with ribotype-80 (Methanomicrobiaceae) and ribotype-88 (Methanobacteriaceae). Ribotype-280 (Methanosaetaceae and rice cluster V), ribotype-375 (rice cluster III), ribotype-389 and ribotype-820, on the other hand, were not much different among the different soil aggregate size fractions. However, the differences were not significant relative to the errors encountered during the extraction of polymerase chain reaction (PCR)-amplifiable DNA from soil. In conclusion, soil aggregate size and incubation time showed a strong effect on the function but only a small effect on the structure of the methanogenic microbial community.

Effect of soil aggregate size on methanogenesis and archaeal community structure in anoxic rice field soil.

In anoxically incubated slurries of Italian rice field soil, CH(4) production is initiated after a lag phase during which ferric iron and sulfate are reduced. The production of CH(4) was affected by the size of soil aggregates used for the preparation of the soil slurry. Rates of CH(4) production were lowest with small aggregates (<50 and 50-100 µm), were highest with aggregates of 200-2000 µm size and were intermediate with aggregates of 2000-15000 µm size. The different amounts of CH(4) accumulated were positively correlated to the concentrations of acetate, propionate and caproate that transiently accumulated in the slurries prepared from different aggregate sizes and also to the organic carbon content. The addition of organic debris that was collected from large-size aggregates to the aggregate size fractions <200 and <50 µm resulted in an increase of CH(4) production to amounts that were comparable to those measured in unamended aggregates of 200-2000 µm size, indicating that CH(4) production in the different aggregate size fractions was limited by substrate. The distribution of archaeal small-subunit rRNA genes in the different soil aggregate fractions was analyzed by terminal restriction fragment length polymorphism which allowed seven different archaeal ribotypes to be distinguished. Ribotype-182 (consisting of members of the Methanosarcinaceae and rice cluster VI), ribotype-389 (rice cluster I and II) and ribotype-820 (undigested DNA, rice cluster IV and members of the Methanosarcinaceae) accounted for >20, >30 and >10% of the total, respectively. The other ribotypes accounted for <10% of the total. The relative quantity of the individual ribotypes changed only slightly with incubation time and was almost the same among the different soil aggregate fractions. Ribotype-389, for example, slightly decreased with time, whereas ribotype-182 slightly increased. At the end of incubation, the relative quantity of ribotype-182 seemed to be slightly higher in soil fractions with larger than with smaller aggregates, whereas it was the opposite with ribotype-80 (Methanomicrobiaceae) and ribotype-88 (Methanobacteriaceae). Ribotype-280 (Methanosaetaceae and rice cluster V), ribotype-375 (rice cluster III), ribotype-389 and ribotype-820, on the other hand, were not much different among the different soil aggregate size fractions. However, the differences were not significant relative to the errors encountered during the extraction of polymerase chain reaction (PCR)-amplifiable DNA from soil. In conclusion, soil aggregate size and incubation time showed a strong effect on the function but only a small effect on the structure of the methanogenic microbial community.

Specificity of chloroform, 2-bromoethanesulfonate and fluoroacetate to inhibit methanogenesis and other anaerobic processes in anoxic rice field soil

Chloroform (CHCl 3), 2-bromoethanesulfonate (BES) and fluoroacetate have frequently been used as methanogenic inhibitors in rice field soil and in other environments, but their effects on other microbial processes have not received sufficient attention. Therefore, we comparatively determined the effects of CHCl 3, BES and fluoroacetate on different microbial processes in rice field soil slurry upon incubation under anoxic conditions: on the reduction of the electron acceptors nitrate, ferric iron, sulfate; on the production of CH 4 and CO 2; on the temporal change of the electron donors H 2, acetate and propionate; and on the turnover of [2- 14 C ]acetate during the early reduction phase (day 7), and during the later methanogenic phase (day 30). The results demonstrate: (1) fluoroacetate inhibited acetate consumption by all microorganisms, (2) BES generally inhibited CH 4 production, and (3) CHCl 3 not only inhibited methanogenesis, but partially also acetate-dependent sulfate reduction, and perhaps H 2-dependent homoacetogenesis. The specificity of the different inhibitors resulted in characteristic patterns of the temporal change of electron donors and acceptors and of CH 4. The pattern of propionate change was consistent with production by fermenting bacteria and consumption by sulfate reducers either using sulfate or methanogens as electron acceptor. Sulfate reducers were also found to be important for acetate consumption during the early phase of soil anoxia. Later on, however, methanogenic acetate consumption was much more pronounced. The application of inhibitors with different specificity was helpful for elucidating the flow of carbon and electrons during degradation of organic matter in anoxic rice field soil.

Fate of 15N labelled nitrate and ammonium in a fertilized forest soil

The possibility that long-term atmospheric nitrogen pollution and fertilization of forest soil may serve as a basis for adaptation for enhanced transformation rates of NO 3 − and NH 4 + in soil bacteria was elucidated in a laboratory bioassay. Bacteria extracted from soils that had been fertilized at various rates for the last 30 yr were characterised with respect to their capability to reduce or oxidise different nitrogen sources. The same soils were used under oxic or anoxic conditions to quantify denitrification, dissimilatory nitrate reduction to ammonium (DNRA) and nitrification. 15NO 3 − and 15NH 4 + were added as tracers to the soils, which were incubated in bottles for 3 to 5 d. Concentrations of 15N 2O in headspace and 15NO 3 − and 15NH 4 + in soil extracts were determined by gas chromatography–mass spectrometry. Total numbers of bacteria were similar in all soils and ranged from 3 to 4×10 8 cells g −1 d.wt. of soil. Between 50 and 70% of the isolated strains were capable of reducing nitrate and the majority of them reduced nitrate to ammonium. About 0.01‰ of all isolates were classified as nitrifiers. Both nitrate reducers and nitrifiers were more common in fertilized soils than in the unfertilized control soil. The foremost fate of added 15NO 3 − and 15NH 4 + in all soils was immobilisation. More than 85% was immobilised in anoxic soils and between 64 and 97% in the oxic soils, with the lowest quantities in fertilized soils. As regards the remaining, non-immobilised N, DNRA dominated over denitrification, as could be expected from the higher frequency of ammonifying bacteria compared with denitrifiers. There was no obvious relationship between NH 4 + produced and the amount of fertilizer applied, whereas denitrification was negatively correlated with the amount of fertilizer applied. Nitrifying activity was low in all soils with no obvious relationship between NO 3 − produced and fertilizer applied. Hence, no correlation was found between the relative abundance of N transforming bacteria and the transformation activity. The N flux followed essentially the same pattern as that seen for product formation. The DNRA flux was higher than that of both denitrification and nitrification. DNRA and denitrification fluxes were highest in the control soil, whereas the nitrification flux was low in all soils. The absence of evidence for adaptation to enhanced rates of transformation of NO 3 − and NH 4 + in soil bacteria exposed to long-term N fertilization is reflected by the low concentration of extractable inorganic N in the fertilized soils. As a consequence of the quantitative importance of immobilization of added N, differences in physiological capacity evolved in soil bacteria to immobilize and mobilise N may determine the rates by which inorganic N is available for plant growth or lost to groundwater and air.

Fermentation pattern of methanogenic degradation of rice straw in anoxic paddy soil.

The anaerobic degradation of different fractions of rice straw in anoxic paddy soil was investigated. Rice straw was divided up into stem, leaf sheath and leaf blade. The different straw fractions were mixed with paddy soil and incubated under anoxic conditions. Fermentation of straw components started immediately and resulted in transient accumulation of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and caproate with much higher concentrations in the presence than in the absence of straw. Also some unidentified compounds with UV absorption could be detected. The maximum concentrations of these compounds were different when using different straw fractions, suggesting differences in the degradation pathway of these straw fractions during the early phase of incubation, i.e. with Fe(III) and sulfate serving as oxidants. When concentrations of the intermediates decreased to background values, CH(4) production started. Rates of CH(4)unamended soil. During the methanogenic phase, the percentage contribution of fermentation products to CH(4) production was determined by inhibition with 2-bromoethanesulfonate (BES). Acetate (48-83%) and propionate (18-28%) were found to be the main intermediates of the carbon flow to CH(4), irrespective of the fraction of the rice straw or its absence. Mass balance calculations showed that 84-89% of CH(4) was formed via acetate in the various incubations. Radiotracer experiments showed that 11-27% of CH(4) was formed from H(2)/CO(2), thus confirming that acetate contributed 73-89% to methanogenesis. Our results show that the addition of rice straw and the fraction of the straw affected the fermentation pattern only in the early phase of degradation, but had no effect on the degradation pathway during the later methanogenic phase.

Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores.

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 microM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-microm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the alpha and beta subclasses of Proteobacteria as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.

Fermentation pattern of methanogenic degradation of rice straw in anoxic paddy soil

The anaerobic degradation of different fractions of rice straw in anoxic paddy soil was investigated. Rice straw was divided up into stem, leaf sheath and leaf blade. The different straw fractions were mixed with paddy soil and incubated under anoxic conditions. Fermentation of straw components started immediately and resulted in transient accumulation of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and caproate with much higher concentrations in the presence than in the absence of straw. Also some unidentified compounds with UV absorption could be detected. The maximum concentrations of these compounds were different when using different straw fractions, suggesting differences in the degradation pathway of these straw fractions during the early phase of incubation, i.e. with Fe(III) and sulfate serving as oxidants. When concentrations of the intermediates decreased to background values, CH 4 production started. Rates of CH 4 production were higher in incubations with straw from the stem>leaf blade>leaf sheath>unamended soil. During the methanogenic phase, the percentage contribution of fermentation products to CH 4 production was determined by inhibition with 2-bromoethanesulfonate (BES). Acetate (48–83%) and propionate (18–28%) were found to be the main intermediates of the carbon flow to CH 4, irrespective of the fraction of the rice straw or its absence. Mass balance calculations showed that 84–89% of CH 4 was formed via acetate in the various incubations. Radiotracer experiments showed that 11–27% of CH 4 was formed from H 2/CO 2, thus confirming that acetate contributed 73–89% to methanogenesis. Our results show that the addition of rice straw and the fraction of the straw affected the fermentation pattern only in the early phase of degradation, but had no effect on the degradation pathway during the later methanogenic phase.