The sensitivity of water chemistry to climate in a forested,nitrogen-saturated catchment recovering from acidification

Fluxes of major ions and nutrients were measured in the N-saturated mountain forest catchment-lake system of Čertovo Lake (Czech Republic) from 1998 to 2014. The lake has been rapidly recovering from atmospheric acidification due to a 90% decrease in sulphate (SO₄²⁻) deposition since the late 1980s and nitrate (NO₃⁻) contribution to the pool of strong acid anion and leaching of dissolved organic carbon (DOC) have increased. Present concentrations of base cations, phosphorus (P), total organic N (TON), and ionic (Al_i) and organically bound (Al_o) aluminium in tributaries are thus predominantly governed by NO₃⁻ and DOC leaching. Despite a continuing recovery lasting 25 years, the Čertovo catchment is still a net source of protons (H⁺), producing 44 mmol m⁻² yr⁻¹ H⁺ on a catchment-area basis (corresponding to 35 μmol L⁻¹ on a concentration basis). Retention of the deposited inorganic N in the catchment averages 20%, and ammonium consumption (51 mmol m⁻² yr⁻¹) and net NO₃⁻ production (28 mol m⁻² yr⁻¹) are together the dominant terrestrial H⁺ generating processes. In contrast, the importance of SO₄²⁻ release from the soils on terrestrial H⁺ production is continuously decreasing, with an average of 47 mmol m⁻² yr⁻¹ during the study. The in-lake biogeochemical processes reduce the incoming acidity by ∼40%, neutralizing 23 μmol L⁻¹ H⁺ (i.e., 225 mmol m⁻² yr⁻¹ on a lake-area basis). Denitrification and photochemical and microbial decomposition of DOC are the most important in-lake H⁺ consuming processes (50 and 39%, respectively), while hydrolysis of Al_i (from tributaries and photochemically liberated from Al_o) is the dominant in-lake H⁺ generating process. Because the trends in water chemistry and H⁺ balance in the catchment-lake system are increasingly related to variability in NO₃⁻ and DOC leaching, they have become sensitive to climate-related factors (drought, elevated runoff) and forest damage that significantly modify the leaching of these anions. During the study period, increased exports of NO₃⁻ (accompanied by Al_i and base cations) from the Čertovo catchment occurred after a dry and hot summer, after forest damage, and during elevated winter runoff. Increasing DOC export due to decreasing acid deposition was further elevated during years with higher runoff (and especially during events with lateral flow), and was accompanied by P, TON, and Al_o leaching. The climate-related processes, which originally “only” confounded chemical trends in waters recovering from acidification, may soon become the dominant variables controlling water composition in N-saturated catchments.     

Proton production from nitrogen transformation drives stream export of base cations in acid-sensitive forested watersheds

The biogeochemical cycles of nitrogen (N) and base cations (BCs), (i.e., K⁺, Na⁺, Ca²⁺, and Mg²⁺), play critical roles in plant nutrition and ecosystem function. Empirical correlations between large experimental N fertilizer additions to forest ecosystems and increased BCs loss in stream water are well demonstrated, but the mechanisms driving this coupling remain poorly understood. We hypothesized that protons generated through N transformation (PPR_N)—quantified as the balance of NH₄⁺ (H⁺ source) and NO₃⁻ (H⁺ sink) in precipitation versus the stream output will impact BCs loss in acid-sensitive ecosystems. To test this hypothesis, we monitored precipitation input and stream export of inorganic N and BCs for three years in an acid-sensitive forested watershed in a granite area of subtropical China. We found the precipitation input of inorganic N (17.71 kg N ha⁻¹ year⁻¹ with 54% as NH₄⁺–N) was considerably higher than stream exported inorganic N (5.99 kg N ha⁻¹ year⁻¹ with 83% as NO₃⁻–N), making the watershed a net N sink. The stream export of BCs (151, 1518, 851, and 252 mol ha⁻¹ year⁻¹ for K⁺, Na⁺, Ca²⁺, and Mg²⁺, respectively) was positively correlated (r = 0.80, 0.90, 0.84, and 0.84 for K⁺, Na⁺, Ca²⁺, and Mg²⁺ on a monthly scale, respectively, P < 0.001, n = 36) with PPR_N (389 mol ha⁻¹ year⁻¹) over the three years, suggesting that PPR_N drives loss of BCs in the acid-sensitive ecosystem. A global meta-analysis of 15 watershed studies from non-calcareous ecosystems further supports this hypothesis by showing a similarly strong correlation between ∑BCs output and PPR_N (r = 0.89, P < 0.001, n = 15), in spite of the pronounced differences in environmental settings. Collectively, our results suggest that N transformations rather than anions (NO₃⁻ and/or SO₄²⁻) leaching specifically, are an important mediator of BCs loss in acid-senstive ecosystems. Our study provides the first definitive evidence that the chronic N deposition and subsequent transformation within the watershed drive stream export of BCs through proton production in acid-sensitive ecosystems, irrespective of their current relatively high N retention. Our findings suggest the N-transformation-based proton production can be used as an indicator of watershed outflow quality in the acid-sensitive ecosystems.     

Long-term changes of ecosystem services at Solling,Germany: Recovery from acidification,but increasing nitrogen saturation?

Time series of values of ingenious parameters indicating ecosystem services from European beech and Norway spruce ecosystems at Solling, Germany, were evaluated with respect to resilient or adaptive behaviour. Studied indicators comprise the use of monitoring data with up to more than 40 years of observation on deposition of potential acidity, sulphate (SO₄²⁻) budgets, exchangeable base cation pools, Bc/Al ratio in soil solution, nitrogen (N) budgets, foliar nutrition as indicated by the foliar Bc/N ratio, and defoliation. Deposition of potential acidity decreased considerably at both ecosystems. SO₄²⁻ budgets reveal retention of sulphur in the soils affecting acid/base budgets. Exchangeable base cation pools decreased at both ecosystems by about 60%. Bc/Al ratio in soil solution in the mineral soil was mostly below critical limits indicating potential toxic stress to tree roots. N retention in the soils decreased from about 40 kg ha⁻¹ yr⁻¹ in the 1970s to currently very low rates of 0–20 kg ha⁻¹ yr⁻¹ indicating increasing N saturation. Foliar Bc/N ratio decreased at the spruce ecosystem indicating possible nutrient imbalances. Defoliation at both Solling ecosystems is on a high level compared to other forests in Germany, but reveals no distinct relation to soil acidification or N saturation. From the selected indicators, SO₄²⁻ and N budgets reveal resilient behaviour, whereas indicators related to the acid/base status tend to adaptive behaviour.     

Amperometric xanthine biosensors based on electrodeposition of platinum on polyvinylferrocenium coated Pt electrode

Novel xanthine biosensors were successfully fabricated by immobilizing xanthine oxidase on polyvinylferrocenium perchlorate matrix (PVF⁺ClO₄⁻) and platinum electrodeposited polyvinylferrocenium perchlorate matrix. PVF⁺ClO₄⁻ film was coated on Pt electrode at +0.7 V vs. Ag/AgCl by electrooxidation of polyvinylferrocene (PVF). Platinum nanoparticles were deposited on PVF⁺ClO₄⁻ electrode by electrochemical deposition in 2.0 mM H₂PtCl₆ solution at −0.2 V. Xanthine oxidase was incorporated into the polymer matrix via ion exchange process by immersing modified Pt electrodes in the enzyme solution. The amperometric responses of the biosensors were measured via monitoring oxidation current of hydrogen peroxide at +0.5 V. Under the optimal conditions, the linear ranges of xanthine detection were determined as 1.73 × 10⁻³–1.74 mM for PVF⁺XO⁻ and 0.43 × 10⁻³–2.84 mM for PVF⁺XO⁻/Pt. The detection limits of xanthine were 5.20 × 10⁻⁴ mM for PVF⁺XO⁻ and 1.30 × 10⁻⁴ mM for PVF⁺XO⁻/Pt. Moreover, the effects of applied potential, electrodeposition potential, H₂PtCl₆ concentration, amount of electrodeposited Pt nanoparticles, thickness of polymeric film, temperature, immobilization time, xanthine and xanthine oxidase concentrations on the response currents of the biosensors were investigated in detail. The effects of interferents, the operational and storage stabilities of biosensors and the applicabilities to drug samples of the biosensors analysis were also evaluated.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Soil nitrate accumulation explains the nonlinear responses of soil CO2 and CH4 fluxes to nitrogen addition in a temperate needle-broadleaved mixed forest

The responses of soil-atmosphere carbon (C) exchange fluxes to growing atmospheric nitrogen (N) deposition are controversial, leading to large uncertainty in the estimated C sink of global forest ecosystems experiencing substantial N inputs. However, it is challenging to quantify critical load of N input for the alteration of the soil C fluxes, and what factors controlled the changes in soil CO₂ and CH₄ fluxes under N enrichment. Nine levels of urea addition experiment (0, 10, 20, 40, 60, 80, 100, 120, 140 kg N ha⁻¹ yr⁻¹) were conducted in the needle-broadleaved mixed forest in Changbai Mountain, Northeast China. Soil CO₂ and CH₄ fluxes were monitored weekly using the static chamber and gas chromatograph technique. Environmental variables (soil temperature and moisture in the 0–10 cm depth) and dissolved N (NH₄⁺-N, NO₃⁻-N, total dissolved N (TDN), and dissolved organic N (DON)) in the organic layer and the 0–10 cm mineral soil layer were simultaneously measured. High rates of N addition (≥60 kg N ha⁻¹ yr⁻¹) significantly increased soil NO₃⁻-N contents in the organic layer and the mineral layer by 120%-180% and 56.4%-84.6%, respectively. However, N application did not lead to a significant accumulation of soil NH₄⁺-N contents in the two soil layers except for a few treatments. N addition at a low rate of 10 kg N ha⁻¹ yr⁻¹ significantly stimulated, whereas high rate of N addition (140 kg N ha⁻¹ yr⁻¹) significantly inhibited soil CO₂ emission and CH₄ uptake. Significant negative relationships were observed between changes in soil CO₂ emission and CH₄ uptake and changes in soil NO₃⁻-N and moisture contents under N enrichment. These results suggest that soil nitrification and NO₃⁻-N accumulation could be important regulators of soil CO₂ emission and CH₄ uptake in the temperate needle-broadleaved mixed forest. The nonlinear responses to exogenous N inputs and the critical level of N in terms of soil C fluxes should be considered in the ecological process models and ecosystem management.     

Batch and continuous fermentative production of hydrogen with anaerobic sludge entrapped in a composite polymeric matrix

Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrose-based synthetic wastewater was converted to H₂ using batch and continuous cultures. A novel composite polymeric material comprising polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H₂ production. Using the PMMA immobilized cells, the favorable conditions for batch H₂ fermentation were 35 °C, pH 6.0, and an 20 g COD l⁻¹ of sucrose, giving a H₂ production rate of 238 ml h⁻¹ l⁻¹ and a H₂ yield of 2.25 mol H₂ mol sucrose⁻¹. Under these optimal conditions, continuous H₂ fermentation was conducted at a hydraulic retention time (HRT) of 4–8 h, giving the best H₂-producing rate of 1.8 l h⁻¹ l⁻¹ (over seven-fold of the best batch result) at a HRT of 6 h and a H₂ yield of 2.0 mol H₂ mol sucrose⁻¹. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H₂ and CO₂. The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMA-immobilized-cell system developed in this work seems to be a promising H₂-producing process due to the high stability in continuous operations and the capability of achieving a competitively high H₂ production rate under a relatively low organic loading rate.     

Ecosystem functions as indicators for heathland responses to nitrogen fertilisation

Anthropogenic deposition of reactive nitrogen (N) has increased during the 20th century, and is considered an important driver of shifts in ecosystem functions and biodiversity loss. The objective of the present study was to identify those ecosystem functions that best evidence a target ecosystem’s sensitivity to N deposition, taking coastal heathlands as an example. We conducted a three-year field experiment in heathlands of the island Fehmarn (Baltic Sea, North Germany), which currently are subject to a background deposition of 9 kg N ha⁻¹ yr⁻¹. We experimentally applied six levels of N fertilisation (application of 0, 2.5, 5, 10, 20, and 50 kg N ha⁻¹ yr⁻¹), and quantified the growth responses of different plant species of different life forms (dwarf shrubs, graminoids, bryophytes, lichens) as well as shifts in the C:N ratios of plant tissue and humus horizons. For an applicability of the experimental findings (in terms of heathland management and critical load assessment) fertilisation effects on response variables were visualised by calculating the treatment ‘effect sizes’. The current year’s shoot increment of the dominant dwarf shrub Calluna vulgaris proved to be the most sensitive indicator to N fertilisation. Shoot increment significantly responded to additions of ≥ 5 kg N ha⁻¹ yr⁻¹ already in the first year, whereas flower formation of Calluna vulgaris increased only in the high-N treatments. Similarly, tissue C:N ratios of vascular plants (Calluna vulgaris and the graminoids Carex arenaria and Festuca ovina agg.) only decreased in the highest N treatments (50 and 20 kg N ha⁻¹ yr⁻¹, respectively). In contrast, tissue C:N ratios of cryptogams responded more quickly and sensitively than vascular plants. For example, Cladonia spp. tissue C:N ratios responded to N additions ≥ 5 kg N ha⁻¹ yr⁻¹ in the second study year. After three years we observed an increase in cover of graminoids and a corresponding decrease of cryptogams at N fertilisation rates of ≥ 10 kg N ha⁻¹ yr⁻¹. Soil C:N ratios proved to be an inappropriate indicator for N fertilisation at least within our three-year study period. Although current critical N loads for heathlands (10−20 kg N ha⁻¹ yr⁻¹) were confirmed in our experiment, the immediate and highly sensitive response of the current year’s shoots of Calluna vulgaris suggests that at least some ecosystem functions (e.g. dwarf shrub growth) also might respond to low (i.e. < 10 kg N ha⁻¹ yr⁻¹) but chronic inputs of N.     

Epiphytic lichen indication of nitrogen deposition and climate in the northern rocky mountains,USA

Lichen bioindication can provide economical and spatially extensive monitoring of climate and pollution impacts on ecological communities. We used non-metric multidimensional scaling of lichen community composition and generalized additive models to analyze regional climate and pollution gradients in the northern Rocky Mountains, U.S. Temperature extremes, relative humidity, and N-deposition were strongly related to lichen community composition. Eutrophic species (genera Physcia, Xanthomendoza, and Xanthoria) were associated with high N deposition, low precipitation, and temperature extremes. Estimated N deposition in our study ranged from <0.5 to 4.26 kg N ha⁻¹ year⁻¹ with degradation to lichen communities observed at 4.0 kg N ha⁻¹ year⁻¹, the indicated critical load. The resulting model can track changes in climate and N pollution related to lichen communities over time, identify probable sensitive or impacted habitats, and provide key information for natural resource management and conservation. The approach is broadly applicable to temperate ecosystems worldwide.     

Effects of intraspecific competition on growth and photosynthesis of Atriplex prostrata

We investigated the effect of intraspecific competition on growth parameters and photosynthesis of the salt marsh species Atriplex prostrata Boucher in order to distinguish the effects of density-dependent growth inhibition from salt stress. High plant density caused a reduction of 30% in height, 82% in stem dry mass, 80% in leaf dry mass, and 95% in root dry mass. High density also induced a pronounced 72% reduction in leaf area, 29% decrease in length of mature internodes and 50% decline in net photosynthetic rate. The alteration of net photosynthesis paralleled growth inhibition, decreasing from 7.6 ± 0.9 μmol CO₂ m⁻² s⁻¹ at low density to 3.5 ± 0.4 μmol CO₂ m⁻² s⁻¹ at high density, indicating growth inhibition caused by intraspecific competition is mainly due to a decline in net photosynthesis rate. Plants grown at high density also exhibited a reduction in stomatal conductance from 0.7 ± 0.1 mol H₂O m⁻² s⁻¹ at low density to 0.3 ± 0.1 mol H₂O m⁻² s⁻¹ at high density and a reduction in transpiration rate from 6.0 ± 0.3 mmol H₂O m⁻² s⁻¹ at low density to 4.3 ± 0.3 mmol H₂O m⁻² s⁻¹ at high density. Biomass production was inhibited by an increase in plant density, which reduced the rate of photosynthesis, stomatal conductance and leaf area of plants.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Increasing nitrogen depositions can reduce lichen viability and limit winter food for an endangered Chinese monkey

Increasing economic growth and industrial development in China is starting to impact even remote areas such as the Shennongjia nature reserve, where nitrogen pollution is becoming a major environmental threat. The epiphytic lichen flora is particularly rich in this area and is one of the components of this habitat most sensitive to nitrogen pollution. Since lichens represent an important food resource for the endangered monkey species Rhinopithecus roxellana, a reduction in lichen availability would have harmful consequences for the conservation of its habitat in the Shennongjia Mountains. To investigate the effects of increased nitrogen availability on the local lichen communities, so far scarcely considered, we conducted a one-year field experiment measuring growth, survival, and phosphomonoesterase activity of the widespread species Usnea luridorufa in response to nitrogen (up to 50 kg N ha⁻¹ year⁻¹ deposition) and phosphorus supply. Growth and survival of thalli and propagules of U. luridorufa decreased when treated with N deposition >12.05 kg N ha⁻¹ year⁻¹ and >2.14 kg N ha⁻¹ year⁻¹, respectively. The important role of phosphorus availability in relation to nitrogen supply was demonstrated by the increase in phosphomonoesterase activity with increasing nitrogen availability until a nitrogen toxicity threshold was reached. However, the high concentration of phosphorus in rainwater showed that phosphorus is not a limiting nutrient in the area.The results make a contribution to the knowledge of the negative effects of increased N deposition in the Shennongjia forest ecosystem.     

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Bioconversion of post harvest crop residues and cattle shed manure into value-added products using earthworm Eudrilus eugeniae Kinberg

The post-harvest residues of some local crops, e.g. wheat (Triticum aestivum), millets (Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were subjected to recycle through vermicomposting by using the epigeic earthworm Eudrilus eugeniae Kinberg, under laboratory conditions. The crop residues were amended with animal dung; and three types of vermibeds were prepared: (i) millet straw (S. vulgare + Pennisenum typhoides in equal quantity) + sheep manure (1:2 ratio) (MS), (ii) pulse bran (Vigna radiata) + wheat straw (Triticum aestivum) + cow dung (1:1:2 ratio) (PWC), and (iii) mixed crop residues (mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR + CD). The fourth treatment was cattle shed manure (CSM). At the end, ready vermicompost showed lower organic C content and higher concentrations of other important plant nutrients. Organic C content decreased in the order: MCR + CD (27.6%) > PWC (22.8%) > CMS (22.6%) > MS (19.4%). The ready vermicompost obtained from MCR + CD vermibed showed the maximum increase (% of initial level) in content of total N (143.4%), available P (111.1%) and exchangeable K (100.0%). The end product showed reduction in C:N ration between the ranges of 60.7% (CSM) and 70.3% (MCR + CD), at the end. The composting earthworm E. eugeniae exhibited the highest values of biological parameters: maximum mean individual biomass (1261.25 ± 7.0 mg), biomass gain (955.84 ± 11.03 mg), growth rate (10.62 ± 0.10 mg wt. worm⁻¹ day⁻¹), cocoon numbers (87.67 ± 6.51), and reproduction rate (0.66 ± 0.01 cocoons worm⁻¹ day⁻¹) in CSM container, while MS vermibeds showed the lowest values of these parameters. During experimentation, the maximum mortality for E. eugeniae was recorded in MS (16.67 ± 7.63%) followed by CSM > PWC > MCR + CD. Results indicated that the C:N ratio of the substrate drastically influenced the growth parameters of E. eugeniae, and it showed the close relations with maximum individual biomass gain (R² = 0.96), individual growth rate (R² = 0.82), and reproduction rate (cocoon worm⁻¹ day⁻¹) (R² = 0.72), in different treatments. This study clearly indicates that vermicomposting of crop residues and cattle shed wastes can not only produce a value-added product (vermicomposting) but at the same time acts as best culture medium for large-scale production of earthworms.     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.     

The simultaneous utilization of kinetic analysis and flow cytometry in the assessment of Lactobacillus rhamnosus ATCC 7469 physiological states produced by increasing oxygen limitation levels and lactic acid accumulation

Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h⁻¹ and 0.6 h⁻¹. At 0.45 h⁻¹, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD⁺ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D = 0.20 h⁻¹, 8% at D = 0.30 h⁻¹, 14% at D = 0.45 h⁻¹ and 26% for D = 0.62 h⁻¹, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.     

Two ways to achieve an anammox influent from real reject water treatment at lab-scale: Partial SBR nitrification and SHARON process

A comparative study to produce the correct influent for Anammox process from anaerobic sludge reject water (700–800 mg NH₄⁺-N L⁻¹) was considered here. The influent for the Anammox process must be composed of NH₄⁺-N and NO₂⁻-N in a ratio 1:1 and therefore only a partial nitrification of ammonium to nitrite is required. The modifications of parameters (temperature, ammonium concentration, pH and solid retention time) allows to achieve this partial nitrification with a final effluent only composed by NH₄⁺-N and NO₂⁻-N at the right stoichiometric ratio. The equal ratio of HCO₃⁻/NH₄⁺ in reject water results in a natural pH decrease when approximately 50% of NH₄⁺ is oxidised. A Sequencing batch reactor (SBR) and a chemostat type of reactor (single-reactor high activity ammonia removal over nitrite (SHARON) process) were studied to obtain the required Anammox influent. At steady state conditions, both systems had a specific conversion rate around 40 mg NH₄⁺-N g⁻¹ volatile suspended solids (VSS) h⁻¹, but in terms of absolute nitrogen removal the SBR conversion was 1.1 kg N day⁻¹ m⁻³, whereas in the SHARON chemostat was 0.35 kg N day⁻¹ m⁻³ due to the different hydraulic retention time (HRT) used. Both systems are compared from operational (including starvation experiments) and kinetic point of view and their advantages/disadvantages are discussed.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.