A survey of symbiotic nitrogen fixation by white clover grown on metal contaminated soils

There is conflicting evidence about toxic effects of heavy metals in soil on symbiotic nitrogen fixation. This study was set-up to assess the general occurrence of such effects. Soils with metal concentration gradients were sampled from six established field trials, where sewage sludge or metal salts have been applied, or from a transect in a sludge treated soil. Additional contaminated soils were sampled near metal smelters, in floodplains, in sludge amended arable land and in a metalliferous area. Symbiotic nitrogen fixation was measured with ¹⁵N isotope dilution in white clover (Trifolium repens L.) grown in potted soil that was not re-inoculated, and using ryegrass (Lolium perenne L.) as reference crop. The fraction nitrogen in clover derived from fixation (N_dff) varied from 0 to 88% depending on soil. Pronounced metal toxicity on N_dff was only confirmed in a sludge treated soil where nitrogen fixation was halved from the control value at soil total metal concentration of 737 mg Zn kg⁻¹, 428 mg Cu kg⁻¹ and 10 mg Cd kg⁻¹. The N_dff was significantly reduced by increasing metal concentration in soils from two other sites where N_dff was low throughout and where these effects might be attributed to confounding factors. No significant effects of metals on N_dff were identified in all other gradients even up to elevated total metal concentration (e.g. 55 mg Cd kg⁻¹). The variation of N_dff among all soils (n=48), is mainly explained by the number of rhizobia in the soil (log MPN, log (cells g⁻¹ soil)), whereas correlations with total or soil solution metal concentrations were weak (R²<0.25). The is significantly affected by the presence or absence of the host plant at the sampling site. No effects of metals were identified at even at total Zn concentrations of about 2000 mg Zn kg⁻¹, whereas metal toxicity could be identified at lower most probable number (MPN) values. This survey shows that the metal toxicity on symbiotic nitrogen fixation cannot be generalized and that survival of a healthy population of the microsymbiont is probably the critical factor.     

Influence of selenium on oxidoreductive enzymes activity in soil and in plants

The aim of this greenhouse experiment was the assessment of the influence of H₂SeO₃ at soil concentrations of 0.05, 0.15 and 0.45 mmol kg⁻¹, on the activity of selected oxidoreductive enzymes in wheat (Triticum aestivum). The wheat plants were grown in 2 dm³ pots filled with dust-silt black soil of pH 7.7. Applied H₂SeO₃ caused activation of plant nitrate reductase at all concentrations, but activation of plant polyphenol oxidase at only two lower concentrations. The highest concentration caused inhibition of polyphenol oxidase and peroxidase. Plant catalase activity decreased under the influence of 0.15 and 0.45 mmol kg⁻¹ concentration. After the final analysis Se was quantified in plants and soil. The amounts in plants were: control (unamended soil) 1.95 mg kg⁻¹; I dose (0.05 mmol kg⁻¹) 18.27 mg kg⁻¹; II dose (0.15 mmol kg⁻¹) 33.20 mg kg⁻¹ and III dose (0.45 mmol kg⁻¹) 38.37 mg kg⁻¹, in soil: 0.265 mg kg⁻¹; 3.61 mg kg⁻¹; 10.53 mg kg⁻¹; 30.53 mg kg⁻¹; respectively. Simultaneously, a laboratory experiment was performed, where the activity of soil catalase and peroxidase were tested after 1, 3, 7, 14, 28, 56, and 112 days after Se treatment. Peroxidase activity in soil decreased with increasing Se content, over the whole experiment. The lowest dose of Se caused activation a significant 10% increase in catalase activity, but the influence of others doses was unclear.     

Land use effects on gross nitrogen mineralization, nitrification, and N2O emissions in ephemeral wetlands

Stable ¹⁵N isotope dilution and tracer techniques were used in cultivated (C) and uncultivated (U) ephemeral wetlands in central Saskatchewan, Canada to: (1) quantify gross mineralization and nitrification rates and (2) estimate the relative proportion of N₂O emissions from these wetlands that could be attributed to denitrification versus nitrification-related processes. In-field incubation experiments were repeated in early May, mid-June and late July. Mean gross mineralization and nitrification rates (10.3 and 3.1 mg kg⁻¹ d⁻¹, respectively) did not differ between C and U wetlands on any given date. Despite these similarities, the mean NH₄⁺ pool size in the U wetlands (17.2 mg kg⁻¹) was two to three times that of the C wetlands (6.7 mg kg⁻¹) whereas the mean NO₃⁻ pool size in U wetlands (2.2 mg kg⁻¹) was less than half that of C wetlands (5.8 mg kg⁻¹). Mean N₂O emissions from the C wetlands decreased from 112.8 to 17.0 ng N₂O m² s⁻¹ from May to July, whereas mean U-wetland N₂O emissions ranged only from 31.8 to 51.1 ng N₂O m² s⁻¹ over the same period. This trend is correlated to water-filled pore space in C wetlands, demonstrating a soil moisture influence on emissions. Denitrification is generally considered the dominant emitter of N₂O under anaerobic conditions, but in the C wetlands, only 49% of the May emissions could be directly attributed to denitrification, decreasing to 29% in July. In contrast, more than 75% of the N₂O emissions from the U wetlands arose from denitrification of the soil NO₃⁻ pool throughout the season. These land use differences in emission sources and rates should be taken into consideration when planning management strategies for greenhouse gas mitigation.     

Basal organic phosphorus mineralization in soils under different farming systems

Soil organic P (P_o) mineralization plays an important role in soil P cycling. Quantitative information on the release of available inorganic P (P_i) by this process is difficult to obtain because any mineralized P_i gets rapidly sorbed. We applied a new approach to quantify basal soil P_o mineralization, based on ³³PO₄ isotopic dilution during 10 days of incubation, in soils differing in microbiological activity. The soils originated from a 20 years old field experiment, including a conventional system receiving exclusively mineral fertilizers (MIN), a bio-organic (ORG) and bio-dynamic (DYN) system. Indicators of soil microbiological activity, such as size and activity of the soil microbial biomass and phosphatase activity, were highest in DYN and lowest in MIN. In order to assess P_o hydrolysis driven by phosphatase in sterile soils, a set of soil samples was γ-irradiated. Basal P_o mineralization rates in non-irradiated samples were between 1.4 and 2.5 mg P kg⁻¹ day⁻¹ and decreased in the order DYN>ORG≥MIN. This is an amount lower, approximately equivalent to, or higher than water soluble P_i of MIN, ORG and DYN soils, respectively, but in every soil was less than 10% of the amount of P isotopically exchangeable during one day. This shows that physico-chemical processes are more important than basal mineralization in releasing plant available P_i. Organic P mineralization rates were higher, and differences between soils were more pronounced in γ-irradiated than in non-irradiated soils, with mineralization rates ranging from 2.2 to 4.6 mg P kg⁻¹ day⁻¹. These rates of hydrolysis, however, cannot be compared to those in non-sterile soils as they are affected by the release of cellular compounds, e.g. easily mineralizable P_o, derived from microbial cells killed by γ-irradiation.     

Cyanobacterial inoculation of heated soils: effect on microorganisms of C and N cycles and on chemical composition in soil surface

Physiological groups of soil microorganisms, total C and N and available nutrients were investigated in four heated (350 °C, 1 h) soils (one Ortic Podsol over sandstone and three Humic Cambisol over granite, schist or limestone) inoculated (1.5 μg chlorophyll a g⁻¹ soil or 3.0 μg chlorophyll a g⁻¹ soil) with four cyanobacterial strains of the genus Oscillatoria, Nostoc or Scytonema and a mixture of them.Cyanobacterial inoculation promoted the formation of microbiotic crusts which contained a relatively high number of NH₄⁺-producers (7.4×10⁹ g⁻¹ crust), starch-mineralizing microbes (1.7×10⁸ g⁻¹ crust), cellulose-mineralizing microbes (1.4×10⁶ g⁻¹ crust) and NO₂⁻ and NO₃⁻ producers (6.9×10⁴ and 7.3×10³ g⁻¹ crust, respectively). These crusts showed a wide range of C and N contents with an average of 293 g C kg⁻¹ crust and 50 g N kg⁻¹ crust, respectively. In general, Ca was the most abundant available nutrient (804 mg kg⁻¹ crust), followed by Mg (269 mg kg⁻¹ crust), K (173 mg kg⁻¹ crust), Na (164 mg kg⁻¹ crust) and P (129 mg kg⁻¹ crust). There were close positive correlations among all the biotic and abiotic components of the crusts.Biofertilization with cyanobacteria induced great microbial proliferation as well as high increases in organic matter and nutrients in the surface of the heated soils. In general, cellulolytics were increased by four logarithmic units, amylolytics and ammonifiers by three logarithmic units and nitrifiers by more than two logarithmic units. C and N contents rose an average of 275 g C kg⁻¹ soil and 50 g N kg⁻¹ soil while the C:N ratio decreased up to 7 units. Among the available nutrients the highest increase was for Ca (315 mg kg⁻¹ soil) followed by Mg (189 mg kg⁻¹ soil), K (111 mg kg⁻¹ soil), Na (109 mg kg⁻¹ soil) and P (89 mg kg⁻¹ soil). Fluctuations of the microbial groups as well as those of organic matter and nutrients were positively correlated.The efficacy of inoculation depended on both the type of soil and the class of inoculum. The best treatment was the mixture of the four strains and, whatever the inoculum used, the soil over lime showed the most developed crust followed by the soils over schist, granite and sandstone. In the medium term there were not significant differences between the two inocula amounts tested.These results showed that inoculation of burned soils with alien N₂-fixing cyanobacteria may be a biotechnological means of promoting microbiotic crust formation, enhancing C and N cycling microorganisms and increasing organic matter and nutrient contents in heated soils.     

Microbial activity in pig slurry-amended soils under semiarid conditions

Soil amendment with manures from intensive animal industries is nowadays a common practice that may favorably or adversely affect several soil properties, including soil microbial activity. In this work, the effect of consecutive annual additions of pig slurry (PS) at rates of 30, 60, 90, 120 and 150 m³ ha⁻¹ y⁻¹ over a 4-year period on soil chemical properties and microbial activity was investigated and compared to that of an inorganic fertilization and a control (without amendment). Field plot experiment conducted under a continuous barley monoculture and semiarid conditions were used. Eight months after the fourth yearly PS and mineral fertilizer application (i.e. soon after the fourth barley harvest), surface soil samples (Ap horizon, 0-15 cm depth) from control and amended soils were collected and analysed for pH, electrical conductivity (EC), contents of total organic C, total N, available P and K, microbial biomass C, basal respiration and different enzymatic activities. The control soil had a slightly acidic pH (6.0), a small EC (0.07 dS m⁻¹), adequate levels of total N (1.2 g kg⁻¹) and available K (483 mg kg⁻¹) for barley growth, and small contents of total organic C (13.2 g kg⁻¹) and available P (52 mg kg⁻¹). With respect to the control and mineral fertilized soils, the PS-amended soils had greater pH values (around neutrality or slightly alkaline), electrical conductivities (still low) and contents of available P and K, and slightly larger total N contents. A significant decrease of total organic C was observed in soils amended at high slurry rate (12.3 g kg⁻¹). Compared with the control and mineral treatments, which produced almost similar results, the PS-amended soils were characterized by a higher microbial biomass C content (from 311 to 442 g kg⁻¹), microbial biomass C/total organic C ratio (from 2.3 to 3.6%) and dehydrogenase (from 35 to 173 μg INTF g⁻¹), catalase (from 5 to 24 μmol O₂ g⁻¹ min⁻¹), BAA-protease (from 0.7 to 1.9 μmol  g⁻¹ h⁻¹) and β-glucosidase (from 117 to 269 μmol PNP g⁻¹ h⁻¹) activities, similar basal respirations (from 48 to 77 μg C-CO₂ g⁻¹ d⁻¹) and urease activities (from 1.5 to 2.2 μmol  g⁻¹ h⁻¹), and smaller metabolic quotients (from 6.4 to 7.7 ng C-CO₂ μg⁻¹ biomass C h⁻¹) and phosphatese activities (from 374 to 159 μmol PNP g⁻¹ h⁻¹). For example, statistical analysis of experimental data showed that, with the exception of metabolic quotient and total organic C content, these effects generally increased with increasing cumulative amount of PS. In conclusion, cumulative PS application to soil over time under semiarid conditions may produce not only beneficial effects but also adverse effects on soil properties, such us the partial mineralization of soil organic C through extended microbial oxidation. Thus, PS should not be considered as a mature organic amendment and should be treated appropriately before it is applied to soil, so as to enhance its potential as a soil organic fertilizer.     

Denitrification enzyme activity and potential of subsoils under grazed grasslands assayed by membrane inlet mass spectrometer

The importance of subsoil denitrification on the fate of agriculturally derived nitrate (NO₃) leached to groundwater is crucial for budgeting N in an ecosystem and for identifying areas where the risk of excess NO₃ is reduced. However, the high atmospheric background of di-nitrogen (N₂) causes difficulties in assessing denitrification enzyme activity (DEA) and denitrification potential (DP) in soils directly. Here, we apply Membrane Inlet Mass Spectrometry (MIMS) technique to investigate indirectly DEA and DP in soils by measuring N₂/Ar ratio changes in headspace water over soil. Soils were collected from 0-10, 15-25 and 60-70 cm depths of a grazed ryegrass and grass-clover. The samples were amended with helium-flushed deionized water containing ranges of NO₃ and carbon (glucose-C) and were incubated for six hours in the dark at 21 °C. The peaks for N₂/Ar ratio, declined with increasing soil depth, indicating a reduced substrate requirements to initiate DEA en-masse (15-30 mg NO₃-N alone or with 60-120 mg glucose-C, kg⁻¹ soil). The dissolved N₂O concentrations were very small (0.004-0.269 μg N kg⁻¹ soil) but responded well to the added N and C, showing a reduction in DEA with soil depth. In three separate studies, only subsoils were incubated for 3 days at 12 °C with 20-30 mg NO₃-N ± 40-60 mg glucose-C, kg⁻¹ soil. Denitrification capacity (DC, NO₃ only treatment) was not statistically different to the control (no amendment) within a land use (0.03-0.05 vs. 0.07-0.22 mg N kg⁻¹ soil d⁻¹), the highest being in ryegrass subsoils receiving groundwater. The DP was significantly (P < 0.0001) higher in subsoils under ryegrass than under grass-clover (0.50-0.71 vs. 1.15 mg N kg⁻¹ soil d⁻¹). The rates of DP (NO₃ + glucose-C) increased significantly (P < 0.0001) in unsaturated and saturated subsoils (0.92 and 2.19 mg N kg⁻¹ soil d⁻¹, respectively) of grass-clover, due to the higher reductive state resulting from the 10 day pre-incubation. Available C accelerated denitrification in soils and superseded the temporary elevation in oxidative state due to NO₃ addition. The substrates load differences between the land uses regulated the degree of denitrification rates. Results suggest that both dissolved N₂O measured by gas chromatography and N₂/Ar ratio measured by MIMS to indirectly determine DEA, and the latter to quantify total DC/DP in soils can be used. However, interference of oxygen in the MIMS system should be considered if available C is added or is naturally elevated in soil or groundwater.     

Phosphorus fractionation in lowland tropical rainforest soils in central Panama

Phosphorus availability is commonly assumed to limit productivity in lowland tropical rainforests, yet there is relatively little information on the chemical forms of soil phosphorus in such ecosystems. We used the Hedley sequential fractionation scheme to assess phosphorus chemistry in five soils supporting tropical rainforest on Barro Colorado Island, Republic of Panama. The soils represented a range of orders (Inceptisols, Alfisols, and Oxisols) formed on contrasting geological substrates and topography, but under uniform climate and vegetation. Total phosphorus in surface horizons ranged between 315 and 1114 mg P kg^− 1, being lowest on a soil derived from marine sediments and highest on soils derived from andesite. The majority of the phosphorus occurred in recalcitrant forms, although between 14% and 39% occurred as organic phosphorus. Readily-available phosphate, as extracted by anion-exchange membranes, occurred in small concentrations (4–13 mg P kg^− 1), although labile phosphorus, defined as phosphate extracted by anion-exchange membrane plus inorganic and organic phosphorus extracted by 0.5 M NaHCO₃, accounted for between 4.7% and 11.4% of the total soil phosphorus. Our results indicate a strong control of geology and topography on soil phosphorus in tropical rainforests, which may have important implications for understanding the diversity and distribution of plant species in such ecosystems. Further, some of the most common soils on Barro Colorado Island, including those on the 50 ha forest dynamics plot, are rich in phosphorus despite their relatively advanced stage of pedogenesis.     

Sulphur immobilization and availability in soils assessed using isotope dilution

Increasing recognition of S deficiency in soils has raised the need for understanding processes governing S cycling and availability in soils. However, the quantification of the two main processes of S cycling, i.e. mineralization and immobilization, remains difficult as these processes occur simultaneously. A modified isotope ³⁵SO₄ dilution technique was developed and used to measure the effect of sulphate (SO₄) fertilization on S mineralization and immobilization in planted (pot experiment with ryegrass (Lolium multiflorum L.)) and unplanted soils (incubation). The immobilization and mineralization of S was calculated from the dynamics of stable and labelled S in soil KH₂PO₄ extracts containing an anion exchange membrane that concentrates SO₄ and mainly excludes other S species. The mathematical analysis of the isotope dilution data differs from methods proposed earlier. The radiolabile S in unplanted soil (E value) and in ryegrass (L value) were used as a measure of total available S in soils. Sulphate immobilization rate significantly declined during incubation. Sulphate application reduced gross mineralization but surprisingly reduced SO₄ immobilization. The E value significantly increased during the incubation in all soils as a result of gross mineralization, e.g. from 3.8 mg S kg⁻¹ at day 0 to 11.5 mg S kg⁻¹ at day 43 in the sandy soil with no sulphate addition. A full recovery in the E value of S added in (+S) treatments was achieved. Similarly, radiolabile S in the above-ground ryegrass biomass (L value) increased with S addition, with a full recovery of added S. The E and L values nearly fit a 1:1 line suggesting identical S dynamics in a planted and unplanted soil. The method proposed has operational advantages compared to methods used earlier.     

Zinc toxicity on N2O reduction declines with time in laboratory spiked soils and is undetectable in field contaminated soils

Denitrification assays in soils spiked with zinc salt have shown inhibition of the N₂O reduction resulting in increased soil N₂O fluxes with increasing soil Zn concentration. It is unclear if the same is true for environmentally contaminated soils. Net production of N₂O and N₂ was monitored during anaerobic incubations (25 °C, He atmosphere) of soils freshly spiked with ZnCl₂ and of corresponding soils that were gradually enriched with metals (mainly Zn) in the field by previous sludge amendments or by corrosion of galvanized structures. Total denitrification activity (i.e. the sum of N₂O+N₂ production rate) was not inhibited by freshly added Zn salts up to 1600 mg Zn kg⁻¹, whereas N₂O reduction decreased by 50% (EC₅₀) at total Zn concentrations of 231 mg Zn kg⁻¹ (ZEV soil) and 368 mg Zn kg⁻¹ (TM soil). In contrast, N₂O reduction was not reduced by soil Zn in any of the field contaminated soils, even at total soil Zn or soil solution Zn concentrations exceeding more than 5 times corresponding EC₅₀'s of the freshly spiked soil. The absence of adverse effects in the field contaminated soils was unrelated to soil NO₃ or organic matter concentration. Ageing (2-8 weeks) and soil leaching after spiking reduced the toxicity of Zn on N₂O reduction, either expressed as total Zn or soil solution Zn, suggesting adaptation reactions. However, no full recovery after spiking was identified at the largest incubation period in one soil. In addition, the denitrification assay performed with sewage sludge showed elevated N₂O release in Zn contaminated sludges (>6000 mg Zn kg⁻¹ dry matter) whereas this was not observed in low Zn sludge (<1000 mg Zn kg⁻¹ dry matter) suggesting limits to adaptation reactions in the sludge particles. It is concluded that the use of soils spiked with Zn salts overestimates effects on N₂O reduction. Field data on N₂O fluxes in sludge amended soils are required to identify if metals indeed promote N₂O emissions in sludge amended soils.     

Possibilities to reduce rice straw-induced global warming potential of a sandy paddy soil by combining hydrological manipulations and urea-N fertilizations

Global warming potential (GWP) of sandy paddy soils may be reduced by trade-offs between N₂O, CH₄ and CO₂ emissions. Laboratory experiments using either rice straw (1% or 0.5%) or together with urea-N (25 or 50 mg N kg⁻¹ soil) at various levels of soil water were carried out for 30 days each, to test this assumption. Waterlogging combined with urea-N increased total N₂O emissions, with greater release upon rewaterlogging (7.4 mg N kg⁻¹ soil) than experienced by removing waterlogging only. Rice straw±urea-N either emitted small amounts of N₂O or resulted in negative values at all water levels, including saturated and aerobic. Total CH₄ fluxes declined with the decreased water levels and amount of rice straw (<193 mg C kg⁻¹ soil), and also for CO₂ with the latter (<1340 mg C kg⁻¹ soil), and rewaterlogging had little influence on both. N₂O under rewaterlogged and waterlogged±urea-N, CH₄ under waterlogged with rice straw, and CO₂ for the remainder were the major contributors to GWP. Results show that waterlogging following aerobic decomposition of rice straw (1%) with urea-N, applied either at the beginning or at the end of the aerobic conditions, could decrease GWP by 56-64% and 32-42% over the sole addition of rice straw (1% and 0.5%) under waterlogged and saturated conditions, respectively.     

Differences in isotopic fractionation of nitrogen in water-saturated and unsaturated soils

Temporal variations in δ¹⁵N of NH₄⁺ and NO₃⁻ in water-saturated and unsaturated soils were examined in a laboratory incubation study. Ammonium sulfate (δ¹⁵N=−2.6‰) was added to 25 g samples of soil at concentrations of 160 mg N kg⁻¹. Soils were then incubated under unsaturated (50% of water holding capacity at saturation, WHC) or saturated (100% of WHC) water conditions for 7 and 36 d, respectively. During 7 d incubation of unsaturated soil, the NH₄⁺-N concentration decreased from 164.8 to 34.4 mg kg⁻¹, and the δ¹⁵N of NH₄⁺ increased from −0.4 to +57.2‰ through nitrification, as evidenced by corresponding increase in NO₃⁻-N concentration and lower δ¹⁵N of NO₃⁻ (product) than that of NH₄⁺ (substrate) at each sampling time. In saturated soil, the concentration of NH₄⁺-N decreased gradually from 162.4 to 24.2 mg kg⁻¹, and the δ¹⁵N values increased from +0.8 to +21.0‰ during 36 d incubation. However, increase in NO₃⁻ concentration was not observed due to loss of NO₃⁻ through concurrent denitrification in anaerobic sites. The apparent isotopic fractionation factors (α_s/p) associated with decrease in NH₄⁺ concentration were 1.04 and 1.01 in unsaturated and saturated soils, respectively. Since nitrification is likely to introduce greater isotope fractionation than microbial immobilization, the higher value for unsaturated soil probably reflected faster nitrification under aerobic conditions. The lower value for saturated soil suggests that immobilization and subsequent remineralization of NH₄⁺ were relatively more dominant than nitrification under the anaerobic conditions.     

Effect of di-(2-ethylhexyl) phthalate (DEHP) on microbial biomass C and enzymatic activities in soil

The effects of di-(2-ethylhexyl) phthalate (DEHP) at five different doses from 10 to 1000 mg kg⁻¹ soil on biological properties were investigated over a period of 56 days. Meanwhile, the dissipation of DEHP was also monitored. The results indicated that the microbial biomass C (C_mic) fluctuated at around 70 mg kg⁻¹ soil for the control, whereas the C_mic varied significantly for the soil samples contaminated by DEHP. The catalase activities in all five treatments were stimulated at most time, and the activities of phosphatase in the soils treated by DEHP with 500 mg kg⁻¹ or 1000 mg kg⁻¹ were significantly higher than the other treatments from the 20th day. Urease was more sensitive and inhibited significantly during the initial period of incubation. Additionally, the dose–response relationship of invertase was presented in the later phase of incubation. The activities of urease and invertase might indicate soil perturbations caused by the introduction of DEHP. The dissipation of DEHP was found to follow the pseudo first-order kinetics behavior.     

A single application of Cu to field soil has long-term effects on bacterial community structure, diversity, and soil processes

Long-term diversity-disturbance responses of soil bacterial communities to copper were determined from field-soils (Spalding; South Australia) exposed to Cu in doses ranging from 0 through to 4012 mg Cu kg⁻¹ soil. Nearly 6 years after application of Cu, the structure of the total bacterial community showed change over the Cu gradient (PCR-DGGE profiling). 16S rRNA clone libraries, generated from unexposed and exposed (1003 mg Cu added kg⁻¹ soil) treatments, had significantly different taxa composition. In particular, Acidobacteria were abundant in unexposed soil but were nearly absent from the Cu-exposed sample (P<0.05), which was dominated by Firmicute bacteria (P<0.05). Analysis of community profiles of Acidobacteria, Bacillus, Pseudomonas and Sphingomonas showed significant changes in structural composition with increasing soil Cu. The diversity (Simpsons index) of the Acidobacteria community was more sensitive to increasing concentrations of CaCl-extractable soil Cu (Cu_Ext) than other groups, with decline in diversity occurring at 0.13 Cu_Ext mg kg⁻¹ soil. In contrast, diversity in the Bacillus community increased until 10.4 Cu_Ext mg kg⁻¹ soil, showing that this group was 2 orders of magnitude more resistant to Cu than Acidobacteria. Sphingomonas was the most resistant to Cu; however, this group along with Pseudomonas represented only a small percentage of total soil bacteria. Changes in bacterial community structure, but not diversity, were concomitant with a decrease in catabolic function (BioLog). Reduction in function followed a dose-response pattern with Cu_Ext levels (R²=0.86). The EC₅₀ for functional loss was 0.21 Cu_Ext mg kg⁻¹ soil, which coincided with loss of Acidobacteria diversity. The microbial responses were confirmed as being due to Cu and not shifts in soil pH (from use of CuSO₄) as parallel Zn-based field plots (ZnSO₄) were dissimilar. Changes in the diversity of most bacterial groups with soil Cu followed a unimodal response - i.e. diversity initially increased with Cu addition until a critical value was reached, whereupon it sharply decreased. These responses are indicative of the intermediate-disturbance-hypothesis, a macroecological theory that has not been widely tested in environmental microbial ecosystems.     

Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils

The concentrations of Zn, Cd, Pb and Cu in earthworm tissues were compared with the total and DTPA-extractable contents of these heavy metals in contaminated soils. Samples were taken from a pasture polluted by waste from a metallurgic industry over 70 y ago. Three individuals of Aporrectodea caliginosa and Lumbricus rubellus and soil samples were collected at six points along a gradient of increasing pollution. Total metal contents of earthworms, soil, and metals extracted by DTPA from the soil were measured. Total heavy metal contents of the soils ranged from 165.7 to 1231.7 mg Zn kg⁻¹, 2.7 to 5.2 mg Cd kg⁻¹, 45.8 to 465.5 mg Pb kg⁻¹ and 30.0 to 107.5 mg Cu kg⁻¹. Their correlations with metals extracted by DTPA were highly significant. Contents of the metals in earthworm tissues were higher in A. caliginosa than in L. rubellus, with values ranging from 556 to 3381 mg Zn kg⁻¹, 11.6 to 102.9 mg Cd kg⁻¹, 1.9 to 182.8 mg Pb kg⁻¹ and 17.9 to 35.9 mg Cu kg⁻¹ in A. caliginosa, and from 667.9 to 2645 mg Zn kg⁻¹, 7.7 to 26.3 mg Cd kg⁻¹, 0.5 to 37.9 mg Pb kg⁻¹ and 16.0 to 37.6 mg Cu kg⁻¹ in L. rubellus, respectively. Correlations between body loads in earthworms with either total or DTPA-extractable contents of soil metals were significant, except for Cd in L. rubellus and Cu in A. caliginosa. Considering its simple analytical procedure, DTPA-extractable fraction may be preferable to total metal content as a predictor of bio-concentrations of heavy metals in earthworms. Biota-to-Soil Accumulation Factor (BSAF) of these four metals are Cd>Zn>Cu>Pb, with range of mean values between: Cd (6.18-17.02), Zn (1.95-7.91), Cu (0.27-0.89) and Pb (0.08-0.38) in A. caliginosa, and Cd (3.64-6.34), Zn (1.5-6.35), Cu (0.29-0.87) and Pb (0.04-0.13) in L. rubellus. The BSAF of Ca, Fe and Mn are Ca>Mn>Fe, with mean values of: Ca (0.46-1.31), Mn (0.041-0.111), Fe (0.017-0.07) in A. caliginosa and Ca (0.98-2.13), Mn (0.14-0.23), Fe (0.019-0.048) in L. rubellus, respectively. Results of principal component analysis showed that the two earthworm species differ in the pattern of metal bioaccumulation which is related to their ecological roles in contaminated soils.     

Nitrous oxide production of heavy metal contaminated soil

Arsenic (As), lead (Pb), copper (Cu) and zinc (Zn) can be found in large concentrations in mine spills of central and northern Mexico. Interest in these heavy metals has increased recently as they contaminate drinking water and aquifers in large parts of the world and severely affect human health, but little is known about how they affect biological functioning of soil. Soils were sampled in seven locations along a gradient of heavy metal contamination with distance from a mine in San Luis Potosí (Mexico), active since about 1800 AD. C mineralization and N₂O production were monitored in an aerobic incubation experiment. Concentrations of As in the top 0-10 cm soil layer ranged from 8 to 22,992 mg kg⁻¹, from 31 to 1845 mg kg⁻¹ for Pb, from 27 to 1620 mg kg⁻¹ for Cu and from 81 to 4218 mg kg⁻¹ for Zn. There was a significant negative correlation between production rates of CO₂ and concentrations of As, Pb, Cu and Zn, and there was a significant positive correlation with pH, water holding capacity (WHC), total N and soil organic C. There was a significant negative correlation (P<0.05) between production rate of nitrous oxide (N₂O) attributed to nitrification by the inhibition method in soil incubated at 50% WHC and total concentrations of Pb and Zn, and there was a significant positive correlation (P<0.05) with pH and total N content. There was a significant negative correlation (P<0.05) between the production rate of N₂O attributed to denitrification by the inhibition method in soil incubated at 100% WHC and total concentrations of Pb, Cu and Zn, and a significant positive correlation (P<0.01) with pH; there was a significant positive correlation (P<0.05) between the production of N₂O attributed to other processes by the inhibition method and WHC, inorganic C and clay content. A negative value for production rate of N₂O attributed to nitrifier denitrification by the inhibition method was obtained at 100% WHC. The large concentrations of heavy metals in soil inhibited microbial activity and the production rate of N₂O attributed to nitrification by the inhibition method when soil was incubated at 50% WHC and denitrification when soil was incubated at 100% WHC. The inhibitor/suppression technique used appeared to be flawed, as negative values for nitrifier denitrification were obtained and as the production rate of N₂O through denitrification increased when soil was incubated with C₂H₂.     

Immobilization and remineralization of N following addition of wheat straw into soil: determination of gross N transformation rates by N-ammonium isotope dilution technique

N dynamics in soil where wheat straw was incorporated were investigated by a soil incubation experiment using ¹⁵N-labelled nitrate or ¹⁵N-labelled wheat straw. The incubated soils were sampled after 7, 28, 54 days from the incorporation of wheat straw, respectively, and gross rates of N transformations including N remineralization and temporal changes in the amount of microbial biomass were determined.Following the addition of wheat straw into soils, rapid decrease of nitrate content in soil and increase of microbial biomass C and N occurred within the first week from onset of the experiment. Both the gross rates of mineralization and immobilization determined by ¹⁵N-ammonium isotope dilution technique were remarkably enhanced by the addition of wheat straw, and gradually decreased with time. Remineralization rate of N derived from ¹⁵N-labelled nitrate, and mineralization rate of N derived from ¹⁵N-labelled wheat straw was estimated by ¹⁵N isotope dilution technique using non-labelled ammonium. Remineralization rates of N derived from ¹⁵N-labelled nitrate were calculated to be 0.71 mg N kg⁻¹ d⁻¹ after 7 days, 0.55 mg N kg⁻¹ d⁻¹ after 28 days, and 0.29 mg N kg⁻¹ d⁻¹ after 54 days.Nearly 10% of the ¹⁵N-labelled N originally contained in the wheat straw was held in the microbial biomass irrespective of the sampling time. The amount of inorganic N in soil which was derived from ¹⁵N-labelled wheat straw ranged between 1.93 and 2.37 mg N kg⁻¹.Rates of N transformations in soil with ¹⁵N-labelled wheat straw were obtained by assuming that the k value was equal to the ¹⁵N abundance of biomass N, and the obtained values were considered to be valid.     

Immobilized-S, microbial biomass-S and soil arylsulfatase activity in the rhizosphere soil of rape and barley as affected by labile substrate C and N additions

Bulk and rhizosphere soil of rape and barley grown in a calcareous soil were pre-incubated for 7 days at 20 °C with Na₂³⁵SO₄ to partially label soil organic S. The soils were then incubated for 7 days more with increasing levels of two C sources as organic acids (succinic and malic acids) and as glucose (from 0 to 640 mg C kg⁻¹ soil) with or without increasing levels of N (from 0 to 15 mg N kg⁻¹ soil) in the form of ammonium nitrate, in order to mimic rhizodeposition inputs into soil. A second incubation experiment with a single highest dose of the used substrates was undertaken and two destructive soil samplings on days 17 and 35 were carried out. Both incubation experiments showed the intensities of S immobilization in the order: barley rhizosphere>rape rhizosphere>bulk soil. Glucose addition generated positive S priming effects in all studied soils after one week of incubation. Significant correlation coefficients were observed between immobilized-S and microbial biomass-S (r=0.95,p<0.001), arylsulfatase activity (ARS) and microbial biomass-S (r=0.65,p<0.05) on day 17 but not on day 35, whereas significant correlation coefficients were found between arylsulfatase activity and immobilized-S at both days 17 (r=0.79,p<0.01) and 35 (r=0.75,p<0.01). A marked decline of biomass-S noted in substrate-amended treatments at day 35 suggests a quick turnover of this compartment followed by its incorporation into the organic S. Finally, with organic acids high values of ARS per unit of biomass-S were recorded over the two studied dates in the rhizosphere soil of rape. It is concluded that the rhizosphere microbial biomass under rape exhibited more efficient arylsulfatase activity and hence greater turnover of organic S than that under the barley rhizosphere soil.     

Measuring rates of gross and net mineralisation of organic phosphorus in soils

The isotopic dilution method developed by Oehl et al. [2001b. Organic phosphorus mineralisation studies using isotopic dilution techniques. Soil Science Society of America Journal 65, 780-787] to measure gross mineralisation of soil organic phosphorus (P) was tested on a range of low-P sorbing soils. This isotopic dilution method relies on accurate prediction of radiolabel behaviour due to soil physicochemical processes. Based on experimental validation of the extrapolation for isotopic dilution due to physicochemical processes using autoclaved soils, a simple power function was used for extrapolation rather than the more complex equation used in the original method. For several soils, however, a potential overestimation of gross mineralisation by 0.1-2.0 mg P kg⁻¹ d⁻¹ was revealed. In addition, the detection limit of P mineralisation ranged between 0.6 and 2.6 mg P kg⁻¹ d⁻¹. The method is likely to be at the detection limit for soils that are high in available P and low in biological activity. The method was modified with respect to the extrapolation and successfully applied to a soil with relatively high microbial P (18 mg P kg⁻¹) and soil respiration rates (29 mg C kg⁻¹ d⁻¹), revealing gross mineralisation rates of organic P of 0.9-1.2 mg P kg⁻¹ d⁻¹. Measurement of uptake of ³²P by the microbial biomass allowed derivation of a net organic P mineralisation rate of 0.5-0.9 mg P kg⁻¹ d⁻¹.     

Attempts to derive EC50 values for heavy metals from land-applied Cu-, Ni-, and Zn-spiked sewage sludge

We established a field trial to assess the impacts on soil biological properties of application of heavy metal-spiked sewage sludge, with the aim of determining toxicity threshold concentrations of heavy metals in soil. Plots were treated with sludges containing increasing concentrations of Cu, Ni and Zn in order to raise the metal concentrations in the soil by 0-200 mg Cu kg⁻¹, 0-60 mg Ni kg⁻¹ and 0-400 mg Zn kg⁻¹, and were then cultivated and sown in ryegrass-clover pasture and monitored annually for 6 years. All biological properties measured (soil basal respiration, microbial biomass C, and sulphatase enzyme activities), except phosphatase activity, increased in all plots over the duration of the experiment. Consequently, it was only possible to assess effects of heavy metals across time if, each year, all data for each metal were normalised by expressing them as percentages of the activities measured in an un-sludged control plot. When this was done, no significant effects of increasing heavy-metal concentrations on basal respiration, microbial biomass C or respiratory quotient (qCO₂) were observed, although total Cu and soil solution Cu were significantly negatively related to microbial biomass C when it was expressed as a proportion of soil total C. None of the properties measured were affected by increasing Ni concentrations. Phosphatase and sulphatase activities were significantly negatively related to increasing Zn concentrations, but not usually to increasing Cu unless they were expressed as a proportion of total C. A sigmoidal dose-response model was used to calculate EC₂₀ and EC₅₀ values using the normalised data, but generally, the model parameters had very large 95% confidence intervals and/or the fits to the model had small R² values. The factors primarily responsible for confounding these results were site and sample variations not accounted for by the normalisation process and the absence of any data points at metal concentrations beyond the calculated EC₅₀ values. In the few instances where reasonable EC₂₀ values could be calculated, they were relatively consistent across properties, e.g., EC₂₀ for total Zn and phosphatase (330 mg kg⁻¹), total Zn and sulphatase (310 mg kg⁻¹), and EC₂₀ for total Cu and sulphatase (140 mg kg⁻¹) and total Cu and microbial biomass C (140 mg kg⁻¹), when both sulphatase and microbial biomass C were expressed as a proportion of total C. Our results suggest that Cu and Zn at the upper concentrations used in this experiment were possibly having adverse effects on some soil biological properties. However, much higher metal concentrations will be needed to accurately calculate EC₂₀ and EC₅₀ and this may not be easily achievable without many applications of sewage sludge, even if the sludge is spiked with heavy metals.