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.     

Enzyme activities and microbial biomass in coastal soils of India

Soil salinity is a serious problem for agriculture in coastal regions, wherein salinity is temporal in nature. We studied the effect of salinity, in summer, monsoon and winter seasons, on microbial biomass carbon (MBC) and enzyme activities (EAs) of the salt-affected soils of the coastal region of the Bay of Bengal, Sundarbans, India. The average pH of soils collected from different sites, during different seasons varied from 4.8 to 7.8. The average organic C (OC) and total N (TN) content of the soils ranged between 5.2-14.1 and 0.6-1.4 g kg⁻¹, respectively. The electrical conductivity of the saturation extract (ECe) of soils, averaged over season, varied from 2.2 to 16.3 dSm⁻¹. The ECe of the soils increased five fold during the summer season (13.8 dSm⁻¹) than the monsoon season (2.7 dSm⁻¹). The major cation and anion detected were Na⁺ and Cl⁻, respectively. Seasonality exerted considerable effects on MBC and soil EAs, with the lowest values recorded during the summer season. The activities of β-glucosidase, urease, acid phosphatase and alkaline phosphatase were similar during the winter and monsoon season. The dehydrogenase activity of soils was higher in monsoon than in winter. Average MBC, dehydrogenase, β-glucosidase, urease, acid phosphatase and alkaline phosphatase activities of the saline soils ranged from 125 to 346 mg kg⁻¹ oven dry soil, 6-9.9 mg triphenyl formazan (TPF) kg⁻¹ oven dry soil h⁻¹, 18-53 mg p-nitro phenol (PNP) kg⁻¹ oven dry soil h⁻¹, 38-86 mg urea hydrolyzed kg⁻¹ oven dry soil h⁻¹, 213-584 mg PNP kg⁻¹ oven dry soil h⁻¹ and 176-362 mg PNP g⁻¹ oven dry soil h⁻¹, respectively. The same for the non-saline soils were 274-446 mg kg⁻¹ oven dry soil, 8.8-14.4 mg TPF kg⁻¹ oven dry soil h⁻¹, 41-80 mg PNP kg⁻¹ oven dry soil h⁻¹, 89-134 mg urea hydrolyzed kg⁻¹ oven dry soil h⁻¹, 219-287 mg PNP kg⁻¹ oven dry soil h⁻¹ and 407-417 mg PNP kg⁻¹ oven dry soil h⁻¹, respectively. About 48%, 82%, 48%, 63%, 40% and 48% variation in MBC, dehydrogenase activity, β-glucosidase activity, urease activity, acid phosphatase activity and alkaline phosphatase activity, respectively, could be explained by the variation in ECe of saline soils. Suppression of EAs of the coastal soils during summer due to salinity rise is of immense agronomic significance and needs suitable interventions for sustainable crop production.     

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.     

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.     

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.     

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.     

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.     

Prey-predator dynamics in communities of culturable soil bacteria and protozoa: differential effects of mercury

We investigated whether the prey-predator dynamics of bacteria and protozoa were affected by inorganic mercury at concentrations of 0, 3.5 and 15 mg Hg(II) kg soil⁻¹. The amount of bioavailable Hg was estimated using a biosensor-assay based on the mer-lux gene fusion. The numbers of bacterial CFUs on the general medium 1/100 tryptic soy agar (TSA) were significantly decreased when the soil had been amended with Hg. In contrast, no effect was seen on the number of CFUs on the Pseudomonas-specific medium Gould's S1 agar. Protozoan numbers estimated by the most probable number (MPN) method with 1/100 TSB as growth medium were also negatively affected by Hg. The different fractions of protozoa were affected to different degrees suggesting that amoebae were less sensitive than slow-growing flagellates, which again were less sensitive than the fast-growing flagellates. In contrast, Hg did not induce any detectable changes in the diversity of flagellate morphotypes. In the treatment with 15 mg Hg kg⁻¹ a transiently increased number of bacteria was seen at day 6 probably concomitant with a decrease in the numbers of protozoa. This might indicate that Hg affected the prey-predator dynamics in communities of culturable bacteria and protozoa in soil. Furthermore, we showed that the number of Pseudomonas spp. was not affected by Hg whereas the number of bacteria growing on a general medium was.     

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.     

In-situ enrichment and analysis of atrazine-degrading microbial communities using atrazine-containing porous beads

We examined the community composition of microbes that colonized atrazine-containing beads buried in agricultural soils that differed in atrazine treatment history. Bacterial abundance was 5-40-fold greater in atrazine-fortified beads. In beads containing 20 mg atrazine kg⁻¹ buried in soil with a history of atrazine application (conditioned soil), the abundance of Actinobacteria increased approximately 80-fold whereas in control soil, Actinobacteria were enriched only 10-fold and the gamma-Proteobacteria and Planctomycetes increased by 60- and 25-fold, respectively. The gamma-Proteobacteria were enriched by 120- and 230-fold in beads containing 200 mg atrazine kg⁻¹ in conditioned and control soil, respectively. The results demonstrate that BioSep^® beads are a suitable matrix for recruiting a diverse subset of the bacterial community involved in atrazine degradation.     

Seasonality of organic phosphorus mineralization in the rhizosphere of the native savanna grass, Trachypogon plumosus

Savanna ecosystems have low primary productivity, strong seasonality, and acid soils with low phosphorus (P) content. Organic P (Po) comprises around 50% of the total soil P and is plant-available only after mineralization. Rhizosphere processes mediated by plants, microorganisms and arbuscular mycorrhiza (AM) are important for plant P nutrition. We studied P transformation rates, Po-fractionation, acid phosphomonoesterase activity (APA), AM status, dehydrogenase activity (DHA), and bacterial and fungal plate counts in the rhizosphere of the native dominant grass Trachypogon plumosus. We collected samples from three acid savanna soils differing in order and P content (Entisol, Vertisol and Ultisol) at Estación Experimental La Iguana (Northeastern Venezuela) during the dry, rainy and transitional seasons over a 2-year-period. Less available Po fractions (moderately labile, moderately and highly resistant) seem to be involved in short-term P-cycling transformations as they significantly varied with season. During the rainy season plant P content (576-1160 mg P kg⁻¹ dry weight) and APA (44-200 mg PNP kg⁻¹ dry soil) were higher, while microbial number and activity (DHA) were lower. The higher P availability in the Entisol (6-9 mg P kg⁻¹ dry soil) resulted in a better plant nutritional status and inhibited APA. T. plumosus seems to be highly dependent on AM symbiosis (45-71% AM colonized root length, 0.6-8 AM spores g⁻¹ dry soil), especially during the rainy season. Po mineralization processes, mediated by biological associations in the rhizosphere, are crucial for understanding seasonal P-cycling and fertility in acid savanna soils.     

Transfer and loss of naturally-occurring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and 340 mg kg⁻¹ were inoculated with an equal number (1×10⁵ cells g⁻¹ soil) of seven different, well-characterized isolates of R. leguminosarum bv. viciae. Four of the isolates were from an uncontaminated control plot (total Zn 54 mg kg⁻¹) and three were from a metal-contaminated plot (total Zn 340 mg kg⁻¹).After 1 year the population size was between 10⁶ and 10⁷ g⁻¹ soil, and remained at this level in all but the most contaminated soil. In the soil from the most contaminated plot no initial increase in rhizobial numbers was seen, and the population declined after 1 year to <30 cells g⁻¹ soil after 4 years. One isolate originally from uncontaminated soil that had five large plasmids (no. 2-8-27) was the most abundant type re-isolated from all of the soils. Isolates originally from the metal-contaminated soils were only recovered in the most contaminated soil. After 1 year, four isolates with plasmid profiles distinct from those inoculated into the soils were recovered. One isolate in the control soil appeared to have lost a plasmid. Three isolates from heavy metal contaminated soils (one isolate from the soil with total Zn 208 mg kg⁻¹ and two isolates from the soil with total Zn 340 mg kg⁻¹) had all acquired one plasmid. Plasmid transfer was confirmed using the distinct ITS-RFLP types of the isolates and DNA hybridization using probes specific to the transferred plasmid. The transconjugant of 2-8-27 which had gained a plasmid was found in one replicate after 2 years of the most contaminated soil but comprised more than 50% of the isolates. A similar type appeared in a separate replicate of the most contaminated soil after 3 years and persisted in both of these soils until the final sampling after 4 years. After 2 years isolates were recovered from four of the soil replicates with the chromosomal type of 2-8-27 which appeared to have lost one plasmid, but these were not recovered subsequently.Isolate 2-8-27 was among the isolates most sensitive to Zn in laboratory assays, whereas isolate 7-13-1 showed greater zinc tolerance. Acquisition of the plasmid conferred enhanced Zn tolerance to the recipients, but transconjugant isolates were not as metal tolerant as 7-13-1, the putative donor. Laboratory matings between 2-8-27 and 7-13-1 in the presence of Zn resulted in the conjugal transfer of the same small plasmid from 7-13-1 to isolate 2-8-27 and the transconjugant had enhanced metal tolerance. Our results show that transfer of naturally-occurring plasmids among rhizobial strains is stimulated by increased metal concentrations in soil. We further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.     

Facilitation of pentachlorophenol degradation in the rhizosphere of ryegrass (Lolium perenne L.)

The phytoremediation of xenobiotics depends upon plant-microbe interactions in the rhizosphere, but the extent and intensity of these effects are currently unknown. To investigate rhizosphere effects on the biodegradation of xenobiotics, a glasshouse experiment was conducted using a specially designed rhizobox where ryegrass seedlings were grown for 53 days in a soil spiked with pentachlorophenol (PCP) at concentrations of 8.7±0.5 and 18±0.5 mg kg⁻¹ soil. The soil in the rhizobox was divided into six separate compartments at various distances from the root surface. Changes in PCP concentrations with increasing distance from the root compartment of the rhizobox were then assessed. The largest and most rapid loss of PCP in planted soil was at 3 mm from the root zone where total PCP decreased to 0.20 and 0.65 mg kg⁻¹, respectively with the two PCP treatments. The degradation gradient followed the order: near-rhizosphere>root compartment>far-rhizosphere soil zones for both concentrations where ryegrass was grown. In contrast, there was no difference in PCP concentration with distance in the unplanted soil. The increases in both soil microbial biomass carbon and the activities of soil urease and phosphatase were accompanied by the enhanced degradation of PCP, which was higher in the near-rhizosphere than far-rhizosphere soil. The results suggest that the effect of root proximity is important in the degradation of xenobiotics such as PCP in soil.     

Bacterial inoculants affecting nickel uptake by Alyssum murale from low, moderate and high Ni soils

Metal hyperaccumulator plants like Alyssum murale have a remarkable ability to hyperaccumulate Ni from soils containing mostly insoluble Ni. We have shown some rhizobacteria increase the phytoavailability of Ni in soils, thus enhancing Ni accumulation by A. murale. Nine bacterial strains, originally isolated from the rhizosphere of A. murale grown in serpentine Ni-rich soil, were examined for their ability to solubilize Ni in different soils and for their effect on Ni uptake into Alyssum. Microbacterium oxydans AY509223; Rhizobium galegae AY509213; Microbacterium oxydans AY509219; Clavibacter xyli AY509236; Acidovorax avenae AY512827; Microbacterium arabinogalactanolyticum AY509225; M. oxydans AY509222; M. arabinogalactanolyticum AY509226 and M. oxydans AY509221 were added to low, moderate and high Ni-contaminated soils. M. oxydans AY509223 significantly increased Ni extraction by 10 mM Sr(NO₃)₂ from the high and medium soils and had no effect on Ni extraction from the low Ni soils. The other eight bacterial isolates significantly increased Ni extraction from all soils. There were no significant effects of bacterial inoculation on fresh and dry weight of A . murale shoots grown in the low and high Ni soils compared to an unamended control. M. oxydans AY509223 significantly increased Ni uptake of A. murale grown in the low, medium, and high soils by 36.1%, 39.3%, and 27.7%, respectively, compared with uninoculated seeds. M. oxydans AY509223 increased foliar Ni from the same soils from 82.9, 261.3 and 2829.3 mg kg⁻¹ to 129.7, 430.7, and 3914.3 mg kg⁻¹, respectively, compared with uninoculated controls. These results show that bacteria are important for Ni hyperaccumulation and could potentially be developed as an inoculum for enhancing uptake during commercial phytoremediation or phytomining of Ni.     

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.     

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.     

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.     

Rhizosphere priming of soil organic matter by bacterial groups in a grassland soil

Plants often impact the rate of native soil organic matter turnover through root interactions with soil organisms; however the role of root-microbial interactions in mediation of the “priming effect” is not well understood. We examined the effects of living plant roots and N fertilization on belowground C dynamics in a California annual grassland soil (Haploxeralf) during a two-year greenhouse study. The fate of ¹³C-labeled belowground C (roots and organic matter) was followed under planted (Avena barbata) and unplanted conditions, and with and without supplemental N (20 kg N ha⁻¹ season⁻¹) over two periods of plant growth, each followed by a dry, fallow period of 120 d. Turnover of belowground ¹³C SOM was followed using ¹³C-phospholipid fatty acid (PLFA) biomarkers. Living roots increased the turnover and loss of belowground ¹³C compared with unplanted soils. Planted soils had 20% less belowground ¹³C present than in unplanted soils after 2 cycles of planting and fallow. After 2 treatment cycles, unlabeled soil C was 4.8% higher in planted soils than unplanted. The addition of N to soils decreased the turnover of enriched belowground ¹³C during the first treatment season in both planted and unplanted soils, however no effect of N was observed thereafter. Our findings suggest that A. barbata may increase soil C levels over time because root and exudate C inputs are significant, but that increase will be moderated by an overall faster C mineralization rate of belowground C. N addition may slow soil C losses; however, the effect was minor and transient in this system. The labeled root-derived ¹³C was initially recovered in gram negative (highest enrichment), gram positive, and fungal biomarkers. With successive growing seasons, the labeled C in the gram negative and fungal markers declined, while gram positive markers continued to accumulate labeled belowground C. The rhizosphere of A. barbata shifted the microbial community composition, resulting in greater abundances of gram negative markers and lower abundances of gram positive, actinobacteria and cyclopropyl PLFA markers compared to unplanted soil. However, the longer-term utilization of labeled belowground C by gram positive bacteria was enhanced in the rhizosphere microbial community compared with unplanted soils. We suggest that the activities of gram positive bacteria may be major controllers of multi-year rhizosphere-related priming of SOM decomposition.     

Response of microbial activity and microbial community composition in soils to long-term arsenic and cadmium exposure

Arsenic (As) and cadmium (Cd) in soils can affect soil microbial function and community composition and, therefore, may have effects on soil ecosystem functioning. The aim of our study was to assess the effects of long-term As and Cd contamination on soil microbial community composition and soil enzyme activities. We analyzed soils that have been contaminated 25 years ago and at present still show enhanced levels of either As, 18 and 39 mg kg⁻¹, or Cd, 34 and 134 mg kg⁻¹. Soil without heavy metal addition served as control. Polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE) showed that bacterial community composition in As and Cd contaminated soils differed from that in the control soil. The same was true for the microbial community composition assessed by analysis of respiratory quinones. Soil fungi and Proteobacteria appeared to be tolerant towards As and Cd, while other groups of bacteria were reduced. The decline in alkaline phosphatase, arylsulphatase, protease and urease activities in the As- and Cd-contaminated soils was correlated with a decrease of respiratory quinones occuring in Actinobacteria and Firmicutes. Xylanase activity was unaffected or elevated in the contaminated soils which was correlated with a higher abundance of fungal quinones, and quinones found in Proteobacteria.     

Performance of para-nitrophenyl phosphate and 4-methylumbelliferyl phosphate as substrate analogues for phosphomonoesterase in soils with different organic matter content

Phosphomonoesterase (PMEase) activity plays a key role in nutrient cycling and is a potential indicator of soil condition and ecosystem stress. We compared para-nitrophenyl phosphate (pNPP) and 4-methylumbelliferyl phosphate (MUP) as substrate analogues for PMEase in 7 natural ecosystem soils and 8 agricultural top soils with contrasting C contents (8.0-414 g kg⁻¹ C) and pH (3.0-7.5). PMEase activities obtained with pNPP (0.05-5 μmol g⁻¹ h⁻¹) were significantly less than activities obtained with MUP (0.9-13 μmol g⁻¹ h⁻¹), especially in soils with a high organic matter content (>130 g kg⁻¹). Only PMEase activities assayed with MUP correlated significantly with total C and total N (r=0.7, P<0.01 all), and pH (r=−0.71, P<0.01). PMEase activities obtained with the two substrate analogues were correlated when expressed on a C-content basis (r=0.8, P<0.001), but not when expressed on an oven-dry soil weight basis. This indicated that interference by organic matter is related to the quantity rather than to the quality of organic matter. Overall, assaying with MUP was more sensitive compared to assaying with pNPP, particularly in the case of high organic and acid soils.