Criteria to distinguish between natural situations and illegal use of boldenone,boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap?). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L^?1 and 0.4 μg L^?1 on triple quadrupole and 0.1 μg L^?1 and 0.2 μg L^?1 on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

Determination of Cr(VI) and Cr(III) species in parenteral solutions using a nanostructured material packed-microcolumn and electrothermal atomic absorption spectrometry

A sequential on-line preconcentration and separation system for Cr(VI) and Cr(III) species determination was developed in this work. For this purpose, a microcolumn filled with nanostructured α-alumina was used for on-line retention of Cr species in a flow-injection system. The method involves the selective elution of Cr(VI) with concentrated ammonia and Cr(III) with 1 mol L⁻¹ nitric acid for sequential injection into an electrothermal atomic absorption spectrometer (ETAAS).Analytical parameters including pH, eluent type, flow rates of sample and eluent, interfering effects, etc., were optimized. The preconcentration factors for Cr(VI) and Cr(III) were 41 and 18, respectively. The limit of detection (LOD) was 1.9 ng L⁻¹ for Cr(VI) and 6.1 ng L⁻¹ for Cr(III). The calibration graph was linear with a correlation coefficient of 0.999. The relative standard deviation (RSD) was 8.6% for Cr(VI) and 6.1% for Cr(III) (c=10 μg L⁻¹, n=10, sample volume=25 mL). Verification of the accuracy was carried out by analysis of a standard reference material (NIST SRM 1643e “Trace elements in natural water”) with a reported Cr content of 20.40±0.24 μg L⁻¹. Using the proposed methodology the total Cr content, computed as sum of Cr(III) and Cr(VI), in this SRM was 20.26±0.96 μg L⁻¹. The method was successfully applied to the determination of Cr(VI) and Cr(III) species in parenteral solutions. Concentration of Cr(III) species was found to be in the range of 0.29–3.62 μg L⁻¹, while Cr(VI) species was not detected in the samples under study.     

Microalgae fiber optic biosensors for herbicide monitoring using sol–gel technology

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L⁻¹) and the broadest dynamic calibration range (19–860 μg L⁻¹) with IC₅₀ 125 ± 14 μg L⁻¹. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.     

Linoleic and α-linolenic fatty acids affect biomass and secondary metabolite production and nutritive properties of Panax ginseng adventitious roots cultured in bioreactors

The effect of elicitation with linoleic (C18:2) and α-linolenic (C18:3) fatty acids (LLA and α-LNA) was investigated in Panax ginseng C.A. Meyer adventitious roots cultured in 5 l balloon-type bioreactors. Fatty acids were added in culture medium at 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μmol l⁻¹ at day 40, at the end of exponential growth phase. Roots were harvested and assayed at day 47. Elicitation with both LLA and α-LNA enhanced accumulation of total polyphenolics and flavonoids in roots compared with control without elicitation. The highest accumulation of flavonoids was observed at 5.0 μmol l⁻¹ for both elicitors. Total phenolics production reached its highest value of about 4.0 mg g⁻¹ DW under the elicitation with 5.0 μmol l⁻¹ LLA and 5.0–20.0 μmol l⁻¹ α-LNA. Meanwhile, α-LNA was more effective than LLA for increasing biomass and ginsenoside production. The biomass of 11.1 g DW l⁻¹ and maximal total ginsenoside content of 7.9 mg g⁻¹ DW were achieved at 5 μmol l⁻¹ α-linolenic acid. The essential polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) fatty acids were accumulated in roots in response to elicitation while content of palmitic (C16:0) and oleic (C18:1) acids declined. The activities of SOD, G-POD and CAT were enhanced by two elicitors to similar extent while APX activity was preferably stimulated by α-LNA. Our results demonstrate that elicitation with α-linolenic acid stimulates production of biomass and secondary metabolites in bioreactor-cultured P. ginseng adventitious roots.     

Ergosterol contents of some wood-rotting basidiomycete fungi grown in liquid and solid culture conditions

Ergosterol contents of six wood-rotting basidiomycetes were analyzed under different cultivation conditions. Four white-rot and two brown-rot fungi were cultivated in liquid synthetic medium with low nutrient nitrogen (2 mM) and 0.1% glucose, and ergosterol in mycelial biomasses were measured weekly for 35 days. The highest ergosterol content per fungal dry mass in the white-rot fungi was found in Phanerochaete chrysosporium being 2100 μg g⁻¹, while in Ceriporiopsis subvermispora it was 1700 μg g⁻¹, Phlebia radiata 700 μg g⁻¹, and Physisporinus rivulosus 560 μg g⁻¹. In brown-rot fungi the ergosterol content was in Poria placenta 2868 μg g⁻¹ and in Gloeophyllum trabeum 3915 μg g⁻¹. On agar media, P. chrysosporium and P. radiata reached the highest ergosterol value in 7 days, while in wood block cultures the ergosterol contents were quite stable. The conversion factors for ergosterol-to-fungal biomass varied from 48 and 243, which were lower than values for ascomycetous soil fungi reported in the literature.     

Red tide blooms of Cochlodinium polykrikoides in a coastal cove

Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from <100 cells L⁻¹ at the onset of the bloom and reached a maximum density exceeding 3.4 × 10⁶ cells L⁻¹ during the summer of 1981. The bloom was mainly restricted to the mid to inner region of this shallow cove with greatest concentrations localized in surface waters of the southwestern region during summer/fall periods of both years. Highly motile cells consisting of single, double and multiple cell zooids were found as chains of 4 and 8 cells restricted to the late August/September periods. The highest cell densities occurred during periods when annual temperatures were between 19 and 28 °C and salinities between 25 and 30. A major nutrient source for the cove was Crying Brook, located at the innermost region at the head of the cove. Inorganic nitrogen (NH₃ and NO₂ + NO₃) from the brook was continually detectable throughout the study with maximum values of 57.5 and 82.5 μmol L⁻¹, respectively. Phosphate (PO₄-P) was always present in the source waters and rarely <0.5 μmol L⁻¹; silicate always exceeded 30 μmol L⁻¹ with maximum concentrations reaching 226 μmol L⁻¹. Chlorophyll a and ATP concentrations during the blooms varied directly with cell densities. Maximum Chl a levels were 218 mg m⁻³ and ATP-carbon was >20 g C m⁻³. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m⁻³ d⁻¹. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d⁻¹. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.     

The role of particles on the biogeochemical cycling of domoic acid and its isomers in natural water matrices

The adsorption of dissolved domoic acid (DA) and its geometrical isomers was assessed in aqueous solutions containing various types of particles. In one series of experiments carried out in coastal seawater, detectable net adsorption of 100 nM DA occurred only onto natural seawater particles (unfiltered seawater) and 0.5 g L⁻¹ chromatographic silica (18%) in 0.2 μm-filtered seawater. Some net adsorption (<5%) also occurred in the 0.5 g L⁻¹ suspension of estuarine sediment and 0.5 g L⁻¹ solution of humic acid in filtered seawater. No losses were seen in 0.5 g L⁻¹ suspensions of illite, kaolinite, montmorillonite, and silica sand. Biological degradation accounted for small losses (8–10%) in filtered seawater without particles. A second series of experiments using organic-free, <5 μm fractions of kaolinite and montmorillonite in deionized water (DIW) demonstrated that 70% of DA adsorbed onto kaolinite, but only 5% onto montmorillonite. Geometrical isomers of DA (iso-DA D, E, and F) showed negligible adsorption (0–8%) onto a variety of particles in filtered seawater, suggesting that major ions in seawater neutralize electrostatic attractions between particles and DA isomers. These results suggest that DA and its isomers are relatively hydrophilic and not particle reactive. Our data suggest that photochemical and biological degradation of dissolved DA and its isomers appears to occur in bulk surface seawater and its transport to bottom sediments must be mainly biologically driven.     

Phosphorus removal by the Ceratophyllum/periphyton complex in a south Florida (USA) freshwater marsh

Removal of phosphorus (P) by Ceratophyllum demersum L. and associated epiphytic periphyton was quantified by measuring the disappearance of soluble reactive P (SRP) from microcosms during 1-h in situ incubations conducted over a 1-year period. Initial P concentrations in these incubations ranged from 30 to >10,000 μg P L⁻¹. Phosphorus removal was proportional to initial P concentrations and was weakly correlated with solar irradiance and water temperature. Removal rates (0.6–32.8 mg P m⁻² d⁻¹) and k_v coefficients (0.68–1.93 h⁻¹) from experiments run at low initial P concentrations (up to 200 μg P L⁻¹) were comparable to results reported for other macrophytes. Removal rates from experiments run at the highest (>10,000 μg P L⁻¹) initial P concentrations (5300 and 11,100 mg P m⁻² d⁻¹) most likely represented luxury nutrient consumption and were not thought to be sustainable long term. We were unable to determine a V_max for P removal, suggesting that the nutrient-storage capability of the C. demersum/periphyton complex was not saturated during our short-term incubations. Based on N:P molar ratios, the marsh was P limited, while the C. demersum/periphyton complex was either N limited or in balance for N and P throughout this study. However, despite its tissue stoichiometry, the C. demersum/periphyton complex always exhibited an affinity for P. It appeared that the biochemical mechanisms, which mediate P removal, at least on a short-term basis, were more influenced by increases in ambient P levels than by tissue nutrient stoichiometry.     

Phosphine in soils, sludges, biogases and atmospheric implications—a review

This is a review of previously published and unpublished results of research into the occurrence of phosphine (PH₃) in the environment in the form of matrix bound phosphine in soils, aquatic sediments and sludges (range ng kg⁻¹to μg kg⁻¹), free phosphine in formed biogases (range ng m⁻³ to μg m⁻³) and in the atmosphere (range pg m⁻³ to ng m⁻³).The reviewed data support the hypothesis of the existence of a small gaseous link in the phosphorus cycle, which could become important over the long term.Matrix-bound phosphine in soils can be interpreted as a stationary state concentration of phosphine between production and consumption. This phosphine turnover within the soil may be important even if the stationary state concentration (matrix-bound phosphine) is small. Under such circumstances, a slow migration process of phosphine in the interstitial gas sphere of soils is possible. Such a process would influence the balance of phosphorus in agricultural and wetland soil.The detection of easily oxidizable phosphine as a ubiquitous trace gas in the atmosphere can be interpreted as the residue of an important turnover of phosphine between widely distributed emission sources and sinks such as soils and sediments. The atmosphere can carry gaseous phosphorus to remote places.     

Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production

The objective of this study was to determine how fungal morphology influences the volumetric cellulase productivity of Trichoderma reesei cultured in four media with lactose and lactobionic acid as fed-batch in a 7 L stirred tank bioreactor. The use of a cellulose–yeast extract culture medium yielded the highest enzyme production with a volumetric enzyme activity of 69.8 U L⁻¹ h⁻¹, and a maximum fungal biomass of 14.7 g L⁻¹. These findings were associated with the following morphological characteristics of the fungus: total mycelia was 98% of total mean projected area, mean hyphae length of 10 mm, mean hyphae volume of 45.1 mm³, mean hyphae diameter of 7.9 μm, number of branches 9, and number of tips per hypha 29. A positive correlation was found between the total mycelia, the number of tips and the volumetric enzyme productivity, indicating the weight of these variables on the enzyme productivity.     

Comparison on the removal of phthalic acid diesters in a bioreactor landfill and a conventional landfill

The removal of phthalic acid diesters (PAEs) in municipal solid waste (MSW) from two simulated landfill reactors was compared. The results showed that the original concentrations of dimethyl phthalate (DMP), dibutyl phthalate (DBP) and dioctyl phthalate (DOP) in the refuse were 3.3 μg g⁻¹, 18.5 μg g⁻¹ and 0.8 μg g⁻¹, respectively. The concentrations of DMP and DBP in both leachate and refuse decreased greatly during decomposition of the waste in both reactors. The major loss of PAEs from the landfill occurred during an active methanogenic environment with a low concentration of volatile fatty acids (VFA) in the later period. In addition, strong correlations were found between the residual DMP, DBP concentrations and the biologically degradable material (BDM) of the refuse. Finally, PAEs degraded more rapidly in the landfill that was operated in conjunction with the methanogenic reactor when compared to the landfill with direct leachate discharge.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Production and recovery of recombinant protease inhibitor α1-antitrypsin

Human α₁-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L⁻¹ and 1.23 g·L⁻¹, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L⁻¹h⁻¹, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L⁻¹ biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10⁻³ ± 3.43 × 10⁻⁴ g AAT L⁻¹h⁻¹.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs) were produced in carbon-limited, single-stage, fed-batch fermentations of Pseudomonas putida KT2440 by co-feeding nonanoic acid (NA) and glucose (G) to enhance the yield of PHA from NA. An exponential (μ = 0.25 h⁻¹) followed by a linear feeding strategy at a NA:G ratio of 1:1 (w/w) achieved 71 g l⁻¹ biomass containing 56% PHA. Although the same overall PHA productivity (1.44 g l⁻¹ h⁻¹) was obtained when NA alone was fed at the same specific growth rate, the overall yield of PHA from NA increased by 25% (0.66 g PHA g NA⁻¹ versus 0.53 g g⁻¹) with glucose co-feeding. Further increasing glucose in the feed (NA:G = 1:1.5) resulted in a slightly higher yield (0.69 g PHA g NA⁻¹) but lower PHA content (48%) and productivity (1.16 g l⁻¹ h⁻¹). There was very little change in the PHA composition.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle 1. Metabolite profiles of boldenone, boldenone esters and boldione in cattle urine

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Until recently, the control of its illegal use was based either on 17beta-boldenone or 17alpha-boldenone (its main metabolite in cattle) identification in edible tissues, hair, faeces or urine. Recent observations and data tend to demonstrate the natural occurrence (but not ubiquitous) in cattle of these steroids, making the analytical strategy of the control more complicated. We investigated the metabolism of boldenone in cattle after intramuscular and oral treatment of boldenone, boldenone esters and boldione. The central objective was to elucidate the structures of the main metabolites (phase I and phase II) in urine, with main objective to be further in position to compare boldenone urinary profiles of treated and non-treated animals. Nine metabolites have been identified, only four were present whatever the treatment and the administered boldenone source. Nevertheless, all of them have been detected at least once in non-treated animals which did not permit us to use them as biomarkers of an illegal treatment. At last, but not at least, all metabolites were found mainly glucuro-conjugated, and rarely sulfo-conjugated, with the only exception of 17beta-boldenone. Current investigations are showing the absence of 17beta-boldenone sulfoconjugate in non-treated animals; that would permit to distinguish non-treated from treated animals with boldione, boldenone and boldenone esters.     

Annual cycle of CO2 exchange over a reed (Phragmites australis) wetland in Northeast China

Net ecosystem exchange of CO₂ (NEE) was measured during 2005 using the eddy covariance (EC) technique over a reed (Phragmites australis (Cav.) Trin. ex Steud.) wetland in Northeast China (121°54′E, 41°08′N). Diurnal NEE patterns varied markedly among months. Outside the growing season, NEE lacked a diurnal pattern and it fluctuated above zero with an average value of 0.07 mg CO₂ m⁻² s⁻¹ resulting from soil microbial activity. During the growing season, NEE showed a distinct V-like diel course, and the mean daily NEE was −7.48 ± 2.74 g CO₂ m⁻² day⁻¹, ranging from −13.58 g CO₂ m⁻² day⁻¹ (July) to −0.10 g CO₂ m⁻² day⁻¹ (October). An annual cycle was also apparent, with CO₂ uptake increasing rapidly in May, peaking in July, and decreasing from August. Monthly cumulative NEE ranged from −115 ± 24 g C m⁻² month⁻¹ (the reed wetland was a CO₂ sink) in July to 75 ± 16 g C m⁻² month⁻¹ (CO₂ source) in November. The annual CO₂ balance suggests a net uptake of −65 ± 14 g C m⁻² year⁻¹, mainly due to the gains in June and July. Cumulative CO₂ emission during the non-growing season was 327 g C m⁻², much greater than the absolute value of the annual CO₂ balance, which proves the importance of the wintertime CO₂ efflux at the study site. The ratio of ecosystem respiration (R_eco) to gross primary productivity (GPP) for this reed ecosystem was 0.95, indicating that 95% of plant assimilation was consumed by the reed plant or supported the activities of heterotrophs in the soil. Daytime NEE values during the growing season were closely related to photosynthetically active radiation (PAR) (r² > 0.63, p < 0.01). Both maximum ecosystem photosynthesis rate (A_max) and apparent quantum yield (α) were season-dependent, and reached their peak values in July (1.28 ± 0.11 mg CO₂ m⁻² s⁻¹, 0.098 ± 0.027 μmol CO₂ μmol⁻¹ photon, respectively), corresponding to the observed maximum NEE in July. Ecosystem respiration (R_eco) relied on temperature and soil water content, and the mean value of Q₁₀ was about 2.4 with monthly variation ranging from 1.8 to 4.1 during 2005. Annual methane emission from this reed ecosystem was estimated to be about 3 g C m⁻² year⁻¹, and about 5% of the net carbon fixed by the reed wetland was released to the atmosphere as CH₄.     

Cyanobacteria and cyanobacterial toxins in the alkaline crater lakes Sonachi and Simbi, Kenya

The phytoplankton communities and the production of cyanobacterial toxins were investigated in two alkaline Kenyan crater lakes, Lake Sonachi and Lake Simbi. Lake Sonachi was mainly dominated by the cyanobacterium Arthrospira fusiformis, Lake Simbi by A. fusiformis and Anabaenopsis abijatae. The phytoplankton biomasses measured were high, reaching up to 3159 mg l⁻¹ in L. Sonachi and up to 348 mg l⁻¹ in L. Simbi. Using HPLC techniques, one structural variant of the hepatotoxin microcystin (microcystin-RR) was found in L. Sonachi and four variants (microcystin-LR, -RR, -LA and -YR) were identified in L. Simbi. The neurotoxin anatoxin-a was found in both lakes. To our knowledge this is the first evidence of cyanobacterial toxins in L. Sonachi and L. Simbi. Total microcystin concentrations varied from 1.6 to 12.0 μg microcystin-LR equivalents g⁻¹ DW in L. Sonachi and from 19.7 to 39.0 μg microcystin-LR equivalents g⁻¹ DW in L. Simbi. Anatoxin-a concentrations ranged from 0.5 to 2.0 μg g⁻¹ DW in L. Sonachi and from 0 to 1.4 μg g⁻¹ DW in L. Simbi. In a monocyanobacterial strain of A. fusiformis, isolated from L. Sonachi, microcystin-YR and anatoxin-a were produced. The concentrations found were 2.2 μg microcystin g⁻¹ DW and 0.3 μg anatoxin-a g⁻¹ DW. This is the first study showing A. fusiformis as producer of microcystins and anatoxin-a. Since A. fusiformis occurs in mass developments in both lakes, a health risk for wildlife can be expected.     

Nitrogenous preference of toxigenic Pseudo-nitzschia australis (Bacillariophyceae) from field and laboratory experiments

Field and laboratory experiments were designed to determine the differential growth and toxin response to inorganic and organic nitrogen additions in Pseudo-nitzschia spp. Nitrogen enrichments of 50 μM nitrate (KNO₃), 10 μM ammonium (NH₄Cl), 20 μM urea and a control (no addition) were carried out in separate carboys with seawater collected from the mouth of the San Francisco Bay (Bolinas Bay), an area characterized by high concentrations of macronutrients and iron. All treatments showed significant increases in biomass, with chlorophyll a peaking on days 4–5 for all treatments except urea, which maintained exponential growth through the termination of the experiment. Pseudo-nitzschia australis Frenguelli abundance was 10³ cells l⁻¹ at the start of the experiment and increased by an order of magnitude by day 2. Particulate domoic acid (pDA) was initially low but detectable (0.15 μg l⁻¹), and increased throughout exponential and stationary phases across all treatments. At the termination of the experiment, the urea treatment produced more than double the amount of pDA (9.39 μg l⁻¹) than that produced by the nitrate treatment (4.26 μg l⁻¹) and triple that of the control and ammonium treatments (1.36 μg l⁻¹ and 2.64 μg l⁻¹, respectively). The mean specific growth rates, calculated from increases in chlorophyll a and from cellular abundance of P. australis, were statistically similar across all treatments.These field results confirmed laboratory experiments conducted with a P. australis strain isolated from Monterey Bay, CA (isolate AU221-a) grown in artificial seawater enriched with 50 μM nitrate, 50 μM ammonium or 25 μM of urea as the sole nitrogen source. The exponential growth rate of P. australis was significantly slower for cells grown on urea (ca. 0.5 day⁻¹) compared to the cells grown on either nitrate or ammonium (ca. 0.9 day⁻¹). However the urea-grown cells produced more particulate and dissolved domoic acid (DA) than the ammonium- or nitrate-grown cells. The field and laboratory experiments demonstrate that P. australis is able to grow effectively on urea as the primary source of nitrogen and produced more pDA when grown on urea in both natural assemblages and unialgal cultures. These results suggest that the influence of urea from coastal runoff may prove to be more important in the development or maintenance of toxic blooms than previously thought, and that the source of nitrogen may be a determining factor in the relative toxicity of west coast blooms of P. australis.