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Methanogens represent the final decomposition guild in anaerobic degradation of organic matter, occurring in digestive tract of various invertebrates. However, factors determining their community structure and activity in distinct gut sections is still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration culminated in anterior hindgut of E. pulchripes In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tract of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so far unclassified "Methanomethylophilaceae" (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promotes the gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates.ImportanceMethane (CH4) is the second most important atmospheric greenhouse gas after CO2 and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats including digestive tract of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane producing and non-emitting species have been observed in millipedes, but what limits their methanogenic potential is not known. In the present study, we show that physico-chemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also find great similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

Past studies have praised earthworms for improving soil structure and fertility, but criticized earthworms for increasing the leaching of nutrients and soil greenhouse gas emissions. Therefore, in order to maximize the environmental benefits and reduce the environmental costs of earthworms, it is important to determine the factors controlling the structure of earthworm communities at local, landscape and continental scales. We first hypothesized that forested riparian buffer strips (FRBS) within agricultural landscapes would be a refuge for earthworms, due to higher soil moisture and organic matter compared to adjacent agricultural fields (“treatment” = FRBS vs. Field). Within sites, we hypothesized that earthworms would be most abundant where the chemical quality of above- and belowground plant litter is high, or where soil disturbance is low. At the continental scale, we hypothesized that total summer precipitation interacts with regional and local scale factors in controlling earthworm community structure. A field survey was conducted to quantify earthworm species abundances in FRBS and adjacent agricultural fields across Eastern Canada and Central Europe (two “bioregions” differing in rainfall). At each of 77 sites, we collected and identified earthworms from three plots within FRBS and adjacent agricultural fields, and noted the tree species, understory vegetation, drainage class, agricultural crop as well as five soil physicochemical variables (texture, pH, total C, total N and % organic matter). In each bioregion and treatment, we found proportionately more endogeic than anecic or epigeic earthworm species. In Eastern Canada there were proportionately fewer anecic and more epigeic individuals in FRBS than in fields, whereas in Central Europe there were fewer endogeic and more anecic earthworms in FRBS than in fields. We also found significant interactions between bioregion and treatment on total earthworm abundance and biomass, and on soil moisture. More specifically, in Eastern Canada we found higher earthworm abundance and biomass, soil moisture and organic matter in FRBS. Conversely, in Central Europe we found higher earthworm abundance and biomass in fields, no treatment effects on soil moisture, and higher soil organic matter in FRBS. The different earthworm distribution patterns in each bioregion were not related to the types of agricultural crops, but rather to differences in precipitation and soil moisture across bioregions. Within FRBS in Eastern Canada, earthworm abundance in deciduous and mixedwood stands were higher than in coniferous stands; in Central Europe, earthworm abundance was higher in deciduous stands only. Within FRBS in Eastern Canada, the abundance of the prominent endogeic species Apporectodia rosea was correlated with herbaceous plants, notably ferns and graminoids. Conditional regression tree analysis revealed positive relationships between earthworms and soil clay content, pH, moisture and organic matter. Our results suggest that local and landscape patterns in earthworm diversity can be predicted by soil and vegetation attributes, but the relative importance of these factors change across continual scales due to climate. Comparing the distributions of earthworms across different scales provides insights into the potential of different species to spread into new habitats with climate change.

Knowledge is lacking on the factors controlling the structure and spatial distribution of earthworm communities within agroecosystems. We first hypothesized that forested riparian buffer strips (FRBS) within agricultural landscapes would be a refuge for earthworms, as we predicted higher soil moisture and organic matter inputs in FRBS than in adjacent agricultural fields (treatments = FRBS vs. Field). We further hypothesized that earthworms would be most abundant where the chemical quality of above- and belowground plant litter is high, or where soil disturbance is low. We conducted a field survey to quantify earthworm species abundances in FRBS and adjacent agricultural fields in two bioregions, namely Eastern Canada and Central Europe. At each of 77 sites, we collected and identified earthworms from three plots within FRBS and within adjacent agricultural fields. In each plot, we identified the tree species, understory vegetation, soil drainage class, agricultural crop as well as five soil physicochemical properties. In each bioregion and treatment, we found proportionately more endogeic than anecic or epigeic earthworm species. In Eastern Canada, there were proportionately fewer anecic and more epigeic individuals in FRBS than in fields; in Central Europe there were fewer endogeic and more anecic earthworms in FRBS than in fields. We found significant bioregion × treatment interactions on earthworm abundance and soil moisture. More specifically, in Eastern Canada we found higher earthworm abundance and soil moisture in FRBS, whereas in Central Europe we found higher earthworm abundance in fields and no treatment effect on soil moisture. In both bioregions, we found higher organic matter in FRBS than in fields. In Eastern Canada, earthworm abundance in deciduous and mixedwood stands were higher than in coniferous stands; in Central Europe, earthworm abundance was highest in deciduous stands only. Within FRBS in Eastern Canada, the abundance of the prominent endogeic species, Apporectodea rosea, was correlated with herbaceous plants, notably ferns and graminoids. Conditional regression tree analysis revealed positive relationships between earthworms and soil clay content, pH, moisture and organic matter. Our results suggest that local and landscape patterns in earthworm diversity can be predicted by soil and vegetation attributes, however the relative importance of these factors changes across continental scales due to differences in precipitation patterns and soil moisture availability. Comparing the distributions of earthworms across different scales provides insights into the potential of different species to spread into new habitats with climate change.

International audience; Phytomanagement of trace element-contaminated soils can reduce soil toxicity and restore soil ecological functions, including the soil gas exchange with the atmosphere. We studied the emission rate of the greenhouse gases (GHGs) CO2, CH4, and N2O; the potential CH4 oxidation; denitrification enzyme activity (DEA), and glucose mineralization of a Cu-contaminated soil amended with dolomitic limestone and compost, alone or in combination, after a 2-year phytomanagement with a mixed stand of Populus nigra, Salix viminalis, S. caprea, and Amorpha fruticosa. Soil microbial biomass and microbial community composition after analysis of the phospholipid fatty acids (PLFA) profile were determined. Phytomanagement significantly reduced Cu availability and soil toxicity, increased soil microbial biomass and glucose mineralization capacity, changed the composition of soil microbial communities, and increased the CO2 and N2O emission rates and DEA. Despite such increases, microbial communities were evolving toward less GHG emission per unit of microbial biomass than in untreated soils. Overall, the aided phytostabilization option would allow methanotrophic populations to establish in the remediated soils due to decreased soil toxicity and increased nutrient availability.

Nitrogen fixation and photosynthesis provided by microbial phototrophs (cyanobacteria and eukaryotic microalgae) are important processes occurring in Arctic soil crusts. Here, we describe and compare these processes in biological soil crusts from Central Svalbard at different stages of development. The gradient from poorly-developed to well-developed soil crusts was accompanied by the changes in biovolume of microbial phototrophs, nitrogenase and photosynthetic activity. The lowest biovolume of microbial phototrophs was detected in poorly-developed soil crusts as a consequence of the initial stage of soil colonization. The biovolume initially increased during the soil crust development but decreased in well-developed lichenized soil crusts. However, nitrogenase activity decreased from poorly to more developed soil crusts. Diurnal courses of photosynthetic activity differed among the soil crust types showing shifts in diurnal minima and maxima; the poorly-developed soil crust reacted faster to changes in temperature and PAR. In spite of different microclimatic conditions during the measurements, temperature was the main factor influencing photosynthetic activity while the effect of PAR was not significant. Higher temperatures led to inhibition of photosynthetic activity and increased energy dissipation, indicating acclimation/adaptation of the soil crust photosynthetic microorganisms to a cold environment.

Fossorial mammals are supposed to face hypoxic and hypercapnic conditions, but such conditions have been rarely encountered in their natural burrow systems. Gas composition in burrows after heavy rains, deeper burrows and especially nest chambers, where animals usually spend most of the day, could be even more challenging than in shallow burrows. Such situations, however, have been rarely surveyed in the wild. In our study, we determined concentrations of O2, CO2 and CH4 in active burrows and nests of the giant root-rat Tachyoryctes macrocephalus, a large fossorial rodent endemic to the Afroalpine zone of the Bale Mountains in Ethiopia. We were able to determine the precise location of nests by tracking individuals equipped with radio-collars. To the best of our knowledge, this is the first study that analyses air samples taken directly from the nests of actually occupied burrow systems in any free-living fossorial mammal. We found no evidence for environmental hypoxia in the examined burrows and nests (range 19.7–21.6% O2). Concentrations of CO2 in the burrows increased after the burrows were plugged in the evening, but did not reach physiologically problematic levels. The highest CO2 concentrations in burrows were detected in the evening during a wet period (up to 0.44%). In root-rat nest chambers, the highest (but still harmless) CO2 concentrations (up to 1.31%) were detected in the morning (measured in the late dry season only) together with an elevated concentration (up to 13.5 ppm) of CH4. Regular surface activity of giant root-rats, combined with the relatively large dimensions of their nest chambers and tunnels, and the absence of heavy soils, may contribute to harmless atmospheres within their burrow systems.

The release of greenhouse gases from millipede digestive tracts warrants study because of its potential effect on climate change and also as an indicator of microbial processes that transform organic matter during passage through the gut of these animals. Gas chromatography was used to quantify the release of methane (CH4), carbon dioxide (CO2), and nitrous oxide (N2O) from living millipedes in laboratory conditions. The effect of four food types (leaf litter of alder, oak, and maple, and rotten wood) on the release of CH4, CO2, and N2O by 12 species was also assessed. In addition, two julid species were fed pure cellulose to test the ability of these millipedes to obtain energy from cellulose and to determine the effect of this diet on gas production. All of the tested millipede species produced CO2 and some produced CH4. Stable and substantial CH4 emission was restricted to the large millipedes in the tropical orders Spirobolida and Spirostreptida. This asymmetrical phylogenetic distribution of CH4 production may be related to body size and the presence of gut commensals, but these factors may influence each other and depend upon geographic distribution of species. The quality of the food and feeding regime can also affect CH4 production in that CH4 release was significantly higher when millipedes were fed alder leaf litter rather than oak or maple leaf litter. CO2 production from millipedes mainly reflected the metabolic response of the animals. Traces of N2O were only occasionally emitted by millipedes; this release evidently depends on the N content in the food and seems to be restricted to members of the Glomeridae family. Based on gas production, the tested species of millipedes were unable to obtain their energy needs from a diet of pure cellulose.

Estimating future fluxes of CH4 between land and atmosphere requires well-conceived process-based biogeochemical models. Current models do not represent the anaerobic oxidation of methane (AOM) in land surface soils, in spite of increasing evidence that this process is widespread. Our objective was to determine whether AOM, or potential AOM, commonly occurs in 20 hydromorphic soils spanning a wide range of chemical properties. Bulk soil samples were collected under shallow water near the shoreline of 15 recently drained fish ponds in southern Bohemia (Czech Republic), as well as from below the water table at 3 peatland locations in northeast Scotland and 2 acid sulfate soils on the southern coast of Finland. Each soil slurry was incubated under both oxic and anoxic conditions, with or without the addition of alternative electron acceptors (SO42− and NO3−) or H2PO4−. Here, “oxic” and “anoxic” conditions refer to anoxic soil respectively incubated in a headspace containing air or argon. Using the isotope dilution method, we determined the gross production and oxidation rates of CH4 after 2 days incubation under oxic headspace conditions, and after 2, 21 and 60 days incubation under anoxic conditions. Large differences in net CH4 fluxes were observed between soil types and between incubation conditions. AOM was detected in each of the 20 bulk soil samples, which spanned >6 pH units and 2 orders of magnitude in organic C content. Significant positive relationships were found between AOM and gross CH4 production rates under anoxic conditions, resulting in AOM rates that were sometimes higher than CH4 oxidation rates under oxic headspace conditions. There was no relationship between net and gross CH4 production rates, such that 2 soil types could display similar low net rates, yet conceal very large differences in gross rates. The effects of alternative electron acceptors on AOM were idiosyncratic and resulted in no net trend. We did find, however, a negative effect of SO42− and H2PO4− on gross CH4 production rates under anoxic and oxic conditions respectively. Under oxic headspace conditions, CH4 oxidation was related to soil organic C content. Taken collectively, our results suggest that AOM, or potential AOM, is prevalent over a wide range of soil types, that AOM may contribute substantially to CH4 oxidation in soils, and that AOM in soils should be integrated to current process-based CH4 cycling models.

In areas used for cattle overwintering detrimental effects normally associated with grazing are intensified. Among the alterations observed, increases on the N availability and soil pH may highly influence structure of ammonia oxidizing microbes and thus influence nitrification pattern in soil. To evaluate this assumption, we assessed the abundance and diversity of ammonia oxidizing bacteria (AOB) and archaea (AOA) in three sites with different degrees of animal impact (severe, moderate or no impact) of an overwintering pasture by means of qPCR and T-RFLP of amoA genes. In areas where no animal impact could be identified AOA was dominating over AOB. However, AOB abundance increased as the degree of animal impact enhances, becoming most dominant in the severely impacted site. Interestingly, the diversity of AOB was the highest in the severely impacted area, where AOA diversity was the lowest. Obviously the pressure imposed by altered environmental conditions created by cattle husbandry lead to the selection of AOB and AOA populations, adapted to alkaline pH and higher ammonia concentration.

Relationships between CH4, CO2, and N2O emissions were studied in soil that had been freshly amended with large deposits of cattle wastes. Dynamics of CH4, CO2, and N2O emissions were investigated with flux chambers from early April to late June 2011, during the 3 months following cattle overwintering at the site. This 81-day field study was supplemented with soil analyses of available C and N content and measurement of denitrification activity. In a more detailed field investigation, the daily time course of emissions was determined. The field research was complemented with a laboratory experiment that focused on the short-term time course of N2O and CH4 production in artificially created anoxic soil microsites. The following hypotheses were tested: (i) a large input of C (and N and other nutrients) in cattle manure creates conditions suitable for methanogenesis, and therefore overwintering areas can produce large amounts of CH4; (ii) N2O is produced and emitted until the level of mineral N decreases, while the level of CH4 production is low; and (iii) production of CH4 is greater when N immobilization decreases the level of NO3− in soil. N2O emissions were relatively large during the first 3 weeks, then peaked (at ca. 4000 μg N2O N m−2 h−1) and soon decreased to almost zero; the changes were related to the mineral and soluble organic N content in soil. CH4 fluxes were large, though variable, in the first 2 months (600–3000 μg CH4 C m−2 h−1) and were independent of C and N availability. Although time courses differed for CH4 and N2O, a negative relationship between N2O and CH4 emissions was not detected. Contrary to CH4 and N2O fluxes, CO2 emissions progressively increased to ca. 300 mg CO2 C m−2 h−1 at the end of the field study and were closely related to air and soil temperatures. Diurnal measurements revealed significant correlations between temperature and emissions of CH4, N2O, and CO2. Addition of C to soil during anaerobic incubation increased the production and consumption of N2O and supported the emission of CH4. The results suggest that rapid denitrification significantly contributes to the exhaustion of oxidizing agents and helps create microsites supporting methanogenesis in otherwise N2O-producing upland soil. The results also indicate that accurate estimate of gas fluxes in animal-impacted grassland areas requires assessment of both diurnal and long-term changes in CH4, CO2, and N2O emissions.

Summary The well-developed biological soil crusts cover up to 40% of the soil surface in the alpine and subnival zones of the Tibetan Plateau, accounting for a vast area of Asia. We investigated the diversity and biomass of the phototrophic part (Cyanobacteria) of the microbial community inhabiting biological soil crusts and uncrusted soils in their surroundings on the elevation gradient of 5200-5900 m a.s.l. The influence of soil physico-chemical properties on phototrophs was studied. The ability of high-altitude phototrophs to fix molecular nitrogen was also determined under labora- tory conditions. The biological soil crust phototroph community did not differ from that living in uncrusted soil in terms of the species composition, but the biomass is three-to-five times higher. An increasing trend in the cyanobacterial biomass from the biologi- cal soil crusts with elevation was observed, with the genera Nostoc spp., Microcoleus vaginatus and Phor- midium spp. contributing to this increase. Based on the laboratory experiments, the highest nitrogenase activity was recorded in the middle elevations, and the rate of nitrogen fixation was not correlated with the cyanobacterial biomass.

Microscopic soil fungi isolated from arable, grassland and forest soils have been suggested as producers of nitrous oxide (N(2)O). The aim of this work was to screen the capabilities for N(2)O production of microscopic fungi originating in the pasture soils of a cattle overwintering area with three levels of cattle impact intensity. In total, 36 fungal species from 11 genera were isolated during a 2-year study, and production of N(2)O under laboratory conditions was confirmed in 23 species (64%). Species belonging to the genera Fusarium, Penicillium, Monographella, Acremonium, Gibberella, Eurotium, and Pseudallescheria were found to be the most potent N(2)O-producers. Different N(2)O production patterns and wide variations in production rates, ranging from 1 to 150 μg N(2)O-Nd(-1), were observed, resulting in the transformation of 0.2-18.4% of the initial NO(2)(-)-N present in the cultivation medium. The data revealed distinct soil fungal communities in the different sections of the cattle overwintering area, and indicate a significant effect of cattle overwintering on the composition of soil fungal consortia. These observations confirm the importance of soil fungi in total N(2)O fluxes from grazed grassland ecosystems.

In order to simulate the warming effects on Arctic wetlands, three passive open-top chambers (OTCs) and three control cage-like structures (CCSs) equipped with soil temperature and soil volumetric water content (VWC) probes for continuous micro- climatic measurements were installed in a wet hummock meadow, Petuniabukta, Bille- fjorden, central Spitsbergen, in 2009. The warming effects on primary productivity were investigated during summer seasons 2009 and 2010 in cyanobacterial colonies of Nostoccommune s.l., which plays an important role in the local carbon and nitrogen cycles. The microclimatic data indicated that the effect of OTCs was dependent on microtopography. During winter, two short-term snow-thaw episodes occurred, so that liquid water was available for the Nostoc communities. Because of the warming, the OTC hummock bases remained unfrozen three weeks longer in comparison to the CCSs and, in spring, the OTC hummock tops and bases exceeded 0°C several days earlier than CCS ones. Mean summer temperature differences were 1.6°C in OTC and CCS hummock tops, and 0.3°C in the OTC and CCS hummock bases. The hummock tops were drier than their bases; however the VWC difference between theOTCs and CCSs was small. Due to the onlyminor differences in the microclimate of OTC and CCS hummock bases, where the Nostoc colonies were located, no differences in ecophysiological characteristics of Nostoc colonies expressed as photochemistry parameters and nitrogenase activities were detected after two years exposition. Long-term monitoring of Nostoc ecophysiology in a manipulated environment is necessary for understanding their development under climate warming.

The effects of earthworms on soil CH4 emissions remain ambiguous, as previous studies revealed both positive and negative effects on net rates of CH4 production. These differences may be linked to land-use history such as pasturing intensity, as the treading of livestock and the input of faeces will affect physical and chemical soil characteristics that are important to methanogenic and methanotrophic bacteria. In order to concomitantly measure the effects of earthworms on the activities of both of these bacterial groups, we performed an isotope dilution assay to measure gross CH4 production and consumption rates. Samples from three soil types, differing mainly in pasture intensity over the past 15 years, were incubated for 8 weeks in the presence or absence of the vermicomposting species Eisenia andrei. Following the incubation, earthworms had a positive effect on gross process rates in the non-pasture control soil, had no effect in the moderate pasture soil, and had a negative effect in the intensive pasture soil. Phospholipid fatty-acid profiles and chemical characteristics of the fresh soil samples suggested that the non-pasture soil would likely produce less CH4 than the control soil. Likewise, methanogenic specific mcrA gene abundance following the incubation was two orders of magnitude higher in the two pasture soils than in the control soil. The isotope dilution assay revealed, however, higher gross CH4 production rates in the control soil. It was also found that gross CH4 transformation rates were neither correlated to net production rates nor to mcrA gene abundance. We discuss the value and limitations of the isotope dilution assay for understanding the factors and underlying mechanisms governing net CH4 emissions rates from soils.

The aim of the study was to evaluate the impact of cattle overwintering husbandry on composition of upland grassland soil archaeal, bacterial, and fungal communities by comparative fingerprinting (SSU rRNA denaturing gradient gel electrophoresis (DGGE)) of total (tDNA) and extracellular DNA (eDNA) extracted from three differently impacted soils (severely, moderately, and non-impacted soil) and cattle excrements. Cattle excrements carried a significant amount of viable microorganisms and eDNA, and as a result of its high external returns, the amounts of extractable eDNA in soil increased with cattle impact, being positively correlated with soil microbial biomass and activity. The soil eDNA fraction (2.8 to 5.7 μg g−1 dw) significantly contributed to the soil metagenome, representing 18–31 % of soil tDNA. The largest shift in soil community structure was observed for Archaea, followed by fungi and bacteria, indicating that soil bacteria possess the highest resilience to cattle-induced changes. Cattle excrements showed more diverse bacterial than archaeal and fungal communities. The specific DGGE bands of cattle excrements were also observed in community profiles of cattle-impacted soils, confirming the effect of cattle husbandry on composition of soil microbial communities, probably as a result of introduced rumen-borne microbes. Similar changes were also reflected in the eDNA-derived DGGE profiles, suggesting a higher contribution of bacteria to soil extracellular metagenome than Archaea and fungi. This study provided first evidences about the extracellular mobilome in cattle-impacted soils, carrying also genetic information of Archaea, and its susceptibility to impact by outdoor cattle husbandry.

Cattle treading accompanied by a high input of organic matter was previously found to favour methanogenesis in soils at a site used as winter pasture in outdoor cattle husbandry. In this current study, the phylogenetic microarray AnaeroChip revealed high methanogenic diversity in a cattle-impacted soil with predominance of Methanosarcina, and presence of Methanoculleus, Methanobacterium, Methanocalculus, Methanobrevibacter, Methanosaeta, Methanothermobacter, Methanogenium, Methanohalobium, and Methanolobus. The bioturbation effects of an epigeic earthworm, Eisenia andrei, on the methanogenic microbial community in cattle-impacted soil were studied in a 6-month laboratory microcosm experiment. The microarray showed that the methanogenic community was changed by addition of earthworms to the soil. The abundance of 16S rRNA Methanosarcina gene copies decreased two fold in soil with worms compared to soil without worms after 2 months of incubation and decreased three fold after 4 and 6 months of incubation. The biomass of anaerobic microorganisms, as determined by unsubstituted non-ester-linked phospholipid fatty acid analysis, decreased in soil incubated for 4 and 6 months with worms. The abundance of the methyl-coenzyme M reductase (mcrA) gene, which is involved in CH4 production and is present in all methanogens, was not, however, changed by worms, and addition of worms even increased the rate of methane production. This study provides the first data concerning interactions between earthworms and methanogens in cattle-impacted pasture soil. The results of this laboratory microcosm experiment indicate that E. andrei changes the composition of the soil methanogenic community but does not reduce the total abundance of the methanogenic community and methane production rate.

The short-term effects of excessive NH 4 + -N on selected characteristics of soil unaffected (low annual N inputs) and affected (high annual N inputs) by cattle were investigated under laboratory conditions. The major hypothesis tested was that above a theoretical upper limit of NH 4 + concentration, an excess of NH 4 + -N does not further increase NO 3 − formation rate in the soil, but only supports accumulation of NO 2 − -N and gaseous losses of N as N2O. Soils were amended with 10 to 500 μg NH 4 + -N g−1 soil. In both soils, addition of NH 4 + -N increased production of NO 3 − -N until some limit. This limit was higher in cattle-affected soil than in unaffected soil. Production of N2O increased in the whole range of amendments in both soils. At the highest level of NH 4 + -N addition, NO 2 − -N accumulated in cattle-affected soil while NO 3 − -N production decreased in cattle-unaffected soil. Despite being statistically significant, observed effects of high NH 4 + -N addition were relatively weak. Uptake of mineral N, stimulated by glucose amendment, decreased the mineral N content in both soils, but it also greatly increased production of N2O.

The effects of pH on denitrifying enzyme activity (DEA) and on the ratio of the denitrification products, N2O and N2, were determined in three pasture soils differing in cattle impact. The linkage between intrinsic differences in the denitrifying communities and pH effects on relative N2O production was also assessed. Soil pH values were adjusted just before DEA determination to obtain soil slurries with a range of pH values. The intrinsic differences in the denitrifier communities were assessed by measuring the kinetic constants of NO 3 – and N2O reductions. DEA for all three soils was highest at pH 8.4, regardless of native soil pH. Because DEA has typically been measured at native soil pH, our results suggest that DEA might have been underestimated in many previous studies. Further, relative N2O production at different pH values did not differ among the soils, even though the denitrifier communities differed in their intrinsic capability to reduce NO 3 – all the way to N2, suggesting that the ratio of denitrification products (N2O and N2) is pH-specific rather than soil-specific. This suggests that manipulations of soil pH will alter N2O fluxes from agricultural soils.

Soil pH can influence denitrification both proximally and distally. Proximal control by pH involves direct changes in denitrification reductase activity while distal control by pH involves changes in the denitrifier community, which is a key component affecting the denitrification rate. The current study separated the proximal and distal control by pH of the denitrification rate and of the relative proportion of two denitrification gas products (N2O and N2). The potential denitrifying enzyme activity (DEA) was measured in the presence or absence of acetylene in three pasture soils differing in pH management in the field. The pH of these soils was further manipulated just before DEA measurement to determine the effect of short-term changes in pH. DEA was driven by the pH management in the field rather than by current pH resulting from short-term changes in pH. However, the N2O/(N2O + N2) ratio was driven by the effects of the current pH value on the kinetics of N2O production and reduction. The data suggest that even if the pH-induced changes in the structure of denitrifying community can control the absolute denitrification rate (distal control by pH), the community does not influence the proportion of denitrification products, which is regulated solely by the proximal control by pH.

Overwintering cattle outdoors causes soil surface disturbance, substantial increases of soil Ntot, Corg, and P and a shift in pH to alkaline levels. Since fungi predominate in unfertilized soils with acidic pH and have filamentous hyphae, we hypothesized that changes caused by overwintering cattle outdoors (trampling, excreta returns, and changes in soil chemistry) will lead to suppressed species richness, lower biomass, and alter the structure of fungal communities. The research was conducted on an upland pasture used more than 10 years for cattle overwintering. Both culture-dependent and -independent methods were used for the determination of either fungal species composition (cultivation; DGGE) or biomass (numbers of CFU; concentration of fungal PLFA marker 18:2ω6,9). Soils under three different levels of cattle disturbance (S – severe, M – moderate, C – no disturbance/control) were investigated during three subsequent years. In addition, the DGGE analysis of soils was completed by comparison with analysis of fresh cattle excrements (Ex). The composition of fungal communities showed significantly higher richness and a substantial shift in species composition in cattle-disturbed soils (S, M) in comparison to the non-disturbed soil (C). The number of separated DGGE bands was significantly higher in S (30.67 ± 1.63; mean ± SD) and M (25.50 ± 1.64) soils than in the C soil (19.33 ± 1.75). Sequencing of typical bands revealed common fungal genera – Alternaria, Penicillium, Fusarium, Rhizopus, Isaria, and Metarhizium. Profiles of the S soil were enriched by bands of rumen-born anaerobic fungi (Neocallimastix, Cyllamyces) occurring mainly in profiles of excrements, where relatively low band richness (14.33 ± 1.15) was observed. The increasing level of cattle disturbance induced an increase in the biomass of complex fungal community over the three-year experimental period from 3.39 ± 2.11 (mean ± SD) nmol of fungal PLFA per gram of the C soil to 5.87 ± 3.16 in the M soil and 9.21 ± 4.69 in the S soil. Concentrations of soil Ntot and Corg were evaluated as the parameters significantly correlating with biomass as well as composition of the fungal community.

The largest areas of acid sulphate (AS) soils in Europe are located in Finland, where 67,000–130,000 ha of AS soils are in agricultural use. In addition to their acidifying effects on waters, AS soils might be a significant source of greenhouse gases. In this pilot research, carbon and nitrogen content and microbial activity were studied in an AS and a non-AS soil. Large carbon and nitrogen stocks (110 Mg Corg ha−1 and 15 Mg Ntot ha−1) as well as high substrate induced respiration (33 μg CO2–C g−1h−1) were found in the C horizons of the AS soil but not in the non-AS soil. High microbial activity in these horizons of the AS soil was further confirmed by the measurement of dehydrogenase activity, basal respiration, the numbers of culturable bacterial cells, and the ratio of culturable to total numbers of cells. Still, the denitrifying enzyme activity was very low in the anaerobic horizons of the AS soil, indicating the prevalence of microbes other than denitrifiers. We suspect that the microbial community originated with the genesis of AS soil and has been supported by the large stocks of accumulated carbon and mineral nitrogen in the C horizons. If these permanently water-saturated subsoils are exposed to oxygen and their microbial activity consequently increases, large carbon and nitrogen stocks are likely to be mobilised, resulting in increased emission of greenhouse gases. Additional studies of boreal AS soils are needed to assess their potential contribution to increases in greenhouse gas fluxes at the local, regional, and global scales.

Short-term diurnal changes in emissions of CO2 and N2O were determined in a cattle overwintering area during three specific periods of the year. Production of both N2O and CO2, as determined with gas chambers buried in soil and spatially distributed, changed rapidly. The general course of fluxes of the two gases was different. CO2 emissions were basically controlled by temperature. Most gas chambers showed the same trends in CO2 flux, indicating low spatial heterogeneity. In contrast, N2O emissions were much more spatially heterogeneous and each chamber had its own time course of emission; therefore, the relationship between flux and temperature was more complicated for N2O than CO2. For estimating gas emissions over long periods, we strongly recommend the use of frequent emission measurements during periods of high gas fluxes.

The potential for N(2) fixation by heterocystous cyanobacteria isolated from soils of different geographical areas was determined as nitrogenase activity (NA) using the acetylene reduction assay. Morphology of cyanobacteria had the largest influence on NA determined under light conditions. NA was generally higher in species lacking thick slime sheaths. The highest value (1446 nmol/h C(2)H(4) per g fresh biomass) was found in the strain of branched cyanobacterium Hassalia (A Has1) from the polar region. A quadratic relationship between NA and biomass was detected in the Tolypothrix group under light conditions. The decline of NA in dark relative to light conditions ranged from 37 to 100 % and differed among strains from distinct geographical areas. Unlike the NA of temperate and tropical strains, whose decline in dark relative to light was 24 and 17 %, respectively, the NA of polar strains declined to 1 % in the dark. This difference was explained by adaptation to different light conditions in temperate, tropical, and polar habitats. NA was not related to the frequency of heterocysts in strains of the colony-forming cyanobacterium Nostoc. Colony morphology and life cycle are therefore more important for NA then heterocyst frequency. NA values probably reflect the environmental conditions where the cyanobacterium was isolated and the physiological and morphological state of the strain.

The effects of Aporrectodea caliginosa earthworms on both carbon dioxide (CO2) accumulation in and emissions from soil, as well as the simultaneous impact of earthworms on soil microbiological properties were investigated in a microcosm experiment carried out over 5.5 months. Concentration of CO2 in soil air was greater at a depth of 15 cm when compared with a depth of 5 cm, but varied during the season both in control and earthworm-inhabited chambers. Peaks of CO2 concentrations at both depths occurred in both treatments during August, approximately 80 days after the experiment started. Generally, the presence of earthworms increased the CO2 concentration at 15-cm depth. Larger CO2 emissions were consistently recorded in conjunction with higher amounts of CO2 in soil air when chambers were inhabited by earthworms. The total CO2 emissions during the experimental period covering 161 days were estimated at 118 g CO2-C m−2 and 99 g CO2-C m−2 from chambers with and without earthworms respectively. Moreover, the presence of earthworms increased microbial biomass in the centre and at the bottom of chambers, and enhanced both dehydrogenase activity and nitrifying enzyme activity in the soils. We suggest that the effect of earthworms on both the enhanced soil accumulation of CO2 as well as emissions of CO2 was mostly indirect, due to the impacts of earthworms on soil microbial community.

We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny, Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE fingerprinting, based on cluster analysis of soil bacterial and fungal community fingerprints, was made in terms of the resulting information about microbial community structures and their response to different degrees of cattle impact. Although the comparative DGGE analysis with different DGGE systems provided similar clustering of microbial community structures in correlation with the degree of cattle impact, our results suggest the DGGE system to be a factor influencing DGGE analysis. To our knowledge this is the first attempt to investigate the hypothetical impact of the DGGE system due to different technical characteristics, recommending the use of one and the same DGGE apparatus throughout an experiment, if the monitoring of microbial community structures requires multiple gel-to-gel analysis.

The potential for methane production was investigated in 47 species of soil invertebrates. No detectable methane production was found in slugs, earthworms, potworms, oribatid mites, woodlices, springtails, centipedes, ants and soil diptera larvae. However, significant methane production was found in several temperate millipedes including some species from the order Julida ( Leptoiulus trilobatus , Megaphyllum projectum , Megaphyllum unilineatum , Unciger transsilvanicus , Unciger foetidus , Leptoiulus proximus and Julus scandinavius ). On the other hand, methane production was not confirmed in Cylindroiulus boleti which belongs to the same family. Members of the other orders (Glomerida and Polydesmida) did not release methane with the exception of one questionable recorded release in Polydesmus complanatus (Polydesmida, Polydesmidae). Methane producing species of millipedes showed significantly lower weight-specific rates of methane release than the cockroach Periplaneta americana and termite Prorhinotermes simplex . Methane release from millipedes was temperature-dependent; for the temperature range 5–25 °C, a Q 10 between 1.55 and 2.17 was calculated and the relationship between methane release and temperature was described by an exponential curve. Our results suggest that although overall methane fluxes from soil invertebrates under study cannot substantially influence a methane budget in most ecosystems, methane production is significant at least in some millipedes and therefore can impact mesoenvironments and microenvironments inhabited by these invertebrates. This work also confirms that methane production is not only supported by tropic soil invertebrates, but also by temperate species.

Pasture soils used for cattle overwintering may represent significant sources of N 2 O emissions from soils. Therefore, the long-term effect of cattle overwintering on the abundance and activity of a denitrifying community was explored. The study was performed at a cattle overwintering area in South Bohemia (Czech Republic), where three sites differing in the degree of animal impact were selected: severely impacted (SI) and moderately impacted (MI), as well as a control site with no impact (NI). N 2 O flux measurement and soil sampling were performed in spring and fall of 2005. The activity was measured in terms of potential denitrification activity. Bacterial nirK , nirS and nosZ genes were used as functional markers of the denitrifying communities; abundance was analyzed using a real-time PCR assay. Surprisingly, in situ N 2 O emissions were the highest in spring at MI and significantly differed from those at SI and NI, while in autumn, rates of emissions generally decreased. In contrast potential denitrification rates were highest at SI, followed by MI, and the lowest at NI. An overall significant shift in N 2 O/N 2 molar ratio was shown in cattle impacted sites. The highest abundance of all genes measured at both sampling times was found at site SI, whereas at site MI increased numbers were observed only in spring. Our results indicate a strong influence of cattle on the abundance as well as the activity of microbes involved in denitrification.

International audience; There is ample evidence that microbial processes can exhibit large variations in activity on a field scale. However, very little is known about the spatial distribution of the microbial communities mediating these processes. Here we used geostatistical modelling to explore spatial patterns of size and activity of the denitrifying community, a functional guild involved in N-cycling, in a grassland field subjected to different cattle grazing regimes. We observed a non-random distribution pattern of the size of the denitrifier community estimated by quantification of the denitrification genes copy numbers with a macro-scale spatial dependence (6–16 m) and mapped the distribution of this functional guild in the field. The spatial patterns of soil properties, which were strongly affected by presence of cattle, imposed significant control on potential denitrification activity, potential N2O production and relative abundance of some denitrification genes but not on the size of the denitrifier community. Absolute abundance of most denitrification genes was not correlated with the distribution patterns of potential denitrification activity or potential N2O production. However, the relative abundance of bacteria possessing the nosZ gene encoding the N2O reductase in the total bacterial community was a strong predictor of the N2O/(N2 + N2O) ratio, which provides evidence for a relationship between bacterial community composition based on the relative abundance of denitrifiers in the total bacterial community and ecosystem processes. More generally, the presented geostatistical approach allows integrated mapping of microbial communities, and hence can facilitate our understanding of relationships between the ecology of microbial communities and microbial processes along environmental gradients.

Temporal changes of microbial respiration of leaf litter during gut passage by two species of bibionid flies (Penthetria holosericae and Bibio marci) and immediately after defecation were studied as well as the effect of B. marci feeding on microbial respiration in the remaining consumed leaf litter. Respiration in the guts of both species was lower than respiration of the original leaves. Microbial respiration increased after defecation, but respiration was higher in comparison with the original litter only for B. marci. Later, microbial respiration in both species decreased dramatically over several hours. Respiration of the remains of consumed leaves, from which the excrements were removed, was significantly higher than the respiration of non-consumed leaves. Scanning electron microscopy indicated that plant cells in the unconsumed part of the litter were massively broken-up by feeding activity and potentially exposed to microbial colonisation from the surrounding soil and microflora coming from the excrements deposited on the consumed litter. Long term (11 months) exposure of leaf litter and excrements produced from this litter showed that the decomposition of excrements was significantly slower than that of leaves. These findings indicate that bibionid feeding activity causes a short term increase in microbial mineralization of litter, but slows it down in the long term.

Rates and patterns of nitrogen transformation differ in divergently managed pasture soils. In pastures with low nutrient inputs, N is utilized efficiently and it is assimilated by plants and soil microorganisms for synthesis of biomass. In more intensive pastures, characterized with higher N inputs, significant amounts of N can be lost from the ecosystem in various forms. Two soils of a cattle overwintering area with different levels of cattle disturbance were supplied with a solution of KNO3 in various levels corresponding in range to 0–500 kg N ha−1. Emissions of N2O were measured during 24 h after a NO3−-N application. We hypothesized that under a low disturbance small additions of up to 5 kg NO3−-N are used by plants and soil microbes without an increase in N2O emissions, while a pasture adapted to a moderate disturbance will increase N2O emissions. Results showed that in both soils, the addition of N always increased N2O emissions, while emissions were more pronounced in soil at the location with a higher intensity of cattle traffic. Contrary to our hypothesis, however, NO3––N was not fully metabolized in the soil with low disturbance by the cattle. Probable explanations of such a result were lower intensity of N transformations in this soil and low utilisation of N by grass. Our results suggest that under certain conditions relatively low nitrate-N inputs can also stimulate N2O fluxes from soils.

Cattle overwintering areas common in central Europe may represent significant point sources of the important greenhouse gases, nitrous oxide (N 2 O) and carbon dioxide (CO 2 ). A 2-year field study was carried out in order to estimate the emissions of N 2 O and CO 2 from soil in a cattle overwintering area located in the southwest of the Czech Republic. The measurements were performed at three sampling locations along a gradient of animal impact (severe, moderate, slight) to test the hypothesis that emissions of CO 2 and N 2 O are positively related to the degree of impact. In addition to CO 2 and N 2 O fluxes determined by using non-vented manual closed chambers, soil mineral nitrogen (NH 4 + and NO 3 − ), pH and temperature were determined to assess their regulatory role and impact on gas fluxes. The overwintering area was about 4 ha and it had been used for overwintering of about 90 cows since 1995. Deposition of animal excreta resulted in a significant accumulation of nitrogen in the soil during winter, but most of the N 2 O was emitted during a few short periods in spring and/or in late autumn. Maximum N 2 O fluxes of up to 2.5 mg N 2 O-N m −2 h −1 were recorded at the most impacted location near the animal house, where the highest concentrations of soil mineral nitrogen also occurred. The emissions of CO 2 showed a completely different pattern to those of N 2 O, being correlated with soil temperature; the highest emissions thus occurred in June–July, while very low fluxes were found in winter. Emission values ranged from about 0 to 700 mg C-CO 2 m −2 h −1 . Furthermore, the effect of animal impact on CO 2 emissions was opposite to that on N 2 O fluxes, as the highest CO 2 fluxes were mostly recorded at the least impacted location, where respiration of plants most likely increased overall CO 2 production. The results show that cattle overwintering areas are important sources of greenhouse gases, including N 2 O and CO 2 . Fluxes of these two gases are, however, differently distributed over the year, which also suggests that they are controlled by different environmental and soil factors.

There is little information concerning N2O fluxes in the pasture soil that has received large amounts of nutrients, such as urine and dung, for several years. The aims of this study were to (1) experimentally quantify the relationship between mineral N input and N2O emissions from denitrification, (2) describe the time course of N2O fluxes resulting in N inputs, and (3) find whether there exists an upper limit of the amount of nitrogen escaping the soil in the form of N2O. The study site was a grassland used as a cattle overwintering area. It was amended with KNO3 and glucose corresponding to 10–1,500 kg N and C per hectare, covering the range of nutrient inputs occurring in real field conditions. Using manual permanent chambers, N2O fluxes from the soil were monitored for several days after the amendments. The peak N2O emissions were up to 94 mg N2O–N m−2 h−1, 5–8 h after amendment. No upper limit of N2O emissions was detected as the emissions were directly related to the dose of nutrients in the whole range of amendments used, but the fluxes reflected the soil and environmental conditions, too. Thus, in three different experiments performed during the season, the total cumulative losses of N2O–N ranged from 0.2 to 5.6% of the applied 500kg % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX!% MathType!MTEF!2!1!+-% feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaciOtaiGac+% eadaWgaaWcbaGaaG4maaqabaGcdaahaaWcbeqaaiabgkHiTaaakiab% gkHiTiGac6eaaaa!3B6A! $$\operatorname{N} \operatorname{O} _{3} ^{ - } - \operatorname{N} $$ ha−1. Splitting of high nutrient doses lowered the rate of N2O fluxes after the first amendment, but the effect of splitting on the total amount of N2O–N released from the soil was insignificant, as the initial lower values of emissions in the split variants were compensated for by a longer duration of gas fluxes. The results suggest that the cattle-impacted soil has the potential to metabolize large inputs of mineral nitrogen over short periods (∼days). Also, the emission factors for % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn % hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr % 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9 % vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x % fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaciOtaiGac+ % eadaWgaaWcbaGaaG4maaqabaGcdaahaaWcbeqaaiabgkHiTaaakiaa % ykW7cqGHsislciGGobaaaa!3CF5! $$ \operatorname{N} \operatorname{O} _{3} ^{ - } \, - \operatorname{N} $$ did not exceed values reported in literature.

The grassland area where cattle overwinter was identified as an important point source of N2O due to soil compaction and excrement deposition. The rates of N2O emission were mostly directly related to the intensity of animal impact. However, laboratory measurements had shown a much greater potential for N2O production from soil in a severely impacted site than indicated by field measurements, possibly due to factors affecting the mole fraction of N2O. The 15N gas-flux method was used to measure the emissions of N2O and N2 at three sites along a gradient of animal impact. Over the experimental period (72 h), the loss of NO3−-N as N gases was 60%, 12% and 3%, and the mole fraction of N2O was 0.04, 0.15 and 0.75 for the severe, moderate and control treatments, respectively. We hypothesize that soil pH, which is enhanced in impacted sites (from 5.7 up to 7.8), may control mole fraction of N2O in such a way that under alkaline conditions most of the nitrogen escapes as N2.

The study compared the effects of mowing, mulching, and no-treatment, applied to a mountain grassland over five years, on microbial transformation of soil organic matter (SOM). Microbial biomass, microbial respiration, cellulose decomposition and mineralization were measured in the laboratory eight times during the three-year experiment. In addition, soil phosphatase activity and factors limiting microbial growth were assessed once to complete the results. Mowing increased soil microbial biomass and carbon use efficiency, which supported carbon sequestration in soil. In contrast, mulching led to a decrease in microbial biomass and microbial metabolic efficiency due to the limitation of easily decomposable carbon. This was a consequence of changes in temperature and light conditions under the mulch layer, which suppressed plant growth and rhizodeposition. Processes causing organic matter transformation in the mulched grassland were similar to those of the untreated grassland. Annual mowing appears to be most suitable for maintenance of SOM content and sustainability of montane grasslands.

The effect of soil aeration status on carbon partitioning of a labelled organic substrate (14C-[U]-glucose) into CO2, microbial biomass, and extra-cellular metabolites is described. The soil was incubated in a continuous flow incubation apparatus under four different aeration conditions: (1) permanently aerobic, (2) permanently anaerobic, (3) shifted from anaerobic to aerobic, and (4) shifted from aerobic to anaerobic. The soil was pre-incubated for 10 days either under aerobic or under anaerobic conditions. Afterwards, glucose was added (315 μg C g−1) and the soils were incubated for 72 h according to four treatments: aerobic or anaerobic conditions maintained, aerobic conditions shifted to anaerobic conditions and anaerobic conditions shifted to aerobic conditions. Carbon partitioning was measured 0, 8, 16, 24, 48 and 72 h after the glucose addition. In permanently aerobic conditions, the largest part of the consumed glucose was built into microbial biomass (72%), much less was mineralised to CO2 (27%), and only a negligible portion was transformed to soluble extra-cellular metabolites. Microbial metabolism was strongly inhibited when aeration conditions were changed from aerobic to anaerobic, with only about 35% of the added glucose consumed during the incubation. The consumed glucose was transformed proportionally to microbial biomass and CO2. In permanently anaerobic conditions, 42% of the consumed glucose was transformed into microbial biomass, 30% to CO2, and 28% to extra-cellular metabolites. After a shift of anaerobic to aerobic conditions, microbial metabolism was not suppressed and the consumed glucose was transformed mainly to microbial biomass (75%) and CO2 (23%). Concomitant mineralisation of soil organic carbon was always lower in anaerobic than in aerobic conditions.

The relationships between the fluxes of nitrous oxide (N 2 O) and carbon dioxide (CO 2 ), and their concentrations in the soil air, three different measures of potential denitrification, soil moisture, soil temperature and precipitation were investigated in soils from beneath ryegrass ( Lolium multiflorum Lam.), red clover ( Trifolium pratense L.) and mixture of ryegrass–red clover stands on a gleic cambisol. Investigations were carried out in order to test the hypothesis that the measure(s) of potential denitrification are good predictor(s) of N 2 O fluxes and thus may be used in empirical models of N 2 O emission. Potential denitrification characteristics used in this study involved (i) short-term denitrifying enzyme activity (DEA), (ii) long-term denitrification potential (DP), both determined in soils amended with nitrate and glucose, and (iii) denitrification rate (DR) measured using intact soil cores. Flux measurements were made using cylindrical chambers (internal diameter 31 cm, volume 0.015 m 3 ). The fluxes of N 2 O and CO 2 and many other characteristics showed large spatial and temporal variability. Emissions of N 2 O from the grass plots were closely related to N 2 O concentrations in the soil atmosphere at 22.5 cm depth. Most soil properties did not correlate with N 2 O fluxes. It was concluded that DP was not a good predictor for N 2 O flux. DEA did not show significant relationship with N 2 O flux, but it is suggested that if determined in representative, large soil samples, DEA could be a predictor of N 2 O fluxes; this assumption needs, however, verification. The only potential denitrification characteristic which was significantly related to N 2 O emission both in grass and clover treatments was DR, which was determined in soil cores.

Soil microbial properties were studied from localities on a transect along the Yenisei River, Central Siberia. The 1000 km-long transect, from 56°N to 68°N, passed through tundra, taiga and pine forest characteristic of Northern Russia. Soil microbial properties were characterized by dehydrogenase activity, microbial biomass, composition of microbial community (PLFAs), respiration rates, denitrification and N mineralization rates. Relationships between vegetation, latitude, soil quality (pH, texture), soil organic carbon (SOC) and the microbial properties were examined using multivariate analysis. In addition, the temperature responses of microbial growth (net growth rate) and activity (soil respiration rate) were tested by laboratory experiments. The major conclusions of the study are as follows: 1. Multivariate analysis of the data revealed significant differences in microbial activity. SOC clay content was positively related to clay content. Soil texture and SOC exhibited the dominant effect on soil microbial parameters, while the vegetation and climatic effects (expressed as a function of latitude) were weaker but still significant. The effect of vegetation cover is linked to SOC quality, which can control soil microbial activity. 2. When compared to fine-textured soils, coarse-textured soils have (i) proportionally more SOC bound in microbial biomass, which might result in higher susceptibility of SOC transformation to fluctuation of environmental factors, and (ii) low mineralization potential, but with a substantial part of the consumed C being transformed to microbial products. 3. The soil microbial community from the northernmost study region located within the permafrost zone appears to be adapted to cold conditions. As a result, microbial net growth rate became negative when temperature rose above 5 °C and C mineralization then exceeded C accumulation. (Less)

Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse the production and consumption of important greenhouse gases. In this study, we investigate changes in archaeal and bacterial communities of four Czech grassland soils affected by outdoor cattle husbandry. Two show short-term (3 years; STI) and long-term impact (17 years; LTI), one is regenerating from cattle impact (REG) and a control is unaffected by cattle (CON). Cattle manure (CMN), the source of allochthonous microbes, was collected from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition of archaeal and bacterial communities in each soil type and CMN. Both short- and long- term cattle impact negatively altered archaeal and bacterial diversity, leading to increase of homogenization of microbial communities in overwintering soils over time. Moreover, strong shifts in the prokaryotic communities were observed in response to cattle overwintering, with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g. Thaumarchaeota, Acidobacteria, and α-Proteobacteria) dominated in CON and expressed strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria, and Bacteroidetes) were common in LTI showing opposite trends. Crenarchaeota were also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes, Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae suggested increased abundance of soil-borne microbes under altered environmental conditions. The observed changes in prokaryotic community composition may have driven corresponding changes in soil functioning.

Respiratory denitrification is controlled by a number of environmental and soil factors including pH, but some of the interactions between pH and denitrification in soil are still uncertain. The objective of the present study was to assess the relationship between natural soil pH and denitrifying enzyme activity (DEA), to investigate the impact of two different techniques used for determining of potential denitrification, namely short-term DEA and the long-term denitrification potential (DP), on resulting pH optimum, especially with respect to the length of measurement, and to elucidate the effect of soil pH on the nature of denitrification products (N2O, N2). For this purpose, five mineral soils similar in a texture but differing in pH, were investigated. In addition to examining the soils in their natural pH values, pHs were adjusted by amendment of sulphuric acid or sodium hydroxide solutions prior to DEA or DP determination. The study showed that there was no simple relationship between DEA and soil pH, as very high DEA was found both in acid and alkaline soil. The study also indicated that the populations of soil denitrifiers were adapted to prevailing natural soil pH in the sense that they exhibited the highest DEA at or near natural soil pH (in experiments where the pH was adjusted to values from about 4 to 11 before DEA determination). Suppression of nitrate reductase activity by introducing nitrite instead of nitrate as an electron acceptor had no effect on this interaction between the DEA and soil pH. If the soil incubation was prolonged under optimum conditions for denitrification (surplus of nitrate or nitrite and available carbon, very low partial pressure of O2) and DP was estimated, the optimum pH for evolution of denitrification products was shifted towards neutrality. It is proposed that this is either due to the development of a community of denitrifiers which can grow better at the neutral pH or due to accommodation of the existing populations to new conditions. The shift in pH optimum occurred after about 12 h, but it took about 1–2 days to fully appear. In agreement with previous studies, it was confirmed that at pH above 7, N2 is much more important denitrification product than N2O. The study also suggests that the expressions like optimum pH for denitrification should be avoided, as different denitrification characteristics obtained using different methodology can be quite differently related to the soil reaction. Such general expression should be specified by introducing the particular denitrification parameters to which it is being applied.

Methane production by intestinal methanogenic Archaea and their community structure were compared among phylogenetic lineages of millipedes. Tropical and temperate millipedes of 35 species and 17 families were investigated. Species that emitted methane were mostly in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular phylogenetic distribution of methane production correlated with the presence of the methanogen-specific mcrA gene. The study brings the first detailed survey of methanogens' diversity in the digestive tract of millipedes. Sequences related to Methanosarcinales, Methanobacteriales, Methanomicrobiales and some unclassified Archaea were detected using molecular profiling (DGGE). The differences in substrate preferences of the main lineages of methanogenic Archaea found in different millipede orders indicate that the composition of methanogen communities may reflect the differences in available substrates for methanogenesis or the presence of symbiotic protozoa in the digestive tract. We conclude that differences in methane production in the millipede gut reflect differences in the activity and proliferation of intestinal methanogens rather than an absolute inability of some millipede taxa to host methanogens. This inference was supported by the general presence of methanogenic activity in millipede faecal pellets and the presence of the 16S rRNA gene of methanogens in all tested taxa in the two main groups of millipedes, the Helminthophora and the Pentazonia.

The biological health of soil is an important aspect of soil quality because of the many critical functions performed by organisms in soil. Various indicators of soil quality have been proposed, but measurements of microbial biomass are most commonly used. During decomposition of plant residues in soil the relative intensities of the O-alkyl-C signal decreases and the alkyl-C signal increases in nuclear magnetic resonance (NMR) spectra. This leads to the suggestion that the alkyl-C to O-alkyl-C ratio of a soil may indicate the degree of decomposition. Consequently, the overall resource quality of soil C as a substrate for heterotrophic microorganisms may be inversely related to the alkyl-C to O-alkyl-C ratio. Our hypothesis is that a relationship exists between the size of the soil microbial community (microbial biomass) and the quality of soil carbon as a resource for microorganisms. New data have been combined with previously published data to show that there was a significant, negative correlation between the biomass C to total C (Cmic, to Corg) ratio and the alkyl-C to O-alkyl-C ratio (p < 0.01), which supports our hypothesis.

The response of the microbial community to changes in aeration status, from oxic to anoxic and from anoxic to oxic, was determined in arable soil incubated in a continuous flow incubation apparatus. Soil incubated in permanently oxic (air) and/or anoxic (O2-free N2) conditions was used as the control. Before experiments soil was preincubated for 6 days, then aeration status was changed and glucose added. Glucose concentration, extractable C, CO2 production, microbial biomass, pH and redox potential were determined 0, 4, 8, 12, 16, 24, 36 and 48 h after change of aeration status. If oxic conditions were changed to anoxic, the amount of glucose consumed was reduced by about 60%, and CO2 production was 10 times lower at the end of incubation compared to the control (permanently oxic conditions). Microbial biomass increased by 114% in glucose-amended soil but did not change in unamended soil. C immobilization prevailed over C mineralization. Redox potential decreased from +627 mV to –306 mV. If anoxic conditions were changed to oxic, consumption of glucose and CO2 evolution significantly increased, compared to permanently anoxic conditions. Microbial biomass did not change in glucose-amended soil, but decreased by 78% in unamended soil. C mineralization was accelerated. Redox potential increased from +238 to +541 mV. The rate of glucose consumption was low in anoxic conditions if soil was incubated in pure N2 but increased significantly when incubation was carried out in a CO2/N2 mixture.

Microbial processes were investigated in the soil of a declining, more eutrophic (Rožmberk West) and a healthy looking, less eutrophic (Rožmberk East) freshwater reed stand. Soil was sampled monthly from June to September 1997. Glucose induced carbon dioxide (CO2) production in oxic and anoxic conditions, methane (CH4) production, nitrification and denitrification activities were measured in laboratory conditions in suspensions prepared from homogenised soil samples. Within a stand the proportion of anaerobic (as opposed to aerobic) microbial activity was greatest in June. Potential methanogenesis was highest in June and decreased later in both stands. Methane production was approximately the same in June at both stands but it was higher at Rožmberk East than at Rožmberk West stand in later months. Denitrifying activity was higher in August than July at both stands. Nitrifying activity was undetectable at both stands over the entire study period. Generally Rožmberk West was more anaerobic than Rožmberk East, with lower redox potential, higher amounts of oxygen-consuming organic matter and a lower ratio of CO2 production in oxic conditions to CO2 production in anoxic conditions. Microbial activity was apparently restricted at Rožmberk West stand in comparison to Rožmberk East. The shift from aerobic to anaerobic microbial metabolism and a coinciding restriction of metabolic activities at Rožmberk West are thought to be indicative of a strengthened oxygen stress in the soil, associated with accumulation of metabolites toxic to both the microorganisms and the reed. Possible links between eutrophication, microbial characteristics and reed performance are discussed.

The effect of increasing soil CO2 concentration was studied in six different soils. The soils were incubated in ambient air (0.05 vol.% CO2) or in air enriched with CO2 (up to 5.0 vol.% CO2). Carbon dioxide evolution, microbial biomass, growth or death rate quotients and glucose decay rate were measured at 6, 12 and 24 h of CO2 exposure. The decrease in soil respiration ranged from 7% to 78% and was followed by a decrease in microbial biomass by 10–60% in most cases. High CO2 treatments did not affect glucose decay rate but the portion of Cgluc mineralized to CO2 was lowered and a larger portion of Cgluc remained in soils. This carbon was not utilized by soil microorganisms.

Acid sulphate (AS) soils along the Baltic coasts contain significant amounts of organic carbon and nitrogen in their subsoils. The abundance, composition, and activity of microbial communities throughout the AS soil profile were analysed. The data from a drained AS soil were compared with those from a drained non-AS soil and a pristine wetland soil from the same region. Moreover, the potential production of methane, carbon dioxide, and nitrous oxide from the soils was determined under laboratory conditions. Direct microscopic counting, glucose-induced respiration (GIR), whole cell hybridisation, and extended phospholipid fatty acid (PLFA) analysis confirmed the presence of abundant microbial communities in the topsoil and also in the deepest Cg2 horizon of the AS soil. The patterns of microbial counts, biomass and activity in the profile of the AS soil and partly also in the non-AS soil therefore differed from the general tendency of gradual decreases in soil profiles. High respiration in the deepest Cg2 horizon of the AS soil (5.66 μg Cg(-1)h(-1), as compared to 2.71 μg Cg(-1)h(-1) in a top Ap horizon) is unusual but reasonable given the large amount of organic carbon in this horizon. Nitrous oxide production peaked in the BCgc horizon of the AS and in the BC horizon of the non-AS soil, but the peak value was ten-fold higher in the AS soil than in the non-AS soil (82.3 vs. 8.6 ng Ng(-1)d(-1)). The data suggest that boreal AS soils on the Baltic coast contain high microbial abundance and activity. This, together with the abundant carbon and total and mineral nitrogen in the deep layers of AS soils, may result in substantial gas production. Consequently, high GHG emissions could occur, for example, when the generally high water table is lowered because of arable farming.

While the importance of anaerobic methane oxidation has been reported for marine ecosystems, the role of this process in soils is still questionable. Grasslands used as pastures for cattle overwintering show an increase in anaerobic soil micro-sites caused by animal treading and excrement deposition. Therefore, anaerobic potential methane oxidation activity of severely impacted soil from a cattle winter pasture was investigated in an incubation experiment under anaerobic conditions using 13C-labelled methane. We were able to detect a high microbial activity utilizing CH4 as nutrient source shown by the respiration of 13CO2. Measurements of possible terminal electron acceptors for anaerobic oxidation of methane were carried out. Soil sulfate concentrations were too low to explain the oxidation of the amount of methane added, but enough nitrate and iron(III) were detected. However, only nitrate was consumed during the experiment. 13C-PLFA analyses clearly showed the utilization of CH4 as nutrient source mainly by organisms harbouring 16:1ω7 PLFAs. These lipids were also found as most 13C-enriched fatty acids by Raghoebarsing et al. (2006) after addition of 13CH4 to an enrichment culture coupling denitrification of nitrate to anaerobic oxidation of methane. This might be an indication for anaerobic oxidation of methane by relatives of "Candidatus Methylomirabilis oxyfera" in the investigated grassland soil under the conditions of the incubation experiment.