Bioremediation of diesel-oil-contaminated alpine soils at low temperatures

Bioremediation of two diesel-oil-contaminated alpine subsoils, differing in soil type and bedrock, was investigated in laboratory experiments at 10 °C after supplementation with an inorganic fertilizer. Initial diesel oil contamination of 4000 mg kg⁻¹ soil dry matter (dm) was reduced to 380–400 mg kg⁻¹ dm after 155 days of incubation. In both soils, about 30 % of the diesel oil contamination (1200 mg kg⁻¹ dm) was eliminated by abiotic processes. The residual decontamination (60 %–65 %) could be attributed to microbial degradation activities. In both soils, the addition of a cold-adapted diesel-oil-degrading inoculum enhanced biodegradation rates only slightly and temporarily. From C/N and N/P ratios (determined by measuring the contents of total hydrocarbons, NH₄ ⁺ N, NO₃ ⁻ N and PO₄ ³⁻ P) of soils␣it could be deduced that there was no nutrient deficiency during the whole incubation period. Soil biological activities (basal respiration and dehydrogenase activity) corresponded to the course of biodegradation activities in the soils. Received: 9 September 1996 / Accepted: 7 December 1996     

Optimization of culture conditions for production of cellulases and xylanases by Scytalidium thermophilum using Response Surface Methodology

Summary Scytalidium thermophilum type culture Humicola insolens MTCC 4520 isolated from composting soil was optimized for production of cellulolytic and hemicellulolytic enzymes (endoglucanase, Avicel-adsorbable endoglucanase, FPase, β-glucosidase, xylanase and mannanase) by solid-state fermentation (SSF). Initial experiments showed that culture medium containing rice straw and wheat bran (1:3) as carbon source prepared in a synthetic basal medium supported maximal enzyme production at 45 °C. Further optimization of enzyme production was carried out using Box-Behnken design of experiments to study the influence of process variables (inoculum level, (NH₄)₂SO₄ and pH) on enzyme production. The response surface plots revealed the conditions for obtaining optimal enzyme levels. The models computed for R ² value ranged between 95% and 98.7% indicating they are appropriate and can be useful to predict the effect of inoculum level, (NH₄)₂SO₄ and pH on enzyme production. Under optimized conditions 62.5 ± 0.50, 23.0 ± 0.58, 3.0 ± 0.50, 151.00 ± 8.194, 196 ± 5.033 and 4.9 ± 0.32 (units/g substrate) of endoglucanase (EG), Avicel-adsorbable endoglucanase (AAEG), FPase, β-glucosidase, xylanase and mannanase were produced, respectively. Isoelectric focusing (IEF) of the crude extract showed that S. thermophilum produced six different EG isoforms, of which the EG corresponding to pI values of 8.4, 7.9 and 6.5 showed affinity for Avicel, thereby indicating the presence of a cellulose-binding domain (CBD). Furthermore, seven isoforms of β-glucosidase and ten multiple forms of xylanase distributed over a wide range of pI were also detected.     

Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi

New information on N uptake and transport of inorganic and organic N in arbuscular mycorrhizal fungi is reviewed here. Hyphae of the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) were shown to transport N supplied as ¹⁵N-Gly to wheat plants after a 48 h labelling period in semi-hydroponic (Perlite), non-sterile, compartmentalised pot cultures. Of the ¹⁵N supplied to hyphae in pot cultures over 48 h, 0.2 and 6% was transported to plants supplied with insufficient N or sufficient N, respectively. The increased ¹⁵N uptake at the higher N supply was related to the higher hyphal length density at the higher N supply. These findings were supported by results from in vitro and monoxenic studies. Excised hyphae from four Glomus isolates (BEG 84, 107, 108 and 110) acquired N from both inorganic (¹⁵NH₄ ¹⁵NO₃, ¹⁵NO₃ ⁻ or ¹⁵NH₄ ⁺) and organic (¹⁵N-Gly and ¹⁵N-Glu, except in BEG 84 where amino acid uptake was not tested) sources in vitro during short-term experiments. Confirming these studies under sterile conditions where no bacterial mineralisation of organic N occurred, monoxenic cultures of Glomus intraradices Schenk and Smith were shown to transport N from organic sources (¹⁵N-Gly and ¹⁵N-Glu) to Ri T-DNA transformed, AM-colonised carrot roots in a long-term experiment. The higher N uptake (also from organic N) by isolates from nutrient poor sites (BEG 108 and 110) compared to that from a conventional agricultural field implied that ecotypic differences occur. Although the arbuscular mycorrhizal isolates used contributed to the acquisition of N from both inorganic and organic sources by the host plants/roots used, this was not enough to increase the N nutritional status of the mycorrhizal compared to non-mycorrhizal hosts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol

 Pseudomonas sp. strain M285 immobilized on diatomaceous earth beads was used to remove 3,5,6-trichloro-2-pyridinol (TCP) from industrial wastewater. Batch studies showed that immobilized Pseudomonas sp. strain M285 mineralized [2,6-¹⁴C]TCP rapidly; about 75% of the initial radioactivity was recovered as ¹⁴CO₂. Transformation of TCP was inhibited by high concentrations of salt, and addition of osmoprotectants (proline and betaine at 1 mM) did not reduce the adverse effect of salt. TCP-containing wastewater (60–140 mg/l) was passed through columns containing immobilized Pseudomonas sp. strain M285 at increasing flow rates and increasing TCP concentrations; TCP removal of 80%–100% was achieved. Addition of nutrients, such as glucose and yeast extract, retarded TCP degradation. Growing cell cultures were found to be better inocula for immobilization than resting cells. Received: 5 February 1996 / Received last revision: 12 August 1996 / Accepted: 24 August 1996     

Occurrence and characterization of a nucleopolyhedrovirus from <Emphasis Type="Italic">Maruca vitrata</Emphasis> (Lepidoptera,Pyralidae) isolated in Taiwan

A nucleopolyhedrovirus (MaviMNPV) was isolated from diseased larvae of legume pod borer (LPB), Maruca vitrata, at Tainan in Taiwan. Electron microscopical studies on the ultrastructure of MaviMNPV occlusion bodies (OBs) showed several virions (up to 19) with multiple nucleocapsids (up to 6) packaged within a single viral envelope. The diameter of OBs was 0.9 to 1.3 μm with a mean of 1.152±0.116 μm. The complete sequence of the MaviMNPV polyhedrin (Polh) gene contained 735 nucleotides (GenBank accession number DQ399596). Phylogenetic analyses using the complete sequence of the Polh gene of MaviMNPV indicated that this virus clusters with Group I NPVs. The genome size of MaviMNPV estimated with restriction enzymes viz., HindIII, EcoRI, BglII and PstI was 113.41 ± 1.50 kbp. First instar LPB larvae were the most susceptible stage (LC₅₀ 2.053 × 10² OBs/ml) followed by second, third and fourth instars with the median lethal concentrations (LC₅₀s) 1.410 × 10³, 2.390 × 10³ and 2.636 × 10³ OBs/ml, respectively. This is the first record of this virus from this region. The first and second authors have equal contributions in this paper     

Evidence of adaptive tolerance to nickel in isolates of Cenococcum geophilum from serpentine soils

Selection for metal-tolerant ecotypes of ectomycorrhizal (ECM) fungi has been reported in instances of metal contamination of soils as a result of human activities. However, no study has yet provided evidence that natural metalliferous soils, such as serpentine soils, can drive the evolution of metal tolerance in ECM fungi. We examined in vitro Ni tolerance in isolates of Cenococcum geophilum from serpentine and non-serpentine soils to assess whether isolates from serpentine soils exhibited patterns consistent with adaptation to elevated levels of Ni, a typical feature of serpentine. A second objective was to investigate the relationship between Ni tolerance and specific growth rates (μ) among isolates to increase our understanding of possible tolerance/growth trade-offs. Isolates from both soil types were screened for Ni tolerance by measuring biomass production in liquid media with increasing Ni concentrations, so that the effective concentration of Ni inhibiting fungal growth by 50% (EC₅₀) could be determined. Isolates of C. geophilum from serpentine soils exhibited significantly higher tolerance to Ni than non-serpentine isolates. The mean Ni EC₅₀ value for serpentine isolates (23.4 μg ml⁻¹) was approximately seven times higher than the estimated value for non-serpentine isolates (3.38 μg ml⁻¹). Although there was still a considerable variation in Ni sensitivity among the isolates, none of the serpentine isolates had EC₅₀ values for Ni within the range found for non-serpentine isolates. We found a negative correlation between EC₅₀ and μ values among isolates (r = −0.555). This trend, albeit only marginally significant (P = 0.06), indicates a potential trade-off between tolerance and growth, in agreement with selection against Ni tolerance in “normal” habitats. Overall, these results suggest that Ni tolerance arose among serpentine isolates of C. geophilum as an adaptive response to Ni exposure in serpentine soils. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Québec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha^-1 P-superphosphate, or with strain 68 plus 70 kg ha^-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha^-1 P-superphosphate, or with strain P31 plus 35 kg ha^-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.     

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 10⁵ cells ml⁻¹, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l⁻¹) KNO₃ 0.41, Na₂HPO₄ 0.03, MgSO₄ · 7H₂O 0.246, CaCl₂ · 2H₂O 0.11, (in mg l⁻¹) Fe(III)citrate · H₂O 2.62, CoCl₂ · 6H₂O 0.011, CuSO₄ · 5H₂O 0.012, Cr₂O₃ 0.075, MnCl₂ · 4H₂O 0.98, Na₂MoO₄ · 2H₂O 0.12, SeO₂ 0.005 and (in μg l⁻¹]) biotin 25, thiamine 17.5 and B₁₂ 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis. Received: 10 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Cellular volume regulation in freshwater bivalves

The effect of ambient osmolality on the height of lateral ciliated cells from the gills of two freshwater bivalve species (Dreissena polymorpha, Toxolasma texasensis) was directly observed microscopically. The addition of 1 mmol · l⁻¹ KCl to an artificial pondwater (APW) superfusion medium resulted in an increase in cell height. When the superfusion solution was made hyperosmotic (∼90 mmol · kg⁻¹ H₂O) by the addition of 45 mmol · l⁻¹ NaCl to APW, the cell height decreased by about 20–30% and there was no evidence of a regulatory volume increase over 20–30 min. In contrast, when 1 mmol · l⁻¹ KCl was added to the hyperosmotic medium the cell height always partially (40–50%) recovered. When the gill tissue was returned to APW following the hyperosmotic treatment the cells returned to the original cell height. Bivalve gills superfused with the hyperosmotic NaCl and KCl solution in the presence of 1 mmol · l⁻¹ ouabain experienced a similar 25% decrease in cell height. When the ouabain-treated tissues were returned to APW the cells swelled, overshooting the original cell height. These results indicate these freshwater bivalves have a limited ability for cellular volume regulation using inorganic ions, but depend on a suitable balance of Na⁺ and K⁺ in the environment to effect regulatory volume changes. Accepted: 17 October 1997     

Effect of Abiotic Stress on Phosphate Solubilization by Biocontrol Fungus <Emphasis Type="Italic">Trichoderma</Emphasis> sp.

This study was undertaken to explore the role of Trichoderma sp. in phosphate (P) solubilization and antagonism against fungal phytopathogens. All fungal isolates (SE₆, KT₆, KT₂₈, and BRT₁₁) and a standard culture of T. harzianum (Th-std) were able to antagonize two fungal phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) of chickpea (Cicer arietinum L.) wilt complex. Transmission electron microscopic studies (TEM) further confirmed ultra-cytological changes in the sclerotia of S. rolfsii parasitized by Trichoderma sp. All fungal cultures exhibited production of NH₃ and siderophore, but only BRT₁₁, SE₆, and Th-std could produce HCN. Among all the cultures tested, isolate KT₆ was found to be most effective for solubilization of ferric phosphate releasing 398.4 μg ml⁻¹ phosphate while isolates BRT₁₁ and SE₆ showed more potential for tricalcium phosphate (TCP) solubilization releasing 449.05 and 412.64 μg ml⁻¹ phosphate, respectively, in their culture filtrates. Part of this study focused on the influence of abiotic stress conditions such as pH, temperature, and heavy metal (cadmium) on phosphate (TCP) solubilizing efficiency. Two selected cultures KT₆ and T. harzianum retained their P solubilizing potential at varying concentrations of cadmium (0–1000 μg ml⁻¹). Isolate KT₆ and standard culture of T. harzianum released 278.4 and 287.6 μg ml⁻¹ phosphate, respectively, at 1000 μg ml⁻¹cadmium. Maximum solubilization of TCP was obtained at alkaline pH and at 28°C temperature. Isolate BRT₁₁ was found most alkalo-tolerant releasing 448.0 μg ml⁻¹ phosphate at pH 9.     

Effect of rhizobia and rock biofertilizers with <Emphasis Type="Italic">Acidithiobacillus</Emphasis> on cowpea nodulation and nutrients uptake in a tableland soil

Chemical fertilizers have been used in the cultivation of plants due to their high solubility and effect on crops yield. Biofertilizers with phosphate rock (PR) and potash rock (KR) plus sulfur inoculated with Acidithiobacillus may improve plant growth and contribute to addition of available P and K in soil. The effectiveness of biofertilizers from phosphate and potash rocks mixed with sulfur and Acidithiobacillus was studied in a Typic Fragiuldult soil of the Brazilian Northeast Tableland. Cowpea (cv. “IPA 206”) was grown with and without rhizobia inoculation. Treatments were: (a) phosphate rock (1000 kg ha⁻¹); (b) Biofertilizers-B_P (250 and 500 kg ha⁻¹); (c) triple superphosphate-TSP (250 kg ha⁻¹); (d) potash rock (1000 kg ha⁻¹); (e) biofertilizer-B_K (250; 500 and 750 kg ha⁻¹); (f) potassium chloride-KCl (250 kg K₂0 ha⁻¹); (g) control without P or K fertilization (P₀K₀). The soil was maintained under water submersion covered with black plastic (solarization process) for a period of 30 days. Biofertilizers (B_p and B_K) and soluble fertilizers increased plant growth and NPK uptake. Biofertilizers reduced soil pH, especially when applied in highest rates. Biofertilizers and TSP+KCl showed the best values of available P and K in soil. Rhizobial inoculation was effective on cowpea, but no nodules were formed by bacteria native from the soil, probably due to the effect of the solarization process. From obtained PK biofertilizers could be used as alternative for cowpea fertilization in Tableland soils.     

Isolation and characterisation of phosphate solubilising microorganisms from the cold desert habitat of <Emphasis Type="Italic">Salix alba Linn.</Emphasis> in trans Himalayan region of Himachal Pradesh

Phosphate solubilising microorganisms (PSM) (bacteria and fungi) associated with Salix alba Linn. from Lahaul and Spiti valleys of Himachal Pradesh were isolated on Pikovskaya (PVK), modified Pikovskaya (MPVK) and National Botanical Research Institute agar (NBRIP) media by spread plating. The viable colony count of P-solubilising bacteria (PSB) and fungi (PSF) was higher in rhizosphere than that of non-rhizosphere. The frequency of PSM was highest on MPVK followed by NBRIP and PVK agar. The maximum proportion of PSM out of total bacterial and fungal count was found in upper Keylong while the least in Rong Tong. The PSB frequently were Gram-positive, endosporeforming, motile rods and belonged to Bacillus sp. The PSF mainly belonged to Penicillium sp., Aspergillus fumigatus, A. niger, A. spp. and non-sporulating sterile. Amongst the isolates with high efficiency for tricalcium phosphate (TCP) solubilisation, seven bacterial and seven fungal isolates dissolved higher amount of P from North Carolina rock phosphate (NCRP) than Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). However, the organisms solubilised higher-P in NBRIP broth than PVK broth. SBC5 (Bacillus sp.) and SBC7 (Bacillus sp.) bacterial isolates exhibited maximun P solubilisation (40 and 33 μg ml⁻¹ respectively) whereas FC28 (Penicillium sp.) isolate (52.3 μg ml⁻¹) amongst fungi while solubilising URP. The amount of P solubilised was positively correlated with the decrease in pH of medium. SBC5 (Bacillus sp.), SBC7 (Bacillus sp.) and SBC4 (Micrococcus) decreased the pH of medium from 6.8 to 6.08 while FC28 (Penicillium sp.) and FC39 (Penicillium sp.) isolates of fungi recorded maximum decrease in pH of medium from 6.8 to 5.96 in NBRIP broth.     

Growth of Indigenous Rhizobium leguminosarum and Rhizobium meliloti in Soils Amended with Organic Nutrients

The ability of indigenous Rhizobium leguminosarum and Rhizobium meliloti to use organic nutrients as growth substrates in soil was assessed by indirect bacteriophage analysis. A total of 17 organic compounds, including 9 carbohydrates, 3 organic acids, and 5 amino acids, were tested (1,000 μg g⁻¹) in three soils with different cropping histories. Four additional soils were screened with a glucose amendment. Nutrient amendments stimulated growth of indigenous rhizobia, allowing subsequent replication of indigenous bacteriophages. Phage populations were enumerated by plating soil extracts on 19 R. leguminosarum and 9 R. meliloti indicator strains, including root nodule isolates from the soils assayed. On the basis of indirect phage analysis, all soils contained native rhizobia similar to one or more of the indicator strains, although not all indicator strains were detected in soil. All organic compounds stimulated growth of indigenous rhizobia, but the growth response varied for each rhizobial strain depending on the nutrient, the nutrient concentration, and the soil. Indigenous rhizobia readily utilized most organic compounds except phenylalanine, glycine, and aspartic acid. The ability of indigenous rhizobia to utilize a wide range of organic compounds as growth substrates in situ indicates their ability to successfully compete with other soil bacteria for nutrients in these soils.     

Bird effects on organic processes in soils from five microhabitats on a nunatak with and without breeding snow petrels in Dronning Maud Land, Antarctica

Five microhabitat types with varying degrees of bird influence were examined. Soils were collected from open polygons, under mosses and bird nests on a nunatak with breeding snow petrels (Pagodroma nivea) and from open polygons and under mosses on a non-bird nunatak. Nutrient levels (total N and P, nitrate, nitrite and ammonia), moisture levels and δ ¹⁵N values were determined and the organic processes of nitrogen fixation (acetylene reduction) and soil respiration (CO₂ flux) were examined. Nests represented the most favourable microhabitat type for soil respiration having the highest nutrient levels and most favourable temperature and moisture regimes. The soils under mosses were also favourable and appear to act as a nutrient sink for nutrients originating from the nests. The open polygons were the least favourable for biological activity. There was little nitrogen fixation in any of the soils except for the soils under mosses from the non-bird nunatak. Fixation is possibly limited in favourable microhabitat types on the bird nunatak by high nitrogen levels. These results were confirmed by the δ ¹⁵N results, which had high values typical of a seabird signal in the soils from the bird nunatak and values near zero, typical of soils containing fixed nitrogen, on the non-bird nunatak. Received: 3 March 1997 / Accepted: 30 March 1998     

Changes and availability of P fractions following 65 years of P application to a calcareous soil in a Mediterranean climate

The fate and availability of P derived from granular fertilisers in an alkaline Calcarosol soil were examined in a 65-year field trial in a semi-arid environment (annual rainfall 325 mm). Sequential P fractionation was conducted in the soils collected from the trial plots receiving 0–12 kg P ha⁻¹crop⁻¹, and the rhizosphere soil after growing wheat (Triticum aestivum L. cv. Yitpi) and chickpea (Cicer arietinum L. cv. Genesis 836) for one or two 60-day cycles in the glasshouse. Increasing long-term P application rate over 65 years significantly increased all inorganic P (Pi) fractions except HCl–Pi. By contrast, P application did not affect or tended to decrease organic P (Po) fractions. Increasing P application also increased Olsen-P and resin-P but decreased the P buffer capacity and sorption maxima. Residual P, Pi and Po fractions accounted for an average of 32, 16 and 52% of total P, respectively. All soil P fractions including residual P in the rhizosphere soil declined following 60-day growth of either wheat or chickpea. The decreases were greater in soils with a history of high P application than low P. An exception was water-extractable Po, which increased following plant growth. Changes in various P fractions in the rhizosphere followed the same pattern for both plant species. Biomass production and P uptake of the plants grown in the glasshouse correlated positively with the residual P and inorganic fractions (except HCl–Pi) but negatively with Po in the H₂O-, NaOH- and H₂SO₄-fractions of the original soils. The results suggest that the long-term application of fertiliser P to the calcareous sandy soil built up residual P and non-labile Pi fractions, but these P fractions are potentially available to crops.     

Insulin and glucagon immunoreactivity during high-intensity exercise under opiate blockade

Eight fit men [maximum oxygen consumption (O_2max) 64.6 (1.9) ml · kg⁻¹ · min⁻¹, aged 28.3 (1.7) years (SE in parentheses) were studied during two treadmill exercise trials to determine the effect of endogenous opioids on insulin and glucagon immunoreactivity during intense exercise (80% O_2max). A double-blind experimental design was used with subjects undertaking the two exercise trials in counterbalanced order. Exercise trials were 20 min in duration and were conducted 7 days apart. One exercise trial was undertaken following administration of naloxone (N; 1.2 mg; 3 ml) and the other after receiving a placebo (P; 0.9% NaCl saline; 3 ml). Prior to each experimental trial a flexible catheter was placed into an antecubital vein and baseline blood samples were collected. Immediately after, each subject received either a N or P bolus injection. Blood samples were also collected after 20 min of continuous exercise (running). Glucagon was higher (P < 0.05), while insulin was lower (P < 0.05), during exercise compared with pre-exercise values in both trials. However, glucagon was higher (P < 0.05) in the P than in the N exercise trial [141.4 (8.3) ng · l⁻¹ vs 127.2 (7.6) ng · l⁻¹]. There were no differences in insulin during exercise between the P and N trials [50.2 (4.3) pmol · l⁻¹ vs 43.8 (5) pmol · l⁻¹]. These data suggest that endogenous opioids may augment the glucagon response during intense exercise. Accepted: 15 June 1996     

Bioremoval of organic and inorganic sulphur from coal samples

The microbial ecology of different Spanish coal samples has been studied. Several bacteria have been isolated from enrichment cultures and characterised and their biodesulphurization abilities evaluated. Using morphological and physiological properties, different isolates have been related to species of the Xanthomonas, Pseudomonas, Chryseomonas and Moraxella genera. Some of the isolates, B(30)15 and T(30)10, gave important levels of organic desulphurization, close to 70%. Other isolates, B(30)7 and B(30)8, were able to remove inorganic sulphur with high efficiencies, over 67%. One of the isolates, B(30)10, metabolically related to Xanthomonas maltophila, was able to remove both organic and inorganic sulphur at neutral pH, with efficiencies of 69% and 68% respectively. The results obtained underline the potential use of some of these strains for industrial coal desulphurization processes. Received: 26 June 1998 / Received revised: 1 October 1998 / Accepted: 2 October 1998     

The root surface phosphatases ofEriophorum vaginatum: Effects of temperature,pH, substrate concentration and inorganic phosphorus

Eriophorum vaginatum L. subsp.spissum (Fern.) Hult., a dominant plant in arctic tundra ecosystems, has acid phosphatase activity evenly distributed along its root surface from the root tip to a distance at least 16 cm from the tip. These root surface phosphatases have optimal activity from pH 3.5 to 4.0; mean soil pH for soil samples collected with roots was 3.69. Apparent energy of activation and Q₁₀ values (14.0 kcal mol⁻¹ and 2.2, respectively) do not provide evidence for temperature acclimation, but substantial phosphatase activity was measured at 1°C. Kinetic parameters determined for this root surface phosphatase were as follows: Km=9.23 mM, Vmax=1.61×10⁻³ μmoles mm⁻²h⁻¹. The presence of inorganic phosphorus in the assay medium did not inhibit root surface phosphatase activity except at very high concentrations (100 mM); even then, only slight inhibition was detected (7 to 19%). A comparison of hydrolysis rates with inorganic phosphate assimilation rates measured forE. vaginatum indicates that organic phosphate hydrolysis may occur at approximately one third the rate of inorganic phosphate absorption. Calculations show that inorganic phosphate produced by root surface phosphatase activity may satisfy 65% of the annual phosphate demand ofE. vaginatum. Since arctic tundra soils are typically higher in dissolved organic phosphorus compounds than in inorganic phosphate, root surface phosphatase activity may make a considerable contribution to the phosphate nutrition of this widespread and abundant arctic plant.