Yessotoxins production during the culture of Protoceratium reticulatum strains isolated from Galician Rias Baixas (NW Spain)

Yessotoxins (YTXs) production along the culture growth of three strains of the dinoflagellate Protoceratium reticulatum isolated from seawater of Galician Rias Baixas, Spain was investigated. Quantification and toxin profile determination in both cells and culture medium along the growth curve were performed by liquid chromatography–mass spectrometry (LC–MS³) analysis. The YTX profile was very similar among strains, the three algal strains produce mainly YTX and also some YTX analogs. Among the strains the maximum toxin production ranged between 416 and 576 ng mL⁻¹. This is the first report about YTX production by P. reticulatum isolated in Galician coast, NW Spain.     

Growth and bioactive secondary metabolites of arctic Protoceratium reticulatum (Dinophyceae)

Harmful algal blooms are mainly caused by marine dinoflagellates and are known to produce potent toxins that may affect the ecosystem, human activities and health. Such events have increased in frequency and intensity worldwide in the past decades. Numerous processes involved in Global Change are amplified in the Arctic, but little is known about species specific responses of arctic dinoflagellates. The aim of this work was to perform an exhaustive morphological, phylogenetical and toxinological characterization of Greenland Protoceratium reticulatum and, in addition, to test the effect of temperature on growth and production of bioactive secondary metabolites. Seven clonal isolates, the first isolates of P. reticulatum available from arctic waters, were phylogenetically characterized by analysis of the LSU rDNA. Six isolates were further characterized morphologically and were shown to produce both yessotoxins (YTX) and lytic compounds, representing the first report of allelochemical activity in P. reticulatum. As shown for one of the isolates, growth was strongly affected by temperature with a maximum growth rate at 15 °C, a significant but slow growth at 1 °C, and cell death at 25 °C, suggesting an adaptation of P. reticulatum to temperate waters. Temperature had no major effect on total YTX cell quota or lytic activity but both were affected by the growth phase with a significant increase at stationary phase. A comparison of six isolates at a fixed temperature of 10 °C showed high intraspecific variability for all three physiological parameters tested. Growth rate varied from 0.06 to 0.19 d⁻¹, and total YTX concentration ranged from 0.3 to 15.0 pg  YTX cell⁻¹ and from 0.5 to 31.0 pg YTX cell⁻¹ at exponential and stationary phase, respectively. All six isolates performed lytic activity; however, for two isolates lytic activity was only detectable at higher cell densities in stationary phase.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Growth and domoic acid content of Pseudo-nitzschia australis isolated from northwestern Baja California,Mexico, cultured under batch conditions at different temperatures and two Si:NO3 ratios

Domoic acid (DA) poisoning in the southern part of the California Current System has been associated typically with blooms of Pseudo-nitzschia australis. The environmental variables that promote growth and DA production in the Mexican part of this system have not been identified. The present study investigated the effect of temperature and two nutrient ratios on the growth characteristics and DA content of two (BTS-1, BTS-2) P. australis strains isolated from the Pacific coast of northern Baja California peninsula, México. Of the different temperatures assayed (10, 12, 14, 15, 18 and 20 °C), the maximum cell abundance was detected at 12 °C for BTS-2 and 14 °C for BTS-1. The highest maximum specific growth rate (1.69 day⁻¹) was measured at 15 °C for BTS-2. With the exception of cells maintained at 15 °C, growth characteristics were similar in P. australis cultured in a high Si:NO₃ (2.5) or low Si:NO₃ (0.5) ratio at each temperature. Dissolved (dDA) and cellular (cDA) DA content measured at the stationary phase of growth was similar in cells cultivated at the different temperatures. No difference in cDA (between 0.11 and 1.87 pg DA cell⁻¹) was observed in cells cultivated at the two nutrient ratios. To evaluate if P. australis accumulates DA (cDA + dDA) at different stages of the culture and not only during the stationary phase of growth, the BTS-1 strain was cultivated at 14 °C and the content of this toxin was measured during culture development. The cultures were maintained at high (HL; 200 μmol quanta m⁻² s⁻¹) and low light (LL; 30 μmol quanta m⁻² s⁻¹) and in the two nutrient ratios to evaluate the effect of these variables on DA content. The photosynthetic performance and pigment concentration were measured as indicators of the physiological condition of the cells. cDA was detected in all culture conditions and during the different stages of growth. The highest DA content was measured during the lag phase of growth and it was present mainly in the medium (dDA = 70.83 pg DA cell⁻¹). Cells cultivated at HL produced more DA than LL cultured cells. P. australis cultured in HL presented lower photosynthetic rates than LL cells and had similar concentrations of photoprotective pigments and the highest maximum photosynthetic rates were detected during the lag phase of growth in all culture conditions. The results demonstrate that P. australis from northern Baja California peninsula presents a narrow temperature range for optimal growth under batch culture conditions. P. australis produce DA at different stages of growth, and DA content was related to the light intensity at which the cells were cultivated.     

The combined effect of salinity and temperature on the growth and toxin content of four Chilean strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated from an outbreak occurring in southern Chile in 2009

The toxic dinoflagellate Alexandrium catenella has been detected in the southern Chile since 1972, causing severe negative impacts on public health and aquaculture activities. Several environmental factors have been determined to affect growth and toxin production in Alexandrium strains. The aim of this study was to determine the effect of four combined conditions of two temperatures (10 and 15 °C) and two salinities (15 and 35 psu) on the growth and the Paralytic Shellfish Poisoning (PSP) toxin content and composition in four Chilean strains of A. catenella (PFB41, PFB42, PFB37 and PFB38), isolated during a summer outbreak occurred in southern Chile in 2009. The growth curves showed a higher effect of the salinity in strains PFB41 and PFB42 than in strains PFB37 and PFB38. The values of growth rates and maximum cell densities ranged from 0.25 to 0.73 div day⁻¹ and 1.1 × 10⁴ to 5.2 × 10⁴ cells mL⁻¹, respectively. All of the strains showed the highest values for both growth parameters at 15 °C and 35 psu. In general, growth parameters were higher at 35 psu independently of the temperature. On the other hand, the total PSP toxin content ranged widely from 3.99 to 239 fmol cell⁻¹. The highest values of PSP toxin content were attained at 10 °C and 35 psu for all of the strains, at both stages of growth. All of the strains displayed different toxin compositions, with neoSTX, GTX4-1, GTX3-2 and GTX5 being the main toxins detected. The results showed significant differences in the absolute values of growth and toxin production parameters among the strains grown under the same culture conditions, and for each strain grown under different combined conditions of temperature and salinity. These findings demonstrate that abiotic factors can differentially affect the population dynamics of the A. catenella toxic genotypes, thus making it extremely difficult to predict the ecological behavior of this species in the field in terms of the intensity of a potential outbreak.     

Production of isomaltulose using Erwinia sp. D12 cells: Culture medium optimization and cell immobilization in alginate

Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. An experimental Design and Response Surface Methodology were applied for the optimization of the nutrient concentration in the culture medium for enzyme production in shaken flasks at 200 rpm and 30 °C. A higher production of glucosyltransferase (7.47 Uml⁻¹) was observed in the culture medium containing sugar cane molasses (160 gl⁻¹), bacteriological peptone (20 gl⁻¹) and yeast extract Prodex Lac SD^® (15 gl⁻¹) after 8 h, at 30 °C. The highest production of glucosyltransferase in the 6.6-l bioreactor (14.6 Uml⁻¹) was obtained in the optimized culture medium after 10 h at 26 °C. When Erwinia sp. D12 cells were immobilized in sodium alginate, it was verified that sodium alginate solution A could be substituted by a cheaper one, sodium alginate solution B. Using a 40% cell suspension and 2% sodium alginate solution B for cell immobilization in a packed-bed reactor, 64.1% conversion of sucrose to isomaltulose was obtained. The packed-bed reactor with immobilized cells plus glutaraldehyde and polyethylenimine solutions remained in a pseudo-steady-state for 180 h.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Effects of phosphorus and nitrogen amendments on the growth of Egeria najas

The effect of nutrient addition on the growth of E. najas was evaluated in a dose response experiment using sand amended with phosphorus (P) and nitrogen (N), and in enrichment trials with N and P amendments to natural sediments. Plants, water and sediment came from lagoons of the Upper Paraná River Floodplain and from Itaipu Reservoir (Brazil). Relative growth rates (RGRs) of E. najas shoots, based on dry mass (DM), varied from 0.03 to 0.060 d⁻¹ for both nutrients. Root:shoot biomass ratios were related to sediment exchangeable P (r = −0.419; P = 0.03) and N (r = −0.54; P = 0.006), however root RGR was not related to sediment nutrient concentrations. When natural sediments were amended with N and P, neither shoot nor root RGRs differed among treatments for substrata from either the reservoir or the floodplain lagoons (P > 0.05). Comparison of nutrient concentrations measured in natural sediments collected from several sites in both the Upper Paraná River Floodplain (range 49–213 μg P g⁻¹ DM; 36–373 μg N g⁻¹ DM) and Itaipu Reservoir (range 43–402 μg P g⁻¹ DM; 7.9–238 μg N g⁻¹ DM) showed that sediment N and P from these systems usually exceeded minimum requirements necessary for E. najas growth, as measured in the dose response experiment. Together, these results indicate that E. najas, at least in early stages of development, responds to sediment nutrient amendments and relies upon bottom sediments to meet its N and P requirements and that for at least two Brazilian ecosystems, growth of this species is not limited by insufficient sediment N or P. Thus, reducing N and P in water is not enough to control E. najas growth in short time periods in these ecosystems.     

Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC₅₀s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell⁻¹ for 13-desmethyl spirolide C, 96.4 pg cell⁻¹ for 12-methylgymnodimine and a potent hemolytic EC₅₀ value of 7.1 × 10³ cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.     

Isolation and bioelectrochemical characterization of novel fungal sources with oxidasic activity applied in situ for the cathodic oxygen reduction in microbial fuel cells

Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatinga's soils and applied during the in situ cathodic oxygen reduction in fuel cells. All strains were cultivated in submerged cultures using an optimized saline medium enriched with 10 g L⁻¹ of glucose, 3.0 g L⁻¹ of peptone and 0.0005 g L⁻¹ of CuSO₄ as enzyme inducer. Parameters of oxidase activity, glucose consumption and microbial growth were evaluated. In-cell experiments evaluated by chronoamperometry were performed and two different electrode compositions were also compared. Maximum current densities of 125.7, 98.7 and 11.5 μA cm⁻² were observed before 24 h and coulombic efficiencies of 56.5, 46.5 and 23.8% were obtained for SIS-31, SIS-21 and SIS-18, respectively. Conversely, maximum power outputs of 328.73, 288.80 and 197.77 mW m⁻³ were observed for SIS-18, SIS-21 and SIS-31, respectively. This work provides the primary experimental evidences that fungi isolated from the Caatinga region in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes to improve electricity generation in MFCs.     

Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment

A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg⁻¹ polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564±32 mg O₂ l⁻¹ d⁻¹ and SB1 oxidised biphenyl at 426±25 mg O₂ l⁻¹ d⁻¹ in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring.     

Comparison of VIDAS CDAB and CDA immunoassay for the detection of Clostridium difficile in a tcdA− tcdB+ C. difficile prevalent area

Enzyme immunoassays for TcdA and/or TcdB are widely used for diagnosis of C. difficile infection. This study compared the performance of the new VIDAS C. difficile Toxin A & B assay (CDAB) with that of the existing VIDAS C. difficile Toxin A II assay (CDA) in a tcdA⁻tcdB⁺ prevalent area. A total of 555 fecal samples were cultured and tested using CDAB and CDA. C. difficile was isolated in 150 samples and the concordance rate was 81.8% (454/555) between CDAB and CDA. PCR assays for tcdA and/or tcdB were used as a confirmatory test on C. difficile strains recovered from culture positive cases (n = 150) and on fecal specimens in culture negative/CDAB positive or equivocal cases (n = 27). The number of tcdA⁺tcdB⁺, tcdA⁻tcdB⁺, and tcdA⁻tcdB⁻ strains on culture positive isolates (n = 150) were 75 (50.0%), 41 (27.3%), and 34 (22.7%), respectively. PCR assays for tcdB gene alone in stool specimens (n = 27) showed positivity in five cases. The sensitivity of VIDAS CDAB was higher than that of VIDAS CDA (65.3% vs. 29.8%), by more than 2-fold. The specificity of CDAB was almost the same as CDA (93.8% vs. 94.5%). Toxigenic culture of C. difficile isolates in culture positive/VIDAS CDAB negative cases (n = 62) additionally detected 22 VIDAS CDAB positive and 9 VIDAS CDAB equivocal cases. The VIDAS CDAB assay detects more tcdA⁺tcdB⁺ strains (60% vs. 45.3%) and tcdA⁻tcdB⁺ strains (70.7% vs. 0%) compared with VIDAS CDA.     

Nitrogen uptake kinetics of Prymnesium parvum (Haptophyte)

The uptake rates of different nitrogen (N) forms (NO₃⁻, urea, and the amino acids glycine and glutamic acid) by N-deficient, laboratory-grown cells of the mixotrophic haptophyte, Prymnesium parvum, were measured and the preference by the cells for the different forms determined. Cellular N uptake rates (ρ_cell, fmol N cell⁻¹ h⁻¹) were measured using ¹⁵N-labeled N substrates. P. parvum showed high preference for the tested amino acids, in particular glutamic acid, over urea and NO₃⁻ under the culture nutrient conditions. However, extrapolating these rates to Baltic Seawater summer conditions, P. parvum would be expected to show higher uptake rates of NO₃⁻ and the amino acids relative to urea because of the difference in average concentrations of these substrates. A high uptake rate of glutamic acid at low substrate concentrations suggests that this substrate is likely used through extracellular enzymes. Nitrate, urea and glycine, on the other hand, showed a non-saturating uptake over the tested substrate concentration (1–40 μM-N for NO₃⁻ and urea, 0.5–10 μM-N for glycine), indicating slower membrane-transport rates for these substrates.     

A nutrient biotic index (NBI) for use with benthic macroinvertebrate communities

Aquatic macroinvertebrates have been among the principal biological communities used for freshwater monitoring and assessment for several decades, but macroinvertebrate biomonitoring has not incorporated nutrient measures into assessment strategies. Two nutrient biotic indices were developed for benthic macroinvertebrate communities, one for total phosphorus (NBI-P), and one for nitrate (NBI-N). Weighted averaging was used to assess the distributions of 164 macroinvertebrate taxa across TP and NO₃⁻ gradients and to establish nutrient optima and subsequent nutrient tolerance values. Both the NBI-P and NBI-N were correlated with increasing mean TP and NO₃⁻ values (r = 0.68 and r = 0.57, respectively, p < 0.0001). A three-tiered scale of eutrophication for TP and NO₃⁻ (oligotrophic: ≤0.0175 mg/l TP, ≤0.24 mg/l NO₃⁻, mesotrophic: >0.0175 to ≤0.065 mg/l TP, >0.24 to ≤0.98 mg/l NO₃⁻, eutrophic: >0.065 mg/l TP, >0.98 mg/l NO₃⁻) was also established through cluster analysis of invertebrate communities using Bray–Curtis (quantitative) similarity. Significant differences (p < 0.0001) were detected between median NBI-P and NBI-N scores among the three trophic states. Therefore, the nutrient biotic indices (NBIs) appear to accurately reflect changes in stream trophic state. Multimetric water quality assessments were also used to identify thresholds of impairment among the three trophic states. Hodges-Lehman estimation indicated that the greatest change in assessment results occurred between the mesotrophic and eutrophic states. The eutrophic state also represented the highest percentage of overall impairment. Therefore, the suggested threshold for nutrient impairment is the boundary between mesotrophic and eutrophic (0.065 mg/l TP and 0.98 mg/l NO₃⁻). The corresponding NBI-P score (6.1) and NBI-N score (6.0) for this threshold incorporate predictive capabilities into the NBIs. The NBI and index score thresholds of impairment will provide monitoring programs with a robust measure of stream nutrient status and serve as a useful tool in enforcing regional nutrient criteria.     

Nutrient-use efficiency in arid-zone forests of the mangroves Rhizophora stylosa and Avicennia marina

Nutrient-use efficiency (NUE) within forests of the mangroves Rhizophora stylosa and Avicennia marina was estimated in arid Western Australia using litter fall rates and rates of leaf CO₂ exchange. Litter fall rates ranged from 9.8 to 34.4 t DW ha⁻¹ y⁻¹ but equated to only 13–41% (mean = 30%) of net canopy primary production. Foliar N:P ratios were in most instances ≥16, suggesting P limitation. NUE for N based on litter fall rates were significantly less (NUE_L = 167–322 g DW g⁻¹ N) than those based on photosynthesis measurements (NUE_P = 234–448 g DW g⁻¹ N), suggesting that NUE estimates for nitrogen based on litter fall data are underestimates. NUE_P estimates for N were significantly greater for R. stylosa than for A. marina. NUE for P were not significantly different, with NUE_L ranging from 2905 to 5053 g DW g⁻¹ P and NUE_P ranging from 1632 to 4992 g DW g⁻¹ P. Both sets of NUE are at the higher end of the range of estimates calculated for most other forests and equivalent to those for wet tropical mangroves. These arid-zone trees live in low-nutrient habitats, but it appears that selection on components of NUE (i.e. traits that reduce nutrient loss) rather than on NUE itself equates to a lack of clear patterns in NUE between different environments, emphasizing the flexible nature of nutrient allocation in woody plants. NUE in R. stylosa correlated inversely with mature leaf N and P content, implying that NUE in this species is maximized by the synthesis of low-nutrient leaves, i.e. a nutrient retention strategy, whereas such does not appear to be the case for A. marina. This strategy translates into a direct advantage in terms of net primary productivity for R. stylosa. This idea is supported by evidence of longer nutrient residence times for R. stylosa than for A. marina.     

Nitrogen uptake kinetics in field populations and cultured strains of Karenia brevis

This study represents the most comprehensive assessment of kinetic parameters for Karenia brevis to date as it encompasses natural populations sampled during three different bloom years in addition to cultured strains under controlled conditions. Nitrogen (N) uptake kinetics for ammonium (NH₄⁺), nitrate (NO₃⁻), urea, an amino acid mixture, individual amino acids (glutamate and alanine), and humic substrates were examined for the toxic red tide dinoflagellate, K. brevis, during short term incubations (0.5–1 h) using ¹⁵N tracer techniques. Experiments were conducted using natural populations collected during extensive blooms along the West Florida Shelf in October 2001, 2002, and 2007, and in cultured strains (CCFWC 251 and CCFWC 267) obtained from the Florida Fish and Wildlife Institute culture collection. Kinetic parameters for the maximum uptake velocity (V_max), half-saturation concentration (K_s), and the affinity constant (α) were determined. The affinity constant is considered a more accurate indicator of substrate affinity at low concentrations. K. brevis took up all organic substrates tested, including N derived from humic substances. Uptake rates of the amino acid mixture and some NO₃⁻ incubations did not saturate even at the highest substrate additions (50–200 μmol N L⁻¹). Based upon the calculated α values, the greatest substrate preference was for NH₄⁺ followed by NO₃⁻ ≥ urea, humic compounds and amino acids. The ability of K. brevis to utilize a variety of inorganic and organic substrates likely helps it flourish under a wide range of nutrient conditions from bloom initiation in oligotrophic waters offshore to bloom maintenance near shore where ambient nutrient concentrations may be orders of magnitude greater.     

Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes

A yellow laccase from the culture filtrate of Trametes hirsuta MTCC-1171 has been purified. The purification methods involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 55.0 kDa. Using 2,6-dimethoxyphenol, 2,2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as the substrates, the K_m, k_cat and k_cat/K_m values of the laccase were found to be 420 μM, 13.04 s⁻¹, 3.11 × 10⁴ M⁻¹ s⁻¹, 225 μM, 13.03 s⁻¹, 1.3 × 10⁵ M⁻¹ s⁻¹ and 100 μM, 13.04 s⁻¹, 5.8 × 10⁴ M⁻¹ s⁻¹, respectively. The pH and temperature optima were 4.5 and 60 °C, respectively while pH and temperature stabilities were pH 4.5 and 50 °C. The activation energy for thermal denaturation of the enzyme was 18.6 kJ/mol/K. The purified laccase has yellow colour and does not show absorption band around 610 nm like blue laccases. The purified laccase transforms toluene, 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator molecules in high yields.     

Short-time response in root morphology of Vallisneria natans to sediment type and water-column nutrient

Rooted submerged macrophytes can absorb significant amounts of nutrients from both sediment and water. We investigated root morphology of Vallisneria natans in mesocosm plastic bins, in response to three types of sediment (sandy loam, clay, and a 50:50 (v/v) mixture of the two sediments) and two levels of water-column nutrient (well water and nutrient medium). Compared to the plants grown in the clay or mixed sediments, root diameter decreased (0.39–0.41 versus 0.36–0.37 mm) but total root length per plant increased (0.87–1.27 versus 1.14–1.62 m) when grown in sandy loam. Increase of nutrient availability in water column led to decreased specific root length (306–339 versus 258–281 m g⁻¹). However, both sediment type and water-column nutrient had no impacts on root number (ranged from 19 to 24 number of roots per plant). Root weight ratio, root:leaf mass ratio and root:leaf length ratio generally decreased with enhanced nutrient availability in sediment or water. Plant growth was affected by sediment type alone (P < 0.05), rather than water-column nutrient (P > 0.05). However, plant N and P contents were significantly impacted by both sediment type (P ≤ 0.001) and water-column nutrient (P < 0.05). Increase of nutrient availability in sediment or water led to increased plant N (ranged from 2.47 to 4.77 mg g⁻¹) and P concentrations (ranged from 42.8 to 62.0 mg g⁻¹). These results indicate that considerable variation in root morphology of V. natans exists in response to the fertility of the sediment it is rooted in.     

Modelling of growth and accumulation of carotenoids in Haematococcus pluvialis as a function of irradiance and nutrients supply

This paper analyzes the feasibility of the autotrophic production of vegetative cells of Haematococcus pluvialis under conditions resembling outdoors. The experimental design simulates in laboratory with artificial light an outdoors circadian cycle similar to solar illumination. The influence of the irradiance and nutrient concentration on the growth rate and carotenoids accumulation in batch cultures is studied. The cultures were not photoinhibited even under the maximum irradiance-level tested (2500 μE m⁻² s⁻¹). Growth was kept nutrient-limited by using nutrients concentration below the standard inorganic medium (10 mM nitrate). When no nutrient-limitation occurs, the growth rate and biomass productivity measured 0.57 day⁻¹ and 0.28 g L⁻¹ day⁻¹, respectively, were similar to the maximum values reported, regardless of the nutritional regime: autotrophic, mixotrophic or heterotrophic. On the other hand, carotenogenesis was only observed under nutrient-limiting conditions when the medium strength was reduced to 0.2- or 0.3-fold of the standard medium. On the other hand, carotenogenesis ceased under severe nutrient deprivation (i.e. nutrient strength of 0.1-fold of the standard medium). The growth rate and the carotenoids accumulation rate were demonstrated to be a function of the average irradiance inside the culture, and of the nutrient content of the medium. A mathematical model for the observed behaviour is proposed. This model was adequate to fit all the experimental data obtained. The values determined for the characteristics parameters are in agreement with those found by other authors. Therefore, the proposed model can be a useful tool for the design and management of Haematococcus cultures, and could allow improving the yield of this production process.