Ecology of aquatic Ranunculus communities under the Mediterranean climate

The Iberian Peninsula encompasses more than 80% of the species richness of European aquatic ranunculi. The floristic diversity of the phytocoenosis characterised by aquatic Ranunculus and the main physical–chemical factors of the water were studied in 43 localities of the central Iberian Peninsula. Four aquatic Ranunculus communities are found in most of the aquatic environments. These are species-poor and have an uneven distribution: three species of Batrachium are heterophyllous and their communities are distributed in different aquatic ecosystems on silicated substrates; one species is homophyllous and its community occurs in various aquatic ecosystems with carbonated waters. In the Mediterranean climate, Ranunculus species are present in different habitats, as shown by the results of all the statistical analyses. Ranunculus trichophyllus communities occur in base-rich waters with a high buffering capacity (2273.44 ± 794.57 mg CaCO₃ L⁻¹) and a high concentration of cations (Ca²⁺, 121 ± 33.12 mg L⁻¹; Mg²⁺, 71.64 ± 82.77 mg L⁻¹), nitrates (2.89 ± 4.80 mg L⁻¹), ammonium (2.19 ± 1.36 mg L⁻¹) and sulphates (216.25 ± 218.54 mg L⁻¹). Ranunculus penicillatus communities are found in flowing waters with a high concentration of phosphates (0.48 ± 0.6 mg L⁻¹) and intermediate buffering capacity (683.66 ± 446.76 mg CaCO₃ L⁻¹). Both Ranunculus pseudofluitans and Ranunculus peltatus communities grow in waters with low buffering capacity (R. pseudofluitans, 385.91 ± 209.2 mg CaCO₃ L⁻¹; R. peltatus, 263.3 ± 180.36 mg CaCO₃ L⁻¹), and a low concentration of cations (R. pseudofluitans: Ca²⁺, 12.57 ± 9.42 mg L⁻¹; Mg²⁺, 3.42 ± 1.67 mg L⁻¹; R. peltatus: Ca²⁺, 15 ± 18.26 mg L⁻¹; Mg²⁺, 6.26 ± 8.89 mg L⁻¹) and nutrients (R. pseudofluitans: nitrates, 0.23 ± 0.2 mg L⁻¹; phosphates, 0.09 ± 0.1 mg L⁻¹; R. peltatus: nitrates, 0.19 ± 0.21 mg L⁻¹; phosphates, 0.09 ± 0.12 mg L⁻¹); the first in flowing waters, the latter in still waters.     

A three-stage experimental constructed wetland for treatment of domestic sewage: First 2 years of operation

Hybrid constructed wetland systems have recently been used to treat wastewaters where high demand for removal of ammonia is required. However, these systems have not been used too often for small on-site treatment systems. This is because in many countries ammonia is not limited in the discharge from small systems. Hybrid systems have a great potential to reduce both ammonia and nitrate concentrations at the same time. In our study we employed a three-stage constructed wetland system consisting of saturated vertical-flow (VF) bed (2.5 m², planted with Phragmites australis), free-drained VF bed (1.5 m², planted with P. australis) and horizontal-flow (HF) bed (6 m², planted with Phalaris arundinacea) in series. All wetlands were originally filled with crushed rock (4-8 mm). However, nitrification was achieved only after the crushed rock was replaced with sand (0-4 mm) in the free-drain wetland. Also, original size of crushed rock proved to be too vulnerable to clogging and therefore, in the first wetlands the upper 40 cm was replaced by coarser fraction of crushed rock (16-32 mm) before the second year of operation started. The system was fed with mechanically pretreated municipal wastewater and the total daily flow was divided into two batches 12 h apart. The evaluation of the results from the period 2007 to 2008 indicated that such a system has a great potential for oxidation of ammonia and reduction of nitrate. The ammonia was substantially reduced in the free-drained VF bed and nitrate was effectively reduced in the final HF bed. The inflow mean NH₄-N concentration of 29.9 mg/l was reduced to 6.5 mg/l with the average removal efficiency of 78.3%. At the same time the average nitrate-N concentration rose from 0.5 to only 2.7 mg/l at the outflow. Removal of BOD₅ and COD amounted to 94.5% and 84.4%, respectively, with respective average outflow concentrations of 10 and 50 mg/l. Phosphorus was removed efficiently despite the fact that the system was not aimed at P removal and therefore no special media were used. Phosphorus removal amounted in 2008 to 65.4%, but the average outflow concentration of 1.8 mg/l is still high. The results of the present study indicate very efficient performance of the hybrid constructed wetlands, but optimal loading parameters still need to be adjusted. The capital cost of the experimental system is comparable to the conventional on-site treatment plant but the operations and maintenance costs are about one third of the conventional plant.     

Distribution of Macrophytes in Relation to Environmental Factors in the Ter River,N. E. Spain

Most of the tested water quality factors increase downstream coinciding with changes in the aquatic vegetation, from typical species of fast-flowing oligotrophic waters to those of slower-flowing eutrophic waters. Several statistic analyses were carried out to relate physical and chemical parameters with plant distribution. Five vegetation types are found in a longitudinal distribution along the river system. They are dominated by bryophytes in the initial parts before the reservoirs and by higher plants in the final parts after the reservoirs.     

Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly enhanced for the reduced S lignin cultivars.     

Disinfection and toxicological assessments of pulsed UV and pulsed-plasma gas-discharge treated-water containing the waterborne protozoan enteroparasite Cryptosporidium parvum

We report for the first time on the comparative use of pulsed-plasma gas-discharge (PPGD) and pulsed UV light (PUV) for the novel destruction of the waterborne enteroparasite Cryptosporidium parvum. It also describes the first cyto-, geno- and ecotoxicological assays undertaken to assess the safety of water decontaminated using PPGD and PUV. During PPGD treatments, the application of high voltage pulses (16 kV, 10 pps) to gas-injected water (N₂ or O₂, flow rate 2.5 L/min) resulted in the formation of a plasma that generated free radicals, ultraviolet light, acoustic shock waves and electric fields that killed ca. 4 log C. parvum oocysts in 32 min exposure. Findings showed that PPGD-treated water produced significant cytotoxic properties (as determined by MTT and neutral red assays), genotoxic properties (as determined by comet and Ames assays), and ecotoxic properties (as determined by Microtox™, Thamnotox™ and Daphnotox™ assays) that are representative of different trophic levels in aquatic environment (p < 0.05). Depending in part on the type of injected gas used, PPGD-treated water became either alkaline (pH ≤ 8.58, using O₂) or acidic (pH ≥ 3.21, using N₂) and contained varying levels of reactive free radicals such as ozone (0.8 mg/L) and/or dissociated nitric and nitrous acid that contributed to the observed disinfection and toxicity. Chemical analysis of PPGD-treated water revealed increasing levels of electrode metals that were present at ≤ 30 times the tolerated respective values for EU drinking water. PUV-treated water did not exhibit any toxicity and was shown to be far superior to that of PPGD for killing C. parvum oocysts taking only 90 s of pulsing [UV dose of 6.29 μJ/cm²] to produce a 4-log reduction compared to a similar reduction level achieved after 32 min PPGD treatment as determined by combined in vitro CaCo-2 cell culture-qPCR.     

Responses of wetland plants to ammonia and water level

Constructed wetland systems receiving animal wastewater may enhance water quality when designed, operated, and maintained properly. In the case of wetlands designed to treat animal waste, system effectiveness may be limited by high ammonia concentrations and inundation, conditions that can adversely affect macrophytic vegetation. We conducted a 4-month greenhouse experiment to assess the impact of ammonia concentration and water level on plants commonly used in constructed wetlands for treating animal waste. We examined the effects of ammonia concentration (0, 50, 100, 200 and 400 mg/l) on the growth and biomass production of Juncus effusus, Sagittaria latifolia, Schoenoplectus tabernaemontani, Typha angustifolia, and Typha latifolia. We also explored interactions between ammonia concentration (0, 50, 100, 200 and 400 mg/l) and water level (flooded and nonflooded conditions) for S. tabernaemontani and T. latifolia. We found that ammonia levels in excess of 200 mg/l inhibited growth for J. effusus, S. latifolia, and T. latifolia after a period of weeks, and levels in excess of 100 mg/l similarly inhibited growth for S. tabernaemontani. Ammonia levels in the range studied had an ambiguous effect on T. angustifolia. Affected species demonstrated similar fertilization/inhibition responses to increased ammonia, but important differences were noted between species. Flooded conditions of 10 cm did not significantly increase ammonia toxicity to S. tabernaemontani or T. latifolia. Our results emphasize the need for careful consideration of the species used in treatment wetlands, and suggest that management of ammonia concentration may enhance plant growth and system function.     

Acclimation and activity of ammonia-oxidizing bacteria with respect to variations in zinc concentration, temperature, and microbial population

Activity of ammonia-oxidizing bacteria (AOB) to simultaneous variation in Zn²⁺ concentration (0.01-3.5 mg/L), temperature (23-33 °C), and AOB concentration (3-30 × 10⁶ gene copies/mL) in a steel industry wastewater treatment plant was evaluated. Two equations were developed to describe the lag period (i.e., AOB acclimation) and ammonia oxidation rate (i.e., growth of the AOB) depending on the variables. AOB concentration and temperature both had significant effects on lag period and the ammonia oxidation rate. Zn²⁺ concentration only had a significant effect on ammonia oxidation rate at 5% α-level. There was a significant interaction between AOB concentration and temperature for both lag period and ammonia oxidation rate. The effects of the variables were not significant when AOB concentration was higher than 2.0 × 10⁷ copies/mL. There was no visible shift or changes in AOB communities based on DGGE analysis with amoA gene primers.     

Impacts of chlorination and heat shocks on growth, pigments and photosynthesis of Phaeodactylum tricornutum (Bacillariophyceae)

The main impacts of cooling water from thermal (nuclear) power plants on aquatic organisms were caused by chlorination and temperature increase. In this study, we investigated the impacts of residual chlorine and short-term heat shocks on growth, pigment contents and photosynthesis of Phaeodactylum tricornutum. Growth of P. tricornutum was completely inhibited; Chlorophyll a and carotenoids contents deceased about 63.3% and 61.4% in 24 h treated with 0.2 mg L^− 1 chlorine. The negative effects of chlorination increased with enhanced concentration and prolonged exposure time. Relative electrode transfer rate (rETR) of P. tricornutum was significantly suppressed when treated with 0.2 mg L^− 1 residual chlorine for 24 h. Furthermore, the effective quantum yield (F_v'/F_m') decreased first but then recovered with prolonged exposure when residual chlorine ranged between 0.1 and 0.2 mg L^− 1. The cells were less sensitive to heat shocks compared with chlorination: the rETR and F_v'/F_m' was suppressed only when the temperature exceeded 35 °C for 1 h. When P. tricornutum was exposed to chlorination combined with heat shocks, the rETR was further inhibited at 35 °C. It indicated that both chlorination and heat shocks had negative impacts on the primary producers living in discharging coastal waters; furthermore, there were synergistic effects of heat shocks on chlorination toxicity.     

Dissolved nitrogen in drinking water resources in Al-Mahareth village of Assir – Saudi Arabia

A water quality study was carried out on ground water wells, which serve as drinking water sources in farming communities in Al-Mahareth village of Assir region of the Kingdom of Saudi Arabia. The objective of this research was to determine concentrations of different forms of nitrogen in drinking water samples. Water samples were collected from these sources every 3 months (from January to December 2008) and analyzed for ammonia, nitrate and nitrite using the Plaintest Photometer Method. Results indicated that the annual mean concentration of nitrate, nitrite and ammonia varied from 23.09 to 25.06 mg/l, 0.006 to 0.36 mg/l and 0.008 to 0.179 mg/l, respectively. An important observation was that, in general, higher nitrate and nitrite concentrations were found during the rainy season compared to the dry season. Concentrations of these potentially toxic substances were below WHO acceptable limits for surface and ground waters, indicating that these water resources appear safe for drinking from a dissolved nitrogen perspective.     

Interactions of Taxol-producing endophytic fungus with its host (Taxus spp.) during Taxol accumulation

Endophytic fungi (Fusarium mairei) culture broth (EFCB) was added to cell suspension cultures of Taxus cuspidata. After 5 days, cultures of T. cuspidata given 4 ml of EFCB produced a maximal yield of 6.11 mg/l paclitaxel, with a release ratio of 75%, 2- and 6.8-fold, respectively, greater than the controls. The active element in EFCB is an exopolysaccharide of ∼79 kD. Endophytic fungi produced 0.19 mg/l of paclitaxel in its producing medium. However, when the supernatant of Taxus cell suspension cultures from day 20 was added to the paclitaxel-producing medium, the biomass of fungi decreased by 24% and the yield of paclitaxel by 45%. In a co-culture system of plant and fungus, the yield of paclitaxel (12.8 mg/l) was >2-fold higher than that in the EFCB-treatment system.     

The influence of water level on the growth and photosynthesis of Hydrocotyle ranunculoides L.fil.

Floating Pennywort (Hydrocotyle ranunculoides L.fil.), a native to North America and naturalized in Central and South America, is an invasive aquatic weed in western Europe and several other regions worldwide. H. ranunculoides settles primarily in stagnant to slow-flowing waters (e.g. ditches, canals, rivers, lakes and ponds). The species prefers sunny and nutrient-rich sites and forms dense interwoven mats, which can quickly cover the surface of infested waters. In this study, the effect of three different water levels on growth of Floating Pennywort was investigated. Plants were cultivated on high-nutrient soils under waterlogged, semi-drained and drained conditions. Highest relative growth rates (RGR) of 0.097±0.004 g g⁻¹ dw d⁻¹ were reached under waterlogged conditions. This was significantly higher than RGR of plants cultivated semi-drained and drained. Floating Pennywort showed some phenological adaptations to drained soil conditions, including significant differences in the relative amounts of leaf, petiole and shoot biomass, whilst the relative amount of root biomass was not significantly influenced by the water level. Furthermore, Floating Pennywort reached under drained conditions lower relative water contents (RWC) of leaves, petioles and shoots, a significant shorter length of internodes, a significant lower extent of shoot porosity (POR), a lower chlorophyll content and an increased Chl_a:Chl_b ratio. In addition, maximum gas exchange of drained cultivated plants is significantly lower, due to strongly decreased leaf conductance under reduced water availability. Overall, H. ranunculoides showed ability to grow under different water levels, but performed best under waterlogged conditions.     

Development of an autofluorescent organophosphates-degrading Stenotrophomonas sp. with dehalogenase activity for the biodegradation of hexachlorocyclohexane (HCH)

Simultaneous biodegradation of hexachlorocyclohexane (HCH) and organophosphates (OPs) by a recombinant Stenotrophomonas sp. was studied in the study. The broad-host-range plasmid pVGAB, harboring enhanced green fluorescent protein gene (egfp) and dehalogenase genes (linA and linB), was constructed and transformed into the OP-degrading strain Stenotrophomonas YC-1 to get the recombinant strain YC-H. Over-expression of dehalogenase (LinA and LinB) and enhanced green fluorescent protein (EGFP) was obtained in YC-H by determining their enzymatic activities and fluorescence intensity. YC-H was capable of rapidly and simultaneously degrading 10 mg/l γ-HCH and 100 mg/l methyl parathion (MP) determined by GC–ECD analysis. A bioremediation assay with YC-H inoculated into fumigated and nonfumigated soil showed that both 10 mg/kg γ-HCH and 100 mg/kg MP could be completely degraded within 32 days. The novel EGFP-marked bacterium could be potentially applied in the field-scale decontamination of HCH and OPs residues in the environment.     

Nitrogen removal from wastewater with a low COD/N ratio at a low oxygen concentration

The goal of the study was to determine the effectiveness of nitrification and denitrification and the kinetics of ammonia removal from a mixture of wastewater and anaerobic sludge digester supernatant in an SBR at limited oxygen concentration. In addition, the COD removal efficiency and sludge production were assessed.In the SBR cycle alternating aerobic and anaerobic phases occurred; in the aeration phase the dissolved oxygen (DO) concentration was below 0.7 mg O₂/L. The low DO concentration did not inhibit ammonia oxidation-nitrification and the efficiency was ca. 96-98%. However, a relatively high COD concentration in the effluent was detected. The values of K_m and V_max, calculated from the Michaelis-Menten equation, were 43 mg N-NH₄/L and 15.64 mg N-NH₄/L h, respectively. Activated sludge production was almost stable (0.62-0.66 g MLVSS/g COD). A high net biomass production resulted from a low specific biomass decay rate of 0.0015 d⁻¹.     

Performance and bacterial compositions of aged refuse reactors treating mature landfill leachate

Aged landfill leachates become more refractory over time and difficulty to treat. Recently, aged refuse bioreactors show great promise in treating leachates. In this study, aged refuse bioreactors were constructed to simulate landfill leachate degradation process. The characteristics of leachate were: COD_cr, ∼2200 mg/L; BOD₅, ∼280 mg/L; total nitrogen, ∼2030 mg/L; and ammonia, ∼1900 mg/L. Results showed that bioreactor could remove leachate pollutants effectively at hydraulic loading of 20 L/m³ d. The removal rate reduced when hydraulic loading doubled or temperature lowered. Effluent recirculation could alleviate the temperature effect. Combining aged refuse and slag biofilters could treat leachate more efficiently. Pyrosequencing analysis indicated that bacteria from Pseudomonas, Lysobacter, Bacillus and δ-proteobacter, Flexibacteraceae were more abundant in the samples. The Shannon index decreased at lower temperature, while evenness and equitability increased with recirculation. We suggest that filter medium and temperature may be the main factors for shaping bacterial community structure.     

Factors controlling vegetation structure in peatland lakes—Two conceptual models of plant zonation

Two conceptual models of plant zonation in peatland lakes are given. The first represents vegetation on slightly sloping substrate (N < 0.2) in shallow and relatively large lakes. The vegetation is not diverse (H′ = 0.0 ± 0.01). The frequency and biomass of the dominant (Sphagnum denticulatum) correlate positively with lake size, and negatively with depth and substrate slope. They are also correlated with water transparency and water color (r = −0.53), concentrations of total organic carbon (r = −0.43), Ca²⁺ (r = 0.40) and humic acids (r = −0.46), and redox potential (r = 0.44). The second model represents vegetation on steep peat walls (N > 0.3) in deep, usually small lakes. Plants occur only on the upper part of the peat wall or form a multispecies curtain hanging from the lip of peat at the top. Species diversity in this scenario is higher (H′ = 0.18 ± 0.17). The curtains usually are composed of mosses such as Warnstorfia exannulata, S. cuspidatum and S. riparium, and vascular plants are rare. The frequency and biomass of bryophytes in this type of structure are related to substrate slope (r = 0.56), lake depth (r = 0.56), Ca²⁺ concentration (r = −0.69) and water color (r = −0.51). In both models, plant biomass is correlated with temperature (r = −0.78), irradiance (r = −0.64) and water oxygenation (r = −0.54).     

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C₁₈ column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r² > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.     

Enhanced algae growth in both phototrophic and mixotrophic culture under blue light

Biomass productivity and fatty acid methyl esters (FAME) derived from intracellular lipid of a Nannochloropsis sp. isolated from Singapore’s coastal waters were studied under different light wavelengths and intensities. Nannochloropsis sp., was grown in both phototrophic and mixotrophic (glycerol as the carbon source) culture conditions in three primary monochromatic light wavelengths, i.e., red, green and blue LEDs, and also in white LED. The maximum specific growth rate (μ) for LEDs was blue > white > green > red. Nannochloropsis sp. achieved a μ of 0.64 and 0.66 d⁻¹ in phototrophic and mixotrophic cultures under blue lighting, respectively. The intracellular fatty acid composition of Nannochloropsis sp. varied between cultures exposed to different wavelengths, although the absolute fatty acid content did differ significantly. Maximum FAME yield from Nannochloropsis sp. was 20.45% and 15.11% of dry biomass weight equivalent under photo- and mixotrophic culture conditions respectively for cultures exposed to green LED (550 nm). However, maximum volumetric FAME yield was achieved for phototrophic and mixotrophic cultures (i.e., 55.13 and 111.96 mg/l, respectively) upon cell exposure to blue LED (470 nm) due to highest biomass productivity. It was calculated that incremental exposure of light intensity over the cell growth cycle saves almost 20% of the energy input relative to continuous illumination for a given light intensity.     

Purification and characterization of a β-1,3-glucomannanase expressed in Pichia pastoris

The glycoside hydrolase β-1,3-glucomannanase is an enzyme that specifically breaks the β-1,3 glycosidic bond of the glucomannan, the main cell wall constituent of some yeasts. In this work, a codon optimized DNA sequence of the MAN5C gene from Penicillium lilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein plMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0 mg/l and 224.0 mg/l, respectively. The purified protein had a specific activity of 14.6 U/mg at 37 °C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for plMAN5C were 50 °C and 4.5, respectively. The recombinant plMAN5C was efficient in lysis of the cell wall of the red yeast Rhodosporidium toruloides to form protoplast. Our work provided an effective system for heterogeneous production of β-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas.     

Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers

Overexpression of recombinant N-acetyl-d-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08 ± 0.02 U/mg, was similar to that of the native protein, 2.13 ± 0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41 ± 10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32 ± 5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.