Potential Denitrification Activity Research Articles

Overwintering management on upland pasture causes shifts in an abundance of denitrifying microbial communities, their activity and N 2O-reducing ability

Pasture soils used for cattle overwintering may represent significant sources of N 2O 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 2O 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 2O 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 2O/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.

Influence of temperature and soil water content on bacterial, archaeal and denitrifying microbial communities in drained fen grassland soil microcosms

In this study, microcosms were used to investigate the influence of temperature (4 and 28 degrees C) and water content (45% and 90% WHC) on microbial communities and activities in carbon-rich fen soil. Bacterial, archaeal and denitrifier community composition was assessed during incubation of microcosms for 12 weeks using terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA and nitrous oxide reductase (nosZ) genes. In addition, microbial and denitrifier abundance, potential denitrification activity and production of greenhouse gases were measured. No detectable changes were observed in prokaryote or denitrifier abundance. In general, cumulatively after 12 weeks more carbon was respired at the higher temperature (3.7 mg CO(2) g(-1) soil), irrespective of the water content, whereas nitrous oxide production was greater under wet conditions (98-336 microg N(2)O g(-1) soil). After an initial lag phase, methane emissions (963 microg CH(4) g(-1) soil) were observed only under warm and wet conditions. T-RFLP analyses of bacterial 16S rRNA and nosZ genes revealed small or undetectable community changes in response to temperature and water content, suggesting that bacterial and denitrifying microbial communities are stable and do not respond significantly to seasonal changes in soil conditions. In contrast, archaeal microbial community structure was more dynamic and was strongly influenced by temperature.

Spatial variations in denitrification activity in wetland sediments explained by hydrology and denitrifying community structure

We determined spatial variations in potential denitrification activity and the controlling hydrological as well as biochemical processes in the sediments of a Swedish treatment wetland. Hydrological processes, including water residence times, were analyzed using a 2D depth-averaged flow model and the denitrifier community structure was analyzed using denaturing gradient gel electrophoreses (DGGE) of nosZ genes, encoding nitrous oxide reductase. In addition, we provide a theoretical basis for evaluation of denitrification rates useful in nitrate-limited conditions. The results demonstrate that potential denitrification rates differed significantly between the sampling locations (CV=0.34). The variations were best described by concentration of nitrogen in sediments and water residence time. DGGE analyses indicated that a few key populations dominated and that the community diversity increased with decreasing nutrient levels and increasing water residence times. Moreover, we found that denitrification rates in terms of Menten and first-order kinetics can be evaluated by fitting a mathematical expression, comparing denitrification and other nitrogen-transforming processes to measured product formation in nitrate-limited experiments.

Soil biogeochemical toxicity end points for sub‐Antarctic islands contaminated with petroleum hydrocarbons

Sub-Antarctic islands have been subjected to petroleum hydrocarbon spills, yet no information is available regarding the toxicity of petroleum hydrocarbons to these subpolar soils. The purpose of the present study was to identify soil biogeochemical toxicity end points for petroleum hydrocarbon contamination in sub-Antarctic soil. Soil from Macquarie Island, a sub-Antarctic island south of Australia, was collected and exposed to 10 concentrations of Special Antarctic Blend (SAB) diesel fuel, ranging from 0 to 50,000 mg fuel/kg soil, for a 21-d period. The sensitivity of nitrification, denitrification, carbohydrate utilization, and total soil respiration to SAB fuel was assessed. Potential nitrification activity was the most sensitive indicator of SAB contamination assessed for nitrogen cycling, with an IC20 (concentration that results in a 20% change from the control response) of 190 mg fuel/ kg soil. Potential denitrification activity was not as sensitive to SAB contamination, with an IC20 of 950 mg fuel/kg soil for nitrous oxide production. Nitrous oxide consumption was unaffected by SAB contamination. Carbohydrate utilization (respiration caused by sucrose) was a more sensitive indicator (IC20, 16 mg fuel/kg soil) of SAB contamination than total respiration (IC20, 220 mg fuel/kg soil). However, total soil respiration was a more responsive measurement end point, increasing soil respiration over a 72-h period by 17 mg of CO2, compared to a change of only 2.1 mg of CO2 for carbohydrate utilization. Our results indicate that IC20s varied between 16 to 950 mg fuel/kg soil for Macquarie Island soil spiked with SAB diesel fuel. These results indicate that current cleanup levels derived from temperate zones may be too liberal for soil contamination in sub-Antarctic islands.

Nitrogen removal capacity of wetlands: sediment versus epiphytic biofilms

Wetlands are important sinks for nutrients and constructed wetlands are current practice for stormwater treatment. For nitrogen, the main removal process is denitrification (microbial reduction of nitrate to nitrogen gas). The bacteria responsible for this process are mostly found in the sediments and in epiphytic biofilms growing on wetland macrophytes. This paper reports on a project which aimed at measuring denitrification potential in sediments and epiphyton in urban wetlands. This study showed that wetland sediments could support high rates of denitrification. Interestingly, the most polluted of the wetlands studied had the highest denitrification potential. The management implication from this result is that indicators of pollution, such as hydrocarbon levels, will not necessarily reflect the ability of a wetland to denitrify. Two of the wetlands were studied in more detail. Here the denitrification potential of the epiphyton on dominant macrophytes and sediments were measured. The results indicated that the potential denitrification activity of the epiphyton was comparable to those measured in the sediments. Hence, biofilms could play a significant part in removing nitrogen loads. This work contributes to a better knowledge of the functioning of wetlands. This will lead to improved design and management of wetlands used for treating stormwater.

Mesocosms Mimic Natural Meadows as regards Greenhouse Gas Fluxes and Potential Activities of Nitrifying and Denitrifying Bacteria

The objective of this study was to determine whether a planted mesocosm mimics a natural habitat in terms of N2O and CH4 fluxes, soil characteristics and potential nitrification and denitrification activities. We compared mesocosms in unchambered open-field plots and in open-top chambers with nonfiltered ambient air with three natural meadows that had similar soil characteristics and species composition. The N2O fluxes in the mesocosms were very similar to the fluxes in the three natural meadows. There were no marked differences in potential nitrification and denitrification activities between the mesocosms and the natural meadows, either. Only the CH4 fluxes differed slightly between the mesocosms and some of the natural meadows. Therefore, it seems that the mesocosms compared rather well to natural habitats. The open-top chambers modified only the soil water content, the values being higher in the unchambered plots than in the chambered plots. These results thus suggest that the open-top chamber experiment enables estimates of greenhouse gas and potential activities of nitrifying and denitrifying bacteria in unfertilized Finnish meadows, in spite of the chamber effects on the soil water content.

Accumulation of Denitrifying Bacteria and Enhancement of Denitrification Activity in Deep Subsurface Soil in Relation to the Geological Profile

We investigated the denitrification activity and the distribution of the denitrifying bacteria of a boring survey site located on a volcanic plateau, where the geological profiles from surface to deep subsurface soil at the groundwater level had been examined. There were differences between the water quality in the Ito pyroclastic flow deposit (Shirasu) layer (44.2 to 54.5 m) and that in the Osumi pumice fall deposit (Pumice) layer (below 54.5 m) corresponding to the impermeable layer of unconfirmed groundwater: The nitrate concentration was less than 1 mg kg−1 in the Shirasu layer and more than 10 mg kg−1 in the Pumice layer (Kubota et al. 2005). Denitrification activity decreased from the surface to the loam layer and was enhanced in the Shirasu layer and the Haraigawa clay impermeable layer at a depth of 65 m. It was observed that the highest potential denitrification activity (103 ng-N2O d−1 g−1) in the impermeable layer was almost equal to that of a Kuroboku surface soil with slurry application. Viable counts of the sonic-samples, which indicated the presence of bacterial group with soil particles attached, increased in the impermeable layer. The ratios of viable or denitrifying bacterial counts in the sonic-samples to those in the wash-samples were significantly higher in the impermeable layer than those in the surface layer. These results suggest that the hydrogeological conditions enhanced the denitrification activity in the impermeable layer, the niches of which might be relatively anaerobic and have a sufficient supply of substrates to enable the denitrifying bacterial populations to multiply.

Denitrification potential in relation to lithology in five headwater riparian zones.

The influence of riparian zone lithology on nitrate dynamics is poorly understood. We investigated vertical variations in potential denitrification activity in relation to the lithology and stratigraphy of five headwater riparian zones on glacial till and outwash landscapes in southern Ontario, Canada. Conductive coarse sand and gravel layers occurred in four of the five riparian areas. These layers were thin and did not extend to the field-riparian perimeter in some riparian zones, which limited their role as conduits for ground water flow. We found widespread organic-rich layers at depths ranging from 40 to 300 cm that resulted from natural floodplain processes and the burial of surface soils by rapid valley-bottom sedimentation after European settlement. The organic matter content of these layers varied considerably from 2 to 5% (relic channel deposit) to 5 to 21% (buried soils) and 30 to 62% (buried peat). Denitrification potential (DNP) was measured by the acetylene block method in sediment slurries amended with nitrate. The highest DNP rates were usually found in the top 0- to 15-cm surface soil layer in all riparian zones. However, a steep decline in DNP with depth was often absent and high DNP activity occurred in the deep organic-rich layers. Water table variations in 2000-2002 indicated that ground water only interacted frequently with riparian surface soils between late March and May, whereas subsurface organic layers that sustain considerable DNP were below the water table for most of the year. These results suggest that riparian zones with organic deposits at depth may effectively remove nitrate from ground water even when the water table does not interact with organic-rich surface soil horizons.

Denitrification enzyme activity of fringe salt marshes in New England (USA).

Coastal salt marshes are a buffer between the uplands and adjacent coastal waters in New England (USA). With increasing N loads from developed watersheds, salt marshes could play an important role in the water quality maintenance of coastal waters. In this study we examined seasonal relationships between denitrification enzyme activity (DEA) in salt marshes of Narragansett Bay, Rhode Island, and watershed N loadings, land use, and terrestrial hydric soils. In a manipulative experiment, the effect of nutrient enrichment on DEA was examined in a saltmeadow cordgrass [Spartina patens (Aiton) Muhl.] marsh. In the high marsh, DEA significantly (p < 0.05) increased with watershed N loadings and decreased with the percent of hydric soils in a 200-m terrestrial buffer. In the low marsh, we found no significant relationships between DEA and watershed N loadings, residential land development, or terrestrial hydric soils. In the manipulation experiment, we measured increased DEA in N-amended treatments, but no effect in the P-amended treatments. The positive relationships between N loading and high marsh DEA support the hypothesis that salt marshes may be important buffers between the terrestrial landscape and estuaries, preventing the movement of land-derived N into coastal waters. The negative relationships between marsh DEA and the percent of hydric soils in the adjacent watershed illustrate the importance of natural buffers within the terrestrial landscape. Denitrification enzyme activity appears to be a useful index for comparing relative N exposure and the potential denitrification activity of coastal salt marshes.

Potential Denitrification Activity in the Landscape of a Western Alaska Drainage Basin

We examined denitrification potentials in six of the major landscape structure (riparian soils of both meandering and braided streams, peat lands, coniferous flats, alder slopes, and groundwater seeps) of the Lake Nerka catchment in southwest Alaska. We found significant potential denitrifying activity in all the soils of the main landscape patch types of the Lake Nerka catchment. The lowest potential denitrifying activity was measured in the peat lands. A highly significant relationship was found between soil organic matter and potential denitrification activity in three landforms— coniferous flats, groundwater seeps, and riparian soils of meandering streams. These three landscape structures also had the highest denitrifying potential. The finer soils of riparian zones along spawning streams, which corresponded to meandering streams, showed a significantly higher potential denitrification activity than the coarse riparian soils along nonspawning streams. These nonspawning streams corresponded to braided streams, where finer sediments were not as prevalent. Therefore, if this high potential denitrification measured in riparian soils of spawning streams is combined with large inputs of nitrate to anaerobic sites, it can result in 15 N signatures that mimic that of the marine-derived nitrogen provided by Pacific salmon in these Alaskan ecosystems.

Denitrification potential and CO2 emission in the northern forest soils of the Enisei meridian (the Siberian IGBP transect)

To estimate the probable contribution of northern forest soils to the global budget of greenhouse microgases, the cryogenic soils along the Yenisei meridian have been studied with respect to their potential denitrification and carbon mineralization activities. It is shown that the forest soils of the boreal zone have a high denitrification potential and, under conditions of a high nitrate nitrogen content, may be a source of nitrogen oxide emission. A significant correlation is observed between N2O and CO2 emissions (r = 0.85, p < 0.001).

Denitrification in a seashore sandy deposit influenced by groundwater discharge

The chemical compositions of ground water and organic matter in sediments were investigated at a sandy shore of Tokyo Bay, Japan to determine the fate of ground water NO3−. On the basis of Cl− distribution in ground water, the beach was classified into freshwater (FR)-, transition (TR)-, and seawater (SW)-zones from the land toward the shoreline. The NO3− and N2O did not behave conservatively with respect to Cl− during subsurface mixing of freshwater and seawater, suggesting NO3− consumption and N2O production in the TR-zone. Absence of beach vegetation indicated that NO3− assimilation by higher plants was not as important as NO3− sink. Low NH4+ concentrations in ground water revealed little reduction of NO3− to NH4+. These facts implied that microbial denitrification and assimilation were the likely sinks for ground water NO3−. The potential activity and number of denitrifiers in water-saturated sediment were highest in the low-chlorinity part of the TR-zone. The location of the highest potential denitrification activity (DN-zone) overlapped with that of the highest NO3− concentration. The C/N ratio and carbon isotope ratio (δ13C) of organic matter in sediment (< 100 -μm) varied from 12.0 to 22.5 and from −22.5 to −25.5‰, respectively. The δ13C value was inversely related to the C/N ratio (r2 = 0.968, n = 11), which was explained by the mixing of organic matters of terrestrial and marine origins. In the DN-zone, the fine sediments were rich in organic matters with high C/N ratios and low δ13C values, implying that dissolved organic matters of terrestrial origin might have been immobilized under slightly saline conditions. A concurrent supply of NO3− and organic matter to the TR-zone by ground water discharge probably generates favorable conditions for denitrifiers. Ground water NO3− discharged to the beach is thus partially denitrified and fixed as microbial biomass before it enters the sea. Further studies are necessary to determine the relative contribution of these processes for NO3− removal.

Vertical changes in denitrification activity and denitrifying bacterial populations in Kuroboku upland soil with slurry application

The vertical changes in the denitrification activity and de nitrifying bacterial populations were investigated in a Kuroboku upland soil with slurry application, where the nitrogen balance had been determined (Niimi and Nishida 1999: Estimation of denitrification by using δ15N values in upland field applied with a large amount of slurry barnyard manure. In 10th Nitrogen Workshop Program and s, IV, p. 41). Averaged cell density of the denitrifying bacterial populations significantly increased in relation to the level of slurry application, 0, 60, 150, and 300 fresh-wt. Mg ha−1. The denitrifying bacterial populations showed a higher correlation with the potential denitrification activity than with the fresh denitrification activity. Averaged contribution of the potential denitrification activity in three soil layers (approximately 5–20, 20–45, and 45–80 cm) to the total activity was 90, 9.5, and 0.4%, respectively. Annual nitrogen loss associated with the denitrification activity in the 0, 60, 150, and 300 Mg ha−1 plots was found to amount to 15.6, 20.0, 41.5, and 94.7 kg-N ha−1 y−1 based on the data from the fresh denitrification activity and to 38.6, 39.1, 163.8, and 611.8 kg-N ha−1 y−1 based on the data from the potential denitrification activity. The above results indicate that by only conducting a surface soil survey, it was possible to obtain a rough estimation of the nitrogen loss associated with the potential denitrification activity in a Kuroboku upland soil with slurry application.

Denitrification activity in sediment surrounding polychaete (Ceratonereis aequisetis) burrows

The effect of burrow-dwelling fauna on sediment denitrification within the Swan River Estuary, Western Australia, was assessed by determining the spatial profile of potential denitrification activity surrounding individual burrows of a polychaete. This activity was described for Ceratonereis aequisetis and compared with uninhabited sediment. Potential porewater denitrification activity was measured as N’2O production in the presence of acetylene (which blocks N2O reduction and NH4+ oxidation) and supplementary NO3-(provided as a substrate for denitrification). Snap-freezing of sediment cores in liquid nitrogen allowed easy sectioning in both the vertical (perpendicular depth from surface sediment) and radial (depth from burrow wall) planes. Overall, potential denitrification activity was significantly greater in inhabited sediment than in uninhabited sediment, although uninhabited sediment had higher surficial (0–10 mm) potential denitrification activity. Potential denitrification activity was also greater closer to the burrow wall (0–9 mm) rather than further into the sediment (9–13 mm). Greater sampling resolution would be required to determine whether a thin oxygenated surface layer (of either the vertical or radial plane) exists in which denitrification is inhibited. Although this study accurately demonstrates the spatial effect of C. aequisetis on sediment potential denitrification, the reported denitrification intensity may not reflect the rate in situ.

Seasonal and vertical changes in denitrification activity and denitrifying bacterial populations in surface and subsurface upland soils with slurry application

The seasonal and vertical changes in the denitrification activity and denitrifying bacterial populations were investigated in surface (approximately 1 m depth) and subsurface (surface to 4 m depth) soils with slurry application where the nitrogen balance had been determined (Niimi and Nishida: 10th Nitrogen Workshop Programme and s, Vol. 4, p. 41, 1999). Slurry application generally enhanced denitrification in the surface soil, although the enhancement was not confirmed in the subsurface soil. Denitrifying bacteria in the surface soil also showed a potential denitrification activity, while, those in the subsurface soil were not related to the potential denitrification activity. Nitrogen loss by denitrification, which was estimated in the subsurface soil, ranged from O.1±O.2 to 58.5±81.0 kg-N ha-1 y-1, while, the loss from the surface soil ranged from 137.4±73.7 to 1,079.1±399.1 kg·N ha-1 y-1. The above results indicate that denitrification in the subsurface soil did not contribute to the reduction of nitrate-N leaching to the groundwater in fields with excessive application of slurry. Soil conditions required to depress the denitrification activity in the subsurface soil were examined.

Soil microbial tests for discriminating between different cropping systems and fertiliser regimes

The aim of this study was to evaluate a set of microbial soil tests for their ability to discriminate between different agricultural practices. For this purpose three sites included in the Swedish Long-Term Soil Fertility Experiments were chosen. The fertility experiments were designed to compare different cropping systems (simulating farming with and without livestock), PK-fertiliser and N-fertiliser regimes. Six different microbial tests were used to derive nine variables describing: (1) basal microbial activity (B-res), (2) potential microbial activities (substrate induced respiration, SIR; potential NH4+ oxidation, PAO; potential denitrification activity, PDA; and alkaline phosphatase activity, Alk-P), (3) specific microbial growth rates (μres and μPDA) and (4) nutrient-limited respiration rates (maximal P-limited respiration, Max-P; and maximal N-limited respiration, Max-N?). Among the individual microbial variables B-res, SIR, μres and μPDA were the best discriminators of the two different cropping systems. All of them, except μPDA, showed some degree of interaction between different treatments. However, the best discriminators between cropping systems were the components [principal component (PC) 1 and 2] from a PC analysis (PCA). In all three soils PC 1 discriminated well between the two cropping systems. In addition, PC 1 and PC 2 reflected the P-fertilisation rate. Max-P alone had the best potential to reflect the microbially available P in the soil and thereby indirectly the plant-available P. The μres was also useful when assessing available P in the soil. The N-fertilisation rate seemed to be the most difficult treatment to assess with the microbial activity variables. In addition, PCA revealed a consistent functional relationship in all three soils between the potential activity variables (SIR, PAO, PDA, and Alk-P).

Biological activity of ancient cultivated soil with buried horizons (the iverskii monastery, XVII century)

The biological activity of an ancient cultivated soil that has been in intense agricultural use since approximately the first half of the XVII century was studied. The potential biological activity of the buried horizon of the antient cultivated soil was higher than that of its modern horizon or that of the noncultivated soil of an adjacent territory occurring under similar lithological and geomorphological conditions. A decrease rate of oxidative processes (decreased rates of CO2 production and CH4 oxidation) and an increased rate of reductive processes (denitrification and nitrogen fixation) were found in the buried horizon. A high potential denitrification activity (with predominant formation of nitrous oxide) was found in the buried horizon; in the upper horizon, the end product was molecular nitrogen.

Reduction of nitrate in clayey subsoils controlled by geochemical and microbial barriers

Vertical distribution of redox zones, concentrations of redox‐sensitive constituents, numbers of aerobic heterotrophic bacteria, and potential denitrification activity were studied in 1‐m cores taken at the transition between the oxidized and reduced layers in two Danish clayey subsoils. Based on the matrix soil colors, a redox sequence of oxidized, suboxic, and reduced zones was identified at both sites. The geochemical composition of the oxidized brown colored zone (to depths of 2.6 and 3.2 m) was characterized by high concentrations of NO3 − and low amounts of total organic carbon, exchangeable forms of NH4 +, Fe2+, and Mn2+, and structural Fe(II) in the clay minerals. In the underlying 20‐ to 30‐cm‐deep suboxic zone, decreasing NO3 − concentrations were observed together with increasing amount of exchangeable forms of Fe2+ and Mn2+, and structural Fe(II). Finally, in the reduced grey zone, NO3 − was no longer present and maximum concentrations of other redox sensitive constituents occurred. Throughout the subsoils, the distribution of exchangeable Fe2+ corresponded most closely to changes in the colors of redox zones. The low‐organic Havrebjerg site displayed geochemical profiles indicating that NO3 − was chemically reduced by structural Fe(II) in the clay minerals of the suboxic zone, and that the Fe(II) formed a geochemical barrier for the downward progression of NO3 −. Aerobic heterotrophic bacteria occurred only in low numbers at this site and potential denitrification activity was very low. In contrast, the Sparresholm site had a significant population of bacteria in the suboxic zone, which also contained a heterogeneous distribution of potential denitrification activity. Specific microsites with facilitated transport of soluble organic substrates are proposed to support the denitrification activity in a heterogeneous distribution, constituting a microbial barrier for downward progression of NO3 − in this subsoil.

Soil attributes as predictors of crop production under standardized conditions

To assess soil fertility or quality three controlling components – its physical, chemical and biological nature – have to be considered. In this study a broad spectrum of agricultural soils from Sweden were cropped with ryegrass in pots under standardized conditions in climate chambers. Measurements of physical, chemical and biological attributes of soil were used to predict C and N yields by simple correlation and the multivariate calibration techniques, principal component analysis combined with multiple linear regression, and partial least squares (PLS) regression. The N yields were typically more accurately predicted than the corresponding C yields. The best single predictor of yields was always total soil N, but estimates produced by multivariate models including organic C, total N, C/N ratio, coarse silt, potential denitrification activity, N mineralization, substrate-induced respiration and sample site humidity were, in all cases, substantially more accurate. Coefficients of correlation between predicted and measured C or N yields ranged between 0.61 and 0.80 with total N as predictor, and between 0.69 and 0.97 with the multivariate models. Both quantitative and qualitative aspects of the organic matter were considered to be important with respect to the predictive ability. Both these aspects were accounted for by the multivariate models. The multivariate technique, PLS regression, facilitated the classification of soils into categories of good, normal or poor fertility in relation to their organic matter content.

Microbial biomass and activities in soil as affected by frozen and cold storage

When monitoring microbial biomass and activities in soil, the storage conditions of the soil samples prior to analysis may be decisive for the results. Studies made on freshly collected soils are generally preferred but these are not always possible for practical reasons, since sampling is often restricted to short periods of the year, e.g. due to climatic conditions. The most commonly used methods to store soils for microbiological analyses are refrigeration or freezing of field moist soil. There are, however, studies that warn against any kind of storage, although other studies do not indicate any drawbacks to it. We have compared the microbial biomass and activities in 12 different, annually frozen, agricultural mineral soils when fresh and when stored at +2±2°C and at −20±2°C for 1 d and for 1, 3, 6 and 13 months. The results showed that the effects of freezing generally were smaller than those of refrigeration. The biomass estimated by chloroform fumigation–extraction and biomass index estimated by substrate induced respiration differed in that chloroform fumigation extracted carbon had decreased with 27% after 3 months at +2°C, while substrate induced respiration showed only small deviations from the results from fresh soils. Basal respiration rate and potential denitrification activity showed a similar pattern, with a pronounced decrease in values for refrigerated soils. The nitrogen mineralisation capacity was the only measure that was greatly influenced by freezing. After 6 months N mineralisation in the frozen soils was 25% higher than that of the fresh soils. Potential ammonium oxidation and the degradation rate of the herbicide linuron were affected only a little or not at all by storage for 13 months. We concluded that storage at −20°C for 13 months does not affect the microflora in annually frozen soils in any decisive way. We have also discussed the possible reasons for the contradictory results between different studies made on storage effects.