The Potential of the Ni-Resistant TCE-Degrading Pseudomonas putida W619-TCE to Reduce Phytotoxicity and Improve Phytoremediation Efficiency of Poplar Cuttings on A Ni-TCE Co-Contamination

To examine the potential of Pseudomonas putida W619-TCE to improve phytoremediation of Ni-TCE co-contamination, the effects of inoculation of a Ni-resistant, TCE-degrading root endophyte on Ni-TCE phytotoxicity, Ni uptake and trichloroethylene (TCE) degradation of Ni-TCE-exposed poplar cuttings are evaluated.

After inoculation with P. putida W619-TCE, root weight of non-exposed poplar cuttings significantly increased. Further, inoculation induced a mitigation of the Ni-TCE phytotoxicity, which was illustrated by a diminished exposure-induced increase in activity of antioxidative enzymes. Considering phytoremediation efficiency, inoculation with P. putida W619-TCE resulted in a 45% increased Ni uptake in roots as well as a slightly significant reduction in TCE concentration in leaves and TCE evapotranspiration to the atmosphere.

These results indicate that endophytes equipped with the appropriate characteristics can assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation. Furthermore, as poplar is an excellent plant for biomass production as well as for phytoremediation, the obtained results can be exploited to produce biomass for energy and industrial feedstock applications in a highly productive manner on contaminated land that is not suited for normal agriculture. Exploiting this land for biomass production could contribute to diminish the conflict between food and bioenergy production.     

Growth and Cadmium Phytoextraction by Swiss Chard,Maize, Rice,Noccaea caerulescens,and Alyssum murale in pH Adjusted Biosolids Amended Soils

Past applications of biosolids to soils at some locations added higher Cd levels than presently permitted. Cadmium phytoextraction would alleviate current land use constraints. Unamended farm soil, and biosolids amended farm and mine soils were obtained from a Fulton Co., IL biosolids management facility. Soils contained 0.16, 22.8, 45.3 mg Cd kg^–1 and 43.1, 482, 812 mg Zn kg^–1 respectively with initial pH 6.0, 6.1, 6.4. In greenhouse studies, Swiss chard (Beta vulgaris var. cicla), a Cd-accumulator maize (inbred B37 Zea mays) and a southern France Cd-hyperaccumulator genotype of Noccaea caerulescens were tested for Cd accumulation and phytoextraction. Soil pH was adjusted from ～5.5–7.0. Additionally 100 rice (Oryza sativa) genotypes and the Ni-hyperaccumulator Alyssum murale were screened for potential phytoextraction use.

Chard suffered phytotoxicity at low pH and accumulated up to 90 mg Cd kg^–1 on the biosolids amended mine soil. The maize inbred accumulated up to 45 mg Cd kg^–1 with only mild phytotoxicity symptoms during early growth at pH > 6.0. N. caerulescens did not exhibit phytotoxicity symptoms at any pH, and accumulated up to 235 mg Cd kg^–1 in 3 months. Reharvested N. caerulescens accumulated up to 900 mg Cd kg^–1 after 10 months. Neither Alyssum nor 90% of rice genotypes survived acceptably.

Both N. caerulescens and B37 maize show promise for Cd phytoextraction in IL and require field evaluation; both plants could be utilized for nearly continuous Cd removal. Other maize inbreds may offer higher Cd phytoextraction at lower pH, and mono-cross hybrids higher shoot biomass yields. Further, maize grown only for biomass Cd maximum removal could be double-cropped.     

The effect of harsh abiotic conditions on the diversity of serpentine plant communities on Lesbos,an eastern Mediterranean island

Background: Diversity patterns of plant communities are related to the environment, including productivity and patchiness of habitat.

Aims: To determine differences in diversity patterns between serpentine and non-serpentine communities.

Methods: A two-year study was conducted in native eastern Mediterranean grasslands. For each year 40 0.25 m² plots were sampled across four pairs of sites, each of which contained a serpentine and an adjacent non-serpentine plant community. Alpha and beta diversity (variation in species composition among plots within localities), species composition and biomass production were determined. Total soil elemental concentrations and pH were also measured.

Results: Serpentine habitats were shown to support a lower alpha diversity relative to non-serpentine habitatas on a per plot basis. Differences in alpha diversity between the two substrates were associated with variation in soil chemistry rather than above-ground biomass production. Serpentine habitats also exhibited lower beta diversity, which was unrelated to variation in biomass production. The two contrasting communities presented distinct species composition.

Conclusions: Differences in diversity patterns between serpentine and non-serpentine communities in the eastern Mediterranean are influenced by soil chemistry rather than biomass production.     

Climate and fragmentation affect forest structure at the southern border of Amazonia

Background: The remaining forests in the extensive contact zone between southern Amazonia (seasonal rain forest) and the Cerrado (savanna) biomes are at risk due to intense land-use and climate change.

Aims: To explore the vulnerability of these transitional forests to changes in land use and climate, we evaluated the effects of fragmentation and climatic variables on forest structure.

Methods: We measured the diameter and height of 14,185 trees with diameter ≥10 cm at 24 forest plots distributed over an area of 25,000 km². For each plot, we obtained data on contemporary fragmentation and climatic variables.

Results: Forest structure variables (height, diameter, height:diameter allometry, biomass) varied significantly both within and among plots. The height, H:D and biomass of trees were positively correlated with annual precipitation and fragment area.

Conclusions: The association between forest structure and precipitation indicates that these forests plots are likely to be vulnerable to dry season intensification anticipated for the southern edge of the Amazon. Additionally, the reduction in the fragment area may contribute to reductions in forest biomass and tree height, and consequently ecosystem carbon stocks. Given the likely susceptibility of these forests, urgent conservation action is needed to prevent further habitat degradation.     

Impact on plant communities by white-tailed deer in Mississippi,USA

Background: The abundance of white-tailed deer (Odocoileus virginianus) in the eastern United States has escalated during the twentieth century, potentially impacting plant communities.

Methods: We measured understorey plant cover and biomass five years after excluding deer from mature forests of three ecological regions in Mississippi, USA. We extended the significance of P values to 0.10 to detect developing impacts.

Results: Deer impacts were limited and varied by ecological region. We recorded 151 species in cover transects. Consistent exclosure treatment effects were detected in two regions where there was greater cover of two deer forages and less cover of three non-forages. Species richness was greater in exclosures in one region, but otherwise species richness and diversity indices did not differ. We recorded 127 species in biomass quadrats. Exclosure treatment effects on biomass were inconsistent. Out of five species with significant differences, three had more biomass in controls, including two deer forages. Except for greater total biomass in controls of one region, there were no differences by growth form or total vegetation for canopy coverage or biomass. Ordination of community canopy cover demonstrated similarity of paired exclosure and controls.

Conclusions: Exclosure treatment effects on canopy cover and species richness in two regions indicated limited negative impacts from deer foraging. A time frame of more than five years may be required for exclusion to allow recovery of vegetation, even with relatively open canopies and a long growing season.     

Inhibitory effects of Eucalyptus saligna leaf litter on grassland species: physical versus chemical factors

Background: In Southern Brazil, large areas of grassland have been replaced by Eucalyptus plantations. Vegetation is scarce under plantations which may be associated with Eucalyptus releasing of allelochemicals.

Aims: To investigate effects of Eucalyptus saligna leaf litter on the colonisation and development of the ground layer by grassland species and if these effects were related to allelopathy.

Methods: We assessed the effects of Eucalyptus litter on the establishment of species of the native grassland community and on seeded test species (Paspalum notatum and Lotus corniculatus – introduced). We tested the impact of the addition of E. saligna leaf litter, artificial leaves (shading and mechanical impediment) and shading. We also tested the phytotoxicity of the soil from E. saligna plantations.

Results: Species richness, diversity, plant height, cover and biomass were lower in E. saligna leaves and artificial leaves treatments than in the control (absence of litter). The test species showed lower biomass and higher mortality in treatment plots with E. saligna leaves, artificial leaves and shading treatments than in the control. Allelochemicals did not accumulate in soil at phytotoxic levels.

Conclusions: Eucalyptus saligna leaf litter suppresses the establishment of grassland vegetation, but the effects are mainly physical. Field evaluations with appropriate controls should be more extensively used in allelopathy investigations.     

Functional group dominance and not productivity drives species richness

Background: There is a lack of consensus about the productivity–richness relationship, with several recent studies suggesting that it is not productivity but other factors that are the important drivers that determine species richness.

Aims: Here, we examine the relationship between productivity, functional group dominance and plant species richness at the plot scale in Tibetan Plateau meadows. These alpine meadows are ideal to examine the species productivity-richness relationship because they have a very high species richness, a large gradient in productivity, and can be dominated by either graminoids (grasses and sedges) or forbs.

Methods: We measured plant species richness and above-ground biomass along a natural gradient of functional group abundance in 44 plots distributed across five natural, winter-grazed but otherwise undisturbed sites in the eastern part of the Qing-Hai Tibetan Plateau, in Gansu province, China in 2008.

Results: Graminoid abundance (i.e. graminoid biomass as percent of the total above-ground biomass) explained 39% of plot differences in species richness while neither productivity nor the biomass of the three most abundant plant species, either individually or combined, were a significant predictor of species richness.

Conclusions: Our results show that within these alpine meadows, a shift from graminoid to forb dominance, rather than the individual dominant species or productivity itself, is strongly correlated with species richness. Thus, differences in functional group abundance can be a strong driver of observed plant species richness patterns.     

Assessment of Heavy Metal Mobility in Dredged Sediments: Porewater Analysis,Single and Sequential Extractions

Dredged sediments from the Leie catchment (Belgium) that were deposited on land between 70 and 10 years ago were characterized according to their physico-chemical properties, total metal concentrations and porewater composition. Actual and potential heavy metal availability was assessed by means of single extractions with 0.01 M CaCl ₂ , 0.05 M EDTA and 0.43 M acetic acid. A modified version of the BCR (Community Bureau of Reference) extraction procedure, adapted to the elevated acid neutralizing capacity of the sediments, was applied. Information on element mobility was assessed and the used methodologies were critically evaluated.

The favorable physico-chemical characteristics of dredged sediments from the catchment of the Leie River (N-Belgium) indicated a rather low actual heavy metal mobility. Based on CaCl ₂ and EDTA extractions, the distribution of Cd, Zn and Ni between the solution and sorbed phase could be described as a function of pH and organic carbon content. Cd and Zn were found to be the elements with the highest potential availability, while Cu and Ni displayed a medium availability. It is stressed that different types of extractions deal with heavy metal mobility in a different way and that the measurement of “key-factors” such as the pH of the extracts can be helpful for the interpretation of the results.     

Assessment of the chitosan-functionalized graphene oxide as a carrier for loading thioguanine,an antitumor drug and effect of urea on adsorption process: Combination of DFT computational and molecular dynamics simulation studies

In this study, the interaction thioguanine (TG) anticancer drug with the functionalized graphene oxide (GO) nanosheet surface is theoretically studied in both gas phase and separately in physiological media using the density functional theory (DFT) calculations. DFT calculations indicated the adsorption and solvation energies are negative for f-GONS/TG complexes which propose the adsorption process of TG molecule onto the f-GONS surface is possible from the energetic viewpoint. QTAIM calculations confirm the nature of partially covalent-partially electrostatic between drug and nanosheet. These results are sorely relevant that an approach for loading of TG molecule is the chemical modification of GO using covalent functionalization which can serve as a nanocarrier to load drug molecules. Moreover, to understand the effect of urea on the nature of the interaction between TG and f-GONS, molecular dynamics (MD) simulation was employed. The results indicated that in the presence of urea the adsorption process gets affected and leads to instability of system, while the affinity of the TG for adsorption onto GO surface is increased in pure water.

Communicated by Ramaswamy H. Sarma     

Solubilization and Plant Uptake of Zinc and Cadmium from Soils Treated with Elemental Sulfur

In growth chamber experiments we studied the potential use of elemental sulfur (S₈) as an acidifying agent to enhance the uptake of Cd and Zn from three different polluted soils by candidate phytoremediation plants (Brassica juncea, Helianthus annuus, Salix viminalis). Two of the three soils were calcareous, the other slightly acidic. One of the calcareous soils had been contaminated by dust emissions from a nearby brass smelter. The pollution of the other two soils had resulted from sewage sludge applications.

Sulfur was added to soils in quantities of 20 to 400 mmol sulfur kg^-1 soil. Plants were grown under fluorescent light in 1.5 l ($OS 13 cm) pots for 28 d.

Within 700 h soil pH decreased significantly in all soils, depending on S₈ dosage. In the acid soil, pH decreased from pH 6.5 to about 4 at the highest treatment level, while pH in one of the calcareous soils dropped even below pH 4. The effect was smaller in the second calcareous soil.

NaNO₃-extractable Cd and Zn increased up to 26-and 13-fold, respectively, in the acid soil, while in the calcareous soils, maximum increases were 9-and 11-fold, respectively.

Increased NaNO₃-extractable concentrations translated well into shoot concentrations (dry matter) in plants. Shoot Zn concentrations in H. annuus, for example, increased from 930 in the controls to 4300 mg kg^-1 in the highest S₈ treatment. However, effects observed in the plants were generally smaller than in the soils. In addition, in some variants growth was negatively affected, resulting in reduced metal removal from the soils.     

Great Cormorants Phalacrocorax carbo feed on larger fish in late winter

Capsule: Great Cormorants Phalacrocorax carbo prey on larger fish in late winter compared to early winter.

Aims: This study aimed to describe Cormorant diet and to discover whether prey size changed during winter.

Methods: The diet of Cormorants was studied using regurgitated pellets collected from roosting places at the upper Elbe River, Czech Republic, during winter (from December to March).

Results: The diet consisted of 24 fish species from 6 fish families. Roach Rutilus rutilus dominated in the diet (over 50% of biomass). Size of fish in the diet increased over time during the whole winter. Except for European Chub Squalius cephalus, the increased size applied for the majority of the most frequently consumed fish species: Roach, European Perch Perca fluviatilis and White Bream Abramis brama. Cormorants consumed mostly fish species of lower commercial and angling value.

Conclusion: Fisheries management should reflect on the fact that competition for larger-sized fish is the highest in late winter and in early spring. For that reason, stocking of potentially vulnerable fish could be delayed to the time when overwintering birds leave the area and recreational fishing could be restricted in late winter.     

Tumor delivery of liposomal doxorubicin prepared with poly-L-glutamic acid as a drug-trapping agent

Context: Poly-l-glutamic acid (PGA) is an anionic polymer with a large number of carboxyl groups that can interact electrostatically with cationic drugs such as doxorubicin (DOX).

Objective: For stable encapsulation of DOX into liposomes, we prepared triethylamine (TEA)-PGA-liposomes using PGA as an internal trapping agent.

Methods: We prepared TEA-PGA-liposomes by remote loading of DOX with a TEA gradient into preformed liposomes prepared with 1, 2, or 4?mg/mL PGA (molecular weights 4800, 9800, and 20 500), and evaluated their biodistribution and antitumor effects on Lewis lung carcinoma (LLC) tumor-bearing mice.

Results: TEA-PGA-liposomes using the higher the molecular weight or concentration of PGA showed a slower release of DOX from the liposomes. TEA-PGA-liposomes prepared with a high concentration of PGA could enhance DOX accumulation in tumors and prolonged DOX circulation in the serum, indicating that DOX may be retained stably in the liposomal interior by interaction with PGA. Furthermore, injection of TEA-PGA-liposomes prepared with 4?mg/mL of PGA₉₈₀₀ or 2?mg/mL PGA₂₀₅₀₀ strongly inhibited tumor growth in LLC tumor-bearing mice.

Conclusions: PGA may be a potential trapping agent for liposomal DOX for tumor drug delivery.     

Water-induced stress influences the relative investment in cleistogamous and chasmogamous flowers of an invasive grass,Microstegium vimineum (Poaceae)

Background: Global climate change has the potential to shape evolutionary trajectories of invasive species via many routes, including through changes in mating systems. Many cleistogamous (CL) plants adjust investment in CL (selfed) vs. chasmogamous (CH, potentially outcrossed) progeny across environmental gradients. However, the details of such adjustments are lacking for highly invasive plant species.

Aims: We used a highly invasive grass, Microstegium vimineum, as a model for understanding how changes in water-induced stress (including potential associated changes in soil nutrient availability) might affect mating systems and thus evolutionary change in invasive species. We predicted that plants would respond to increased water-induced stress through a relative reduction in investment in CL vs. CH reproduction (i.e., a decrease in the CL:CH ratio).

Methods: Under greenhouse conditions, we measured fecundity (number of inflorescences and florets per plant) as well as relative investment in CL vs. CH florets (CL:CH ratios for number of inflorescences, florets per inflorescence, overall florets) in response to three watering treatments approximating mesic (low) to inundated (high) conditions.

Results: Plant biomass was significantly lower in high-watering treatment relative to intermediate and low treatments, indicating that the high-water condition was stressful. Contrary to expectations, stressed plants significantly increased relative investment in CL reproduction, a pattern associated with decreased inflorescence number and increased numbers of CL florets per inflorescence.

Conclusions: We conclude that changes in water-induced stress could strongly influence realised rates of outcrossing in this invasive plant, leading to mating system evolution, and altered invasiveness.     

Evaluation of Heavy Metals Pollution Loadings in the Sediments of the Ave River Basin (Portugal)

Sediment samples from the Ave river basin were collected with the aim of determining metal total pollution contents. Cr, Cu, Fe, Mn, Pb, Zn and VM at 550 °C were determined. Some physico-chemical parameters were also quantified in water samples collected in the water column just above the sediments.

Metal contamination factors (CF) indicated that sediments were not contaminated with Cu and Pb, slightly with Zn and moderately with Cr.

For sediments with high metal pollution loadings, the original BCR (Community Bureau of Reference) metal speciation protocol was also applied. Speciation studies showed that chromium was mainly associated with the oxidisable plus residual fractions (>85%). These results suggest that changes in the physico-chemical properties of the river water (e.g. pH, Eh) should not be accompanied by a significative release of chromium from sediments.

The relationships between chromium speciation fractions, physic-chemical parameters of the sediments and water samples were studied by Principal Component Analysis, and allowed to reduce the dimensionality of the data matrix from 14 to 3 significant components accounting for 89% of the variance. It was found that hydrous Fe/Mn oxides and organic matter are the “carriers” of chromium associated to fractions exchangeable and oxidable.     

Effect of Immobilizing Substances and Salinity on Heavy Metals Availability to Wheat Grown on Sewage Sludge-Contaminated Soil

The objective of the investigation was to evaluate the effect of immobilizing substances and NaCl salinity on the availability of heavy metals: Zn, Cd, Cu, Ni, and Pb to wheat (Triticum aestivum L.). In greenhouse pot experiment, a sewage sludge amended soil was treated with the following immobilizing substances: three clay minerals (Na-bentonite, Ca-bentonite and zeolite), iron oxides (goethite and hematite), and phosphate fertilizers (superphosphate and Novaphos). The pots were planted with wheat and were irrigated either with deionized or saline water containing 1600 mg L^?1 NaCl. Wheat was harvested two times for shoot metal concentrations and biomass measurements. Metal species in soil solution were estimated using the software MINEQL+.

The addition of metal immobilizing substances to the soil significantly decreased metal availability to wheat. The largest reduction in metal bioavailability was found for bentonites. The irrigation with saline water (1600 mg L^?1 NaCl) resulted in a significant increase in metal chloride species (MCl⁺ and MCl₂ ⁰). The highest metal complexation with Cl occurred for Cd, which was about 53% of its total soil solution concentration. The total concentration of Cd (Cd_T) in soil solution increased by 1.6–2.8-fold due to saline water. The NaCl salinity caused a significant increase in uptake and shoot concentration of Cd for two harvests and small but significant increase in shoot Pb concentration for the second harvest. It was concluded that the use of bentonites is the most promising for the reduction of heavy metal availability to plants. Saline water containing 1600 mg L^{? 1} NaCl increased the availability of Cd and Pb to wheat and decreased the efficiency of bentonites to immobilize soluble Cd.     

Experimental evidence from Cantabrian mountain heathlands suggests new recommendations for management of Vaccinium myrtillus L

Background: Management actions in mountain heathlands oriented to increasing the cover of Vaccinium myrtillus promote mowing of Calluna vulgaris to avoid competition. However, such action ignores the fact that plant–plant interactions range from competition to facilitation under different stress conditions.

Aims: To test whether the interactions between these two species are related to competition or facilitation, which would change the perspective for their management in mountain heathlands.

Methods: A total of 40 experimental plots located in a montane heathland (northern Spain) were selected. Calluna or Vaccinium plants were either cut at ground level or not cut, and plots were fertilized with nitrogen or not in a three-factorial design. Vegetative and reproductive shoot mass and length of the current year’s shoots were estimated in two consecutive years for both species. Herbivory on Vaccinium by browsing ungulates was compared in control plots and plots where Calluna had been cut 2 yrs ago.

Results: The two study species co-occurred in 72.4% of the sampled plots, although Calluna was more abundant. Vaccinium growth was greater in plots with Calluna than in plots where Calluna had been cut. Browsing on Vaccinium was also higher in plots were Calluna had been cut, and the effect of fertilization was only significant on the reproductive biomass of Calluna.

Conclusions: Contrary to initial proposals, our results suggest that Vaccinium does not always benefit from the cutting of Calluna in heathlands. Thus, the management of Cantabrian mountain heathlands should consider maintaining Calluna in order to facilitate the growth of Vaccinium.     

The role of stand structure and palm abundance in predicting above-ground biomass at local scale in southern Amazonia

Background: Most work in Amazonia has concentrated on dense lowland evergreen rain forest, a vegetation type with >40% cover. Large parts of southern Amazonia are covered by open evergreen lowland rain forest, physiognomically dominated by high abundance of palms. This vegetation type has received relatively little attention so far. Understanding the key predictors of above-ground biomass (AGB) across scales is important to accurately quantify the impacts of land cover change on the terrestrial carbon budget.

Aims: We assessed the structure of southern Amazonian forests, Brazil, to quantify the relative importance of variation in AGB caused by the abundance/density of palm species and by forest structure.

Methods: We stratified the landscape into homogeneous units in terms of vegetation types and elevation for using as a guide for plot establishment. We used the variation partitioning technique to decompose the relative contribution of forest structure and palm abundance.

Results: The AGB_community (including trees, palms and lianas) and AGB_tree (excluding palms and lianas) significantly decreased with increasing abundance of palms. The Attalea speciosa, a large-leaved palm species, was the most important for explaining the variance of AGB. The total variance of AGB_tree was partially explained by a redundant effect of A. speciosa and trees (28%) and by trees alone (62%), based on models of basal area. The redundant effect, alongside with additional analyses, indicated (1) competition between A. speciosa and small trees and (2) covariation between A. speciosa and large trees.

Conclusions: The abundance of palms plays a minor but significant role in predicting the AGB at the local scale in southern Amazonia.     

Feasibility of Phytoextraction to Clean Up Low-Level Uranium-Contaminated Soil

The potential to phytoextract uranium (U) from a sandy soil contaminated at low levels was tested in the greenhouse. Two soils were tested: a control soil (317 Bq ²³⁸U kg^-1) and the same soil washed with bicarbonate (69 Bq ²³⁸U kg^-1). Ryegrass (Lolium perenne cv. Melvina), Indian mustard (Brassica juncea cv. Vitasso), and Redroot Pigweed (Amarathus retroflexus) were used as test plants.

The annual removal of the soil activity with the biomass was less than 0.1%. The addition of citric acid (25 mmol kg^-1) 1 week before the harvest increased U uptake up to 500-fold. With a ryegrass and mustard yield of 15000 kg ha^-1 and 10000 kg ha^-1, respectively, up to 3.5% and 4.6% of the soil activity could annually be removed with the biomass.

With a desired activity reduction level of 1.5 and 5 for the bicarbonate washed and control soil, respectively, it would take 10 to 50 years to attain the release limit.

A linear relationship between the plant ²³⁸U concentration and the ²³⁸U concentration in the soil solution of the control, bicarbonate-washed, or citric acid-treated soil points to the importance of the soil solution activity concentration in determining U uptake and hence to the importance of solubilising agents to increase plant uptake.

However, citric acid addition resulted in a decreased dry weight production (all plants tested) and crop regrowth (in case of ryegrass).     

Effects of microtopography and water table on Sphagnum palustre L. in subtropical high mountains and implications for peatland restoration

Introduction. Human disturbance has recently led to increasingly serious destruction of Sphagnum L. wetlands in subtropical high mountains, resulting in an urgent need for wetland restoration.

Methods. Through a field experiment conducted in western Hubei Province, China, the effects of four different microtopographic types [concave surface, convex surface, concave and convex surface (CC surface), and flat surface] and water table depth (0 to ?30?cm) on three growth indicators (number of capitula, coverage and biomass) of Sphagnum palustre L. were examined. The objective was to identify the optimal hydrological conditions for S. palustre growth and thus facilitate its rapid recolonisation and restoration of these wetlands.

Key results. The results showed that different microtopographic conditions significantly influenced S. palustre growth. Among them, S. palustre in the CC surface showed the worst growth, while no significant differences existed among the other three microtopographic types. Additionally, as the water table increased, the growth of S. palustre increased, but long-term flooding impeded growth. The water table affected S. palustre growth via effects on its tissue water content.

Conclusions. Microtopographic reshaping was not essential for the success of S. palustre recolonisation, and microtopography that maintained the water table to within ?10?cm of the surface without flooding were best, independent of the microtopographic types. In addition, the growth patterns of S. palustre changed with changes in the environment, which may be related to its long-term adaptation to conditions of a lower water table.     

Effect of lyophilization on liposomal encapsulation of salmon calcitonin

Purpose: The intent of this work was to assess the impact of lyophilization on the encapsulation of salmon calcitonin (sCT) into liposomes.

Methods: Four different liposomal formulations were investigated, i.e. DPPC:Chol:DSPE-PEG₂₀₀₀ (75:20:5 and 65:30:5) and DPPC:Chol (80:20 and 66.7:33.3). Lipid films were prepared and hydrated with loading buffer containing sCT and different concentrations of the cryoprotectant, trehalose dihydrate. The liposomes were lyophilized, reconstituted and extruded to obtain small unilamellar vesicles. Non-encapsulated sCT was separated by gel filtration. Non-lyophilized formulations and liposomes lyophilized without the cryoprotectant were used as controls. Liposomes were analyzed for particle size, polydispersity index, zeta-potential and encapsulation efficiency. ³¹P-NMR (phosphorous nuclear magnetic resonance spectroscopy) was performed on selected formulations.

Results: Post-lyophilization, no significant change in particle sizes and zeta-potentials were noted, regardless of the presence or absence of the cryoprotectant. Encapsulation efficiencies, however, increased following lyophilization, in both PEGylated (lyophilization control batch) and non-PEGylated liposomes (cryoprotectant batches only). ³¹P-NMR revealed the presence of two distinct vesicle populations – liposomes and micelles – in PEGylated formulation. The presence of micelles might be responsible for the observed encapsulation enhancement of sCT in the PEGylated formulation.

Conclusions: Lyophilization resulted in an increase in encapsulation efficiency of sCT in PEGylated liposomes, even in the absence of a cryoprotectant, due to presence of micellar vesicles.