Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose and study of its swelling, metal ion sorption and flocculation behaviour

The present paper reports the graft copolymerization of N-vinylformamide onto sodium carboxymethylcellulose by free radical polymerization using potassium peroxymonosulphate/thiourea redox system in an inert atmosphere. The reaction conditions for maximum grafting have been optimized by varying the reaction variables, including the concentration of N-vinylformamide (12.0 × 10⁻²–28.0 × 10⁻² mol dm⁻³), potassium peroxymonosulphate (4.0 × 10⁻³–12.0 × 10⁻³ mol dm⁻³), thiourea (1.2 × 10⁻³–4.4 × 10⁻³ mol dm⁻³), sulphuric acid (2.0 × 10⁻³–10.0 × 10⁻³ mol dm⁻³), sodium carboxymethylcellulose (0.2–1.8 g dm⁻³) along with time duration (60–180 min) and temperature (25–45° C). Water swelling capacity, metal ion sorption and flocculation studies of synthesized graft copolymer have been performed with respect to the parent polymer. The graft copolymer has been characterized by FTIR spectroscopy and thermogravimetric analysis.     

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.     

Novel chitosan membranes as support for lipases immobilization: Characterization aspects

Membranes of chitosan (QS), chitosan treated with glutaraldehyde (QGA) and chitosan crown ether (QCE) were utilized as carriers for immobilization of Candida antarctica and Candida rugosa lipases. Membrane supports were characterized by several techniques (Raman spectroscopy, elemental analysis by CHN determination and Energy Dispersive X-ray (EDX), water sorption isotherms, and surface area from nitrogen sorption data). To verify the presence of enzymes, some of these techniques were also used for lipase on chitosan biocatalytic systems. Measurements of protein load from Biuret assays and catalytic activity in esterification in nonaqueous media were also made for the immobilized enzymes. Sorption isotherms at 20, 30, 40 and 50 °C for QS, QGA and QCE supports were fitted to the Guggenheim, Anderson and Böer model. GAB monolayer moisture parameter, Xm, varied between 0.029 and 0.051 for QS, 0.039 and 0.058 for QGA and 0.039–0.075 g of water g⁻¹ s.s. for QCE membranes. Elemental analysis and Raman spectra measurements of the lipase, supports and immobilized lipase systems gave evidence of the presence of enzymes on supports. Chitosan supports with internal surface area (m2 g⁻¹) among 3.31 and 1.26 were obtained. Regardless of these low values, acceptable protein load (0.61 to 3.21%) and esterification initial rates were achieved (0.88–2.75 mmol min⁻¹ g of protein⁻¹).     

Synthesis, characterization and applications of graft copolymer (Chitosan-g-N,N-dimethylacrylamide)

An unreported graft copolymer of N,N-dimethylacrylamide (DMA) with chitosan has been synthesized under nitrogen atmosphere using peroxymonosulphate/mandelic acid redox pair. The effect of reaction conditions on grafting parameters i.e. grafting ratio, efficiency, conversion, add on and homopolymer has been studied. Experimental results show that maximum grafting has been obtained at 1.0 g dm⁻³ concentration of chitosan, 30 × 10⁻² mol dm⁻³ concentration of N,N-dimethylacrylamide and 7.0 × 10⁻³ mol dm⁻³ concentration of hydrogen ion. It has also been observed that grafting ratio, add on, conversion and efficiency increase upto 3.2 × 10⁻³ mol dm⁻³ of mandelic acid, 12.0 × 10⁻³ mol dm⁻³ of potassium peroxymonosulphate, 150 min of time and 40 °C of temperature. Grafted polymer has been characterized by FTIR spectroscopy and thermogravimetric analysis. Water swelling capacity of chitosan-g-N,N-dimethylacrylamide has been determined. It has been observed that the graft copolymer is thermally more stable than parent backbone.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III under laboratory culture

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III, two species of causative bloom dinoflagellates in China, were investigated using bi-algal cultures under controlled laboratory conditions. The growth of P. donghaiense and S. trochoidea were significantly suppressed when the initial cell densities were set at 1.9 × 10⁴ cells mL⁻¹ or 1.9 × 10⁵ cells mL⁻¹ for P. donghaiense and 1.0 × 10⁴ cells mL⁻¹ for S. trochoidea when the initial size/density ratio was 1:1 or 10:1, respectively, but no out-competement was observed in either bi-algal culture by the end. The simultaneous assay on the culture filtrate showed that P. donghaiense filtrate prepared at a lower initial density (1.9 × 10⁴ cells mL⁻¹) stimulated the co-cultured S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹, but filtrate at a higher density (1.9 × 10⁵ cells mL⁻¹) depressed its growth. Differently, the filtrate of S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹ significantly suppressed the growth of P. donghaiense at a density of 1.9 × 10⁴ cells mL⁻¹, but had little stimulatory effect on P. donghaiense at a density of 1.9 × 10⁵ cells mL⁻¹compared to the control (P > 0.05). It is likely that these two species of microalgae interact with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. We then quantify their interactions in the bi-algal culture by using a mathematical model. The estimated parameters from the model showed that the inhibition exerted by S. trochoidea on P. donghaiense was about 43 and 24 times stronger than the inhibitory effect that P. donghaiense exerted on S. trochoidea when the initial size/density were 1:1 and 10:1, respectively. S. trochoidea seemed to have a survival strategy that was superior to P. donghaiense in the bi-algal culture under controlled laboratory conditions. We also observed a closely positive relationship between the initial cell density and its effect on the co-cultured microalga by measuring the fluorenscence: filtrate prepared from higher initial cell density had stronger interference on the co-cultured microalga. Moreover, pre-treated under different temperature conditions (30 °C, 60 °C and 100 °C) would significantly changed the effect of culture filtrate on the co-cultured microalga. Result inferred that P. donghaiense or S. trochoidea would release allelochemicals into the bi-algal culture medium and the allelochemicals might be a mixture with temperature-sensitive components in it.     

Biosorption of an Azo Dye by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Fungal Biomasses

Biosorption is an eco-friendly and cost-effective method for treating the dye house effluents. Aspergillus niger and Trichoderma sp. were cultivated in bulk and biomasses used as biosorbents for the biosorption of an azo dye Orange G. Batch biosorption studies were performed for the removal of Orange G from aqueous solutions by varying the parameters like initial aqueous phase pH, biomass dosage, and initial dye concentration. It was found that the maximum biosorption was occurred at pH 2. Experimental data were analyzed by model equations such as Langmuir and Freundlich isotherms, and it was found that both the isotherm models best fitted the adsorption data. The monolayer saturation capacity was 0.48 mg/g for Aspergillus niger and 0.45 mg/g for Trichoderma sp. biomasses. The biosorption kinetic data were tested with pseudo first-order and pseudo second-order rate equations, and it was found that the pseudo second-order model fitted the data well for both the biomasses. The rate constant for the pseudo second-order model was found to be 10–0.8 (g/mg min⁻¹) for Aspergillus niger and 8–0.4 (g/mg min⁻¹) for Trichoderma sp. by varying the initial dye concentrations from 5 to 25 mg/l. It was found that the biomass obtained from Aspergillus niger was a better biosorbent for the biosorption of Orange G dye when compared to Trichoderma sp.     

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae – F52, TM109, and MA1200 – formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7–2.9 × 10⁸ L⁻¹ liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 × 10⁸ blastospores ml⁻¹ on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.     

Biosorption of di(2-ethylhexyl)phthalate by seaweed biomass

Samples of various Sargassum species were collected in the Hong Kong marine environment and used for studies on biosorption of di(2-ethylhexyl)phthalate (DEHP). Batch adsorption experiments were carried out to determine the removal capacity and removal efficiency of the biosorbents. The DEHP removal ability was similar among beached seaweed and three freshly collected Sargassum species. Different physico-chemical factors were evaluated in order to enhance the performance of the biosorbents. Under optimized conditions (25 mg biomass, initial pH 4, 25 °C, 40 mg L^–1 DEHP), the mean removal capacity of beached seaweed and Sargassum siliquastrum was 5.68 and 6.54 mg g^–1, respectively. Examination of the Langmuir and Freundlich adsorption isotherms showed that the biosorption phenomenon by these biosorbents could well be described by these models. Desorption of DEHP was also assessed with methanol, which showed the most satisfactory desorbing ability. Further study in multiple adsorption–desorption of DEHP by the biosorbents demonstrated the reusability of both beached seaweed and S. siliquastrum for biosorption of DEHP.     

Biosorption of uranium by Myxococcus xanthus

This paper deals with uranium biosorption by Myxococcus xanthus biomass in which dry biomass, accumulating up to 2.4 mM of uranium g⁻¹, is demonstrated to be a more efficient biosorbent than wet biomass. For uranium concentrations of 0.1–0.3 mM, between 95.79% and 95.99% of the uranium was taken up from the solution. Dry biomass biosorption was found to be relatively rapid, reaching equilibrium after 5–10 min. In addition, the pH influenced biosorption, pH 4.5 promoting maximum uptake. It was also established that the biosorbed uranium is located on the cellular wall and within the extracellular mucopolysaccharide of this microorganism. Furthermore, using sodium carbonate as a desorbent agent, 80.82% of the biosorbed uranium could be recovered. The results obtained indicate the possible utilization of M. xanthus biomass to solve some problems of the water contaminated by uranium.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.     

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

Pine nut peroxidase immobilized on chitosan crosslinked with citrate and ionic liquid used in the construction of a biosensor

A biosensor based on the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI·Tf₂N) and a novel source of peroxidase (tissue from the pine nuts of Araucaria angustifolia) was constructed. This enzyme was immobilized on chitosan crosslinked with citrate and the biosensor used for the determination of rosmarinic acid by square-wave voltammetry. The peroxidase in the presence of hydrogen peroxide catalyzes the oxidation of rosmarinic acid to quinone and the electrochemical reduction of the product was obtained at a potential of +0.15 V vs. Ag/AgCl. Different analytical parameters influencing the biosensor response, that is, peroxidase units, pH, hydrogen peroxide concentration and parameters for the square-wave voltammetry (frequency, pulse amplitude and scan increment), were investigated. The best performance was observed for the biosensor under the following conditions: 1000 units mL⁻¹ peroxidase, pH 7.0 and 8.3 × 10⁻⁴ mol L⁻¹ hydrogen peroxide with a frequency of 30 Hz, pulse amplitude of 100 mV and scan increment of 5.0 mV. The biosensor gave a linear response to rosmarinic acid over the concentration range of 9.07 × 10⁻⁷ to 4.46 × 10⁻⁶ mol L⁻¹ with a detection limit of 7.25 × 10⁻⁸ mol L⁻¹. The recovery of rosmarinic acid in plant extracts ranged from 97.0% to 109.6% and the determination of this substance in these samples using the biosensor compared favorably with that using the capillary electrophoresis method.     

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.     

Water relations and carbon dioxide exchange of epiphytic lichens in the Namib fog desert

Although there is only negligible rainfall, frequent nocturnal fog, dew and high air humidity support a luxurious lichen vegetation in the coastal zone of the central Namib Desert (Namibia). In earlier publications, we have studied ecophysiological performance of a series of epilithic and terrestrial lichens. Here, we have extended this work to three epiphytic species (Heterodermia namaquana, Ramalina lacera, and Xanthoria turbinata) that inhabit the sparse perennial shrubs growing in this area. Our intention, monitoring lichen CO₂ exchange, their water relations and microclimate conditions, was to determine the functional mechanisms that allow these epiphytes to exist under the special conditions of a fog desert. Measurements were conducted mainly during the spring season.The epiphytic lichens showed response patterns very similar to the epilithic and epigaeic species at the same site. Their metabolism was activated through moistening by dew and/or fog during the night and, in the very early morning, they exhibited the typical brief peak of net photosynthesis (NP) between sunrise and desiccation. The thalli were almost completely dry for the remainder of the day. Average duration of the positive NP during the morning peak was about 3 h. Dew condensation, alone, resulted in activation that provided 58–63% of integrated carbon income (ΣNP) as compared to fog (plus dew). In the late afternoon, there was a tendency for hydration to increase again, due to water vapour uptake at higher air humidity, and this allowed on some days a brief additional period of very low rates of photosynthesis shortly before sunset.Light response of photosynthesis showed “sun-plant” characteristics with saturation around 1000 μmol m⁻² s⁻¹ photosynthetically active photon flux density (PPFD). Light compensation point (LCP) of CO₂ exchange after sunrise was highly dependent on actual water content (WC) for X. turbinata: at low hydration it was ca. 10 μmol m⁻² s⁻¹ PPFD whilst, at high WC, it was almost 80 μmol m⁻² s⁻¹ PPFD. In contrast, LCP of R. lacera was almost independent of WC. This phenomenon was probably due to differences in thallus structure.Maximal attained NP and daily ΣNP both showed a saturation-type response to previous maximal nocturnal WC. Neither parameter was increased substantially when higher maximal thallus WCs were produced by experimental moistening in the night. All three species, despite their different morphologies, performed optimally at the highest nocturnal moistening achieved by natural fog and were not able to make use of higher hydration.The three studied epiphytes were similar in their chlorophyll-related rates of NP. Due to lower chlorophyll content, dry weight and carbon-related NP of X. turbinata was only about one-third of that of the other two species. The average carbon income on days with fog and/or dew hydration during the spring season amounted to 2.4 and 2.1 mgC (gC)⁻¹ day⁻¹ (related to thallus carbon content) for H. namaquana and R. lacera, respectively. This primary production was of similar magnitude to those found for the terrestrial species at the same site.     

Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA

We report on the emergence of Cochlodinium polykrikoides blooms in the Peconic Estuary and Shinnecock Bay, NY, USA, during 2002–2006. Blooms occurred during late summer when temperatures and salinities ranged from 20 to 25 °C and 22 to 30 ppt, respectively. Bloom patches achieved cell densities exceeding 10⁵ ml⁻¹ and chlorophyll a levels exceeding 100 μg l⁻¹, while background bloom densities were typically 10³–10⁴ cells ml⁻¹. Light, scanning electron and ultrathin-section transmission electron microscopy suggested that cells isolated from blooms displayed characteristics of C. polykrikoides and provide the first clear documentation of the fine structure for this species. Sequencing of a hypervariable region of the large subunit rDNA confirmed this finding, displaying 100% similarity to other North American C. polykrikoides strains, but a lower similarity to strains from Southeast Asia (88–90%). Bioassay experiments demonstrated that 24 h exposure to bloom waters (>5 × 10⁴ cells ml⁻¹) killed 100% of multiple fish species (1-week-old Cyprinodon variegates, adult Fundulus majalis, adult Menidia menidia) and 80% of adult Fundulus heteroclitus. Microscopic evaluation of the gills of moribund fish revealed epithelial proliferation with focal areas of fusion of gill lamellae, suggesting impairment of gill function (e.g. respiration, nitrogen excretion, ion balance). Lower fish mortality was observed at intermediate C. polykrikoides densities (10³–10⁴ cells ml⁻¹), while fish survived for 48 h at cell densities below 1 × 10³ cells ml⁻¹. The inability of frozen and thawed-, or filtered (0.2 μm)-bloom water to cause fish mortality suggested that the thick polysaccharide layer associated with cell membranes and/or a toxin principle within this layer may be responsible for fish mortality. Juvenile bay scallops (Argopecten irradians) and American oysters (Crassostrea virginica) experienced elevated mortality compared to control treatments during a 9-day exposure to bloom water (5 × 10⁴ cells ml⁻¹). Surviving scallops exposed to bloom water also experienced significantly reduced growth rates. Moribund shellfish displayed hyperplasia, hemorrhaging, squamation, and apoptosis in gill and digestive tissues with gill inflammation specifically associated with areas containing C. polykrikoides cells. In summary, our results indicate C. polykrikoides blooms have become annual events on eastern Long Island and that bloom waters are capable of causing rapid mortality in multiple species of finfish and shellfish.     

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster^™ DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4±3.0×10⁶ and 5.4±3.0×10² CFU ml⁻¹ cell suspension. The detection limit was about 540 CFU ml⁻¹, which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4±3.0×10⁴ CFU g⁻¹ dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.