Brevetoxin persistence in sediments and seagrass epiphytes of east Florida coastal waters

A bloom of Karenia brevis Davis developed in September 2007 near Jacksonville, Florida and subsequently progressed south through east Florida coastal waters and the Atlantic Intracoastal Waterway (ICW). Maximum cell abundances exceeded 10⁶ cells L⁻¹ through October in the northern ICW between Jacksonville and the Indian River Lagoon. The bloom progressed further south during November, and terminated in December 2007 at densities of 10⁴ cells L⁻¹ in the ICW south of Jupiter Inlet, Florida. Brevetoxins were subsequently sampled in sediments and seagrass epiphytes in July and August 2008 in the ICW. Sediment brevetoxins occurred at concentrations of 11–15 ng PbTx-3 equivalents (g dry wt sediment)⁻¹ in three of five basins in the northern ICW during summer 2008. Seagrass beds occur south of the Mosquito Lagoon in the ICW. Brevetoxins were detected in six of the nine seagrass beds sampled between the Mosquito Lagoon and Jupiter Inlet at concentrations of 6–18 ng (g dry wt epiphytes)⁻¹. The highest brevetoxins concentrations were found in sediments near Patrick Air Force Base at 89 ng (g dry wt sediment)⁻¹. In general, brevetoxins occurred in either seagrass epiphytes or sediments. Blades of the resident seagrass species have a maximum life span of less than six months, so it is postulated that brevetoxins could be transferred between epibenthic communities of individual blades in seagrass beds. The occurrence of brevetoxins in east Florida coast sediments and seagrass epiphytes up to eight months after bloom termination supports observations from the Florida west coast that brevetoxins can persist in marine ecosystems in the absence of sustained blooms. Furthermore, our observations show that brevetoxins can persist in sediments where seagrass communities are absent.     

Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna

The efficacy of an essential oil obtained from Pimpinella anisum fruits and its major compound, trans-Anethole, was tested on the eggs, larvae and adults of Culex quinquefasciatus. While causing no significant mortality on eggs, other tested stages were very sensitive to the essential oil and trans-Anethole. LC₅₀ for the 2nd to 4th instar larvae was estimated as 26–27 μL·L^− 1 and 15–19 μL·L^− 1 for the essential oil and trans-Anethole, respectively. As for the essential oil applied on adults, LC(LD)₅₀ was estimated as 9.3 μL mL^− 1 (spray test), 1.9 μL L^− 1 (fumigation test) and 0.6 μg cm^− 2 (tarsal test), and for trans-Anethole as 8.1 μL mL^− 1 (spray test), 2.1 μL L^− 1 (fumigation test) and 0.4 μg cm^− 2 (tarsal test). The time needed to achieve 50% mortality after application of LC(LD)₉₉ of the essential oil was significantly different; for example, in larvicidal assays it ranged from 15 to 235 min depending on the larval instar, and from 9 to 180 min when applied to adults, depending on the mode of application. It was also found that temperature had an important effect on the larvicidal efficacy of the essential oil, and oviposition deterrent activity was studied.The essential oil and trans-Anethole were toxic for Daphnia magna (62–92% mortality) and significantly reduced its fertility at high concentrations (35–50 μL mL^− 1) and long exposure (48 h). However, no negative effect on Daphnia mortality or fertility was found at shorter exposure times (6 h) and/or lower concentrations (20 μL mL^− 1).Based on the results of this study, we can recommend the essential oil from P. anisum as a suitable active substance for potential botanical insecticides.     

Accumulation and detoxification dynamic of cyanotoxins in the freshwater shrimp Palaemonetes argentinus

The uptake and accumulation of microcystin-LR (MC-LR) in the shrimp Palaemonetes argentinus was investigated using both laboratory and field assays. Shrimps were exposed in aquarium during 1, 2, 3 and 7 days to 1, 10 and 50 μg L⁻¹ MCLR. Accumulation (0.7 ± 0.2 μg MC-LR g⁻¹) was observed after three days exposures to 50 μg L⁻¹ toxin. Then, shrimps were relocated in fresh water (free of MCLR) to verify the detoxification dynamic, showing a drop to 0.18 ± 0.01 μg MCLR g⁻¹ after three days. The activity of glutathione-S-transferase, measured in the microsomal fraction (mGST), was significantly increased during the exposure period, with further increment during the detoxification period. Furthermore, cytosolic GST (sGST) and glutathione reductase (GR) increased their activities during detoxification, while inhibition was observed for catalase (CAT) with no significant changes for glutathione peroxidase (GPx). Current results suggest that GSH conjugation could be an important MC detoxification mechanism in P. argentinus and that MCLR induce oxidative stress in this shrimp.Field exposures were carried out in San Roque Reservoir (Córdoba, Argentina) after a cyanobacteria bloom. Nodularin (Nod) presence was measured for the first time in this waterbody (0.24 ± 0.04 μg L⁻¹), being the first report of Nod in South America freshwaters. Nod was also detected in Palaemonetes argentinus (0.16 ± 0.03 μg g⁻¹) after three weeks of exposure in this reservoir, with the concomitant activation of mGST, sGST and CAT.Although internal doses of Nod were low throughout the exposure, they were enough to cause biochemical disturbances in Palaemonetes argentinus.Further laboratory studies on Nod accumulation and antioxidant responses in Palaemonetes argentinus are necessary to fully understand these field results. P. argentinus should be considered a potential vector for transferring cyanotoxins to higher trophic levels in aquatic environments.     

Mercury levels assessment in hair of riverside inhabitants of the Tapajós River,Pará State,Amazon, Brazil: Fish consumption as a possible route of exposure

The study present evaluated the levels of mercury (Hg) and methylmercury (MeHg) in hair samples of people from Barreiras community, riverside inhabitants of the Tapajós River (Pará, Brazil), an area impacted by clandestine gold mining, as well as we analyzed the levels of Hg and Se (selenium) in nine fish species (carnivores and non-carnivorous) from the Tapajós River, which stand out as the main species consumed by riverside inhabitants, to evaluate a relationship between frequency of fish consumption and Hg concentration, and also to evaluate possible mechanisms of fish protection (or non-protection) to Hg exposure by Se. Furthermore we analyze the water quality to evaluate the environmental trophic state, fact responsible by creating conditions that can potentiate the effects of toxic mercury. Concentrations of Hg and MeHg were analyzed in hair samples of 141 volunteers in different age band. Of those, 84.40% of samples present values above the threshold for biological tolerance, which is 6.00 μg g⁻¹ of total Hg in hair. Total Hg, in men there was a variation of 2.07–24.93 μg g⁻¹, while for women the variation was 4.84–27.02 μg g⁻¹. Consequently, the level of MeHg in men presented a variation of 1.49–19.57 μg g⁻¹, with an average of 11.68 μg g⁻¹, while with women the variation was from 3.73 to 22.35 μg g⁻¹, with an average of 10.38 μg g⁻¹. In fish species, Hg concentrations in carnivorous species had an average of 0.66 μg g⁻¹, higher than that permitted by current legislation, ranging from 0.30 to 0.98 μg g⁻¹, while the non-carnivorous species have values below the recommended by the legislation averaging 0.09 μg g⁻¹, ranging between 0.02 and 0.44 μg g⁻¹. For Se in fish, show that among carnivores, the contents of Se ranged between 0.18 and 0.54 μg g⁻¹ with a mean of 0.34 μg g⁻¹, while for non-carnivores these values were of the order of 0.16–0.56 μg g⁻¹, with an average of 0.32 μg g⁻¹. In surface water quality variables at the sampling points all showed values in accordance with the range established by current legislation. In this regard, the results provided by this study, while not conclusive, are strong indicators that despite not having been shown the relationship between the concentration of mercury in hair and feeding habits along the Tapajós River basin communities showed that a plausible correlation exists between levels of mercury and selenium in fish. This fact may serve as a subsidy to research human health, because in the Amazon, there is still a lot to examine with regards to the full understanding of the Se cycle.     

Algicidal and growth-inhibiting bacteria associated with seagrass and macroalgae beds in Puget Sound,WA, USA

The algicidal and growth-inhibiting bacteria associated with seagrasses and macroalgae were characterized during the summer of 2012 and 2013 throughout Puget Sound, WA, USA. In 2012, Heterosigma akashiwo-killing bacteria were observed in concentrations of 2.8 × 10⁶ CFU g⁻¹ wet in the outer organic layer (biofilm) on the common eelgrass (Zostera marina) in north Padilla Bay. Bacteria that inhibited the growth of Alexandrium tamarense were detected within the biofilm formed on the eelgrass canopy at Dumas Bay and North Bay at densities of ∼10⁸ CFU g⁻¹ wet weight. Additionally, up to 4100 CFU mL⁻¹ of algicidal and growth-inhibiting bacteria affecting both A. tamarense and H. akashiwo were detected in seawater adjacent to seven different eelgrass beds. In 2013, H. akashiwo-killing bacteria were found on Z. marina and Ulva lactuca with the highest densities of ∼10⁸ CFU g⁻¹ wet weight at Shallow Bay, Sucia Island. Bacteria that inhibited the growth of H. akashiwo and A. tamarense were also detected on Z. marina and Z. japonica at central Padilla Bay. Heterosigma akashiwo cysts were detected at a concentration of 3400 cysts g⁻¹ wet weight in the sediment from Westcott Bay (northern San Juan Island), a location where eelgrass disappeared in 2002. These findings provide new insights on the ecology of algicidal and growth-inhibiting bacteria, and suggest that seagrass and macroalgae provide an environment that may influence the abundance of harmful algae in this region. This work highlights the importance of protection and restoration of native seagrasses and macroalgae in nearshore environments, in particular those regions where shellfish restoration initiatives are in place to satisfy a growing demand for seafood.     

A green and efficient procedure for the preconcentration and determination of cadmium,nickel and zinc from freshwater,hemodialysis solutions and tuna fish samples by cloud point extraction and flame atomic absorption spectrometry

Cloud point extraction (CPE) was used to simultaneously preconcentrate trace-level cadmium, nickel and zinc for determination by flame atomic absorption spectrometry (FAAS). 1-(2-Pyridilazo)-2-naphthol (PAN) was used as a complexing agent, and the metal complexes were extracted from the aqueous phase by the surfactant Triton X-114 ((1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol). Under optimized complexation and extraction conditions, the limits of detection were 0.37 μg L⁻¹ (Cd), 2.6 μg L⁻¹ (Ni) and 2.3 μg L⁻¹ (Zn). This extraction was quantitative with a preconcentration factor of 30 and enrichment factor estimated to be 42, 40 and 43, respectively. The method was applied to different complex samples, and the accuracy was evaluated by analyzing a water standard reference material (NIST SRM 1643e), yielding results in agreement with the certified values.     

Brevetoxin exposure,superoxide dismutase activity and plasma protein electrophoretic profiles in wild-caught Kemp's ridley sea turtles (Lepidochelys kempii) in southwest Florida

Because of their vulnerable population status, assessing exposure levels and impacts of toxins on the health status of Gulf of Mexico marine turtle populations is critical. From 2011 to 2013, two large blooms of the red tide dinoflagellate, Karenia brevis, occurred along the west coast of Florida USA (from October 2011 to January 2012 and October 2012 to April 2013). Other than recovery of stranded individuals, it is unknown how harmful algal blooms affected the Kemp's ridley sea turtles (Lepidochelys kempii) inhabiting the affected coastal waters. It is essential to gather information regarding brevetoxin exposure in these turtles to determine if it poses a threat to marine turtle health and survival. From April 2012 to May 2013, we collected blood from 13 immature Kemp's ridley turtles captured in the Pine Island Sound region of the Charlotte Harbor estuary. Nine turtles were sampled immediately after or during the red tide events (bloom group) while four turtles were sampled between the events (non-bloom group). Plasma was analyzed for total brevetoxins (reported as ng PbTx-3 eq/mL), superoxide dismutase (SOD) activity, total protein concentration and protein electrophoretic profiles (albumin, alpha-, beta- and gamma-globulins). Brevetoxin concentrations ranged from 7.0 to 33.8 ng PbTx-3 eq/mL. Plasma brevetoxin concentrations in the nine turtles sampled during or immediately after the red tide events were significantly higher (by 59%, P = 0.04) than turtles sampled between events. No significant correlations were observed between plasma brevetoxin concentrations and plasma proteins or SOD activity, most likely due to the small sample size; however alpha-globulins tended to increase with increasing brevetoxin concentrations in the bloom group. Smaller (carapace length and mass) bloom turtles had higher plasma brevetoxin concentrations than larger bloom turtles, possibly due to a growth dilution effect with increasing size. The research presented here improves the current understanding of potential impacts of environmental brevetoxin exposure on marine turtle health and survival.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology,lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Poly-β-hydroxybutyrate/ectoine co-production by ectoine-excreting strain Halomonas salina

The ectoine-excreting bacterial strain of Halomonas salina was employed in the co-production of poly-β-hydroxybutyrate (PHB) and ectoine (Ect) during a fermentation process (PHB/Ect co-production). An efficient PHB/Ect co-production process was carried out at low NaCl concentration (30 g L⁻¹). It was established using ¹H Nuclear Magnetic Resonance spectroscopy that H. salina produces PHB. The effects of the NaCl concentration, the initial C/N ratio, the phosphate concentration and mixed carbon sources were investigated with respect to PHB/Ect co-production. The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30 g L⁻¹. The optimal conditions for PHB/Ect co-production by the ectoine-excreting strain of H. salina were 30 g L⁻¹ NaCl, with an initial C/N ratio of 15, an initial phosphate concentration of 12 g L⁻¹ and mixed carbon sources of 55 g L⁻¹ glucose and 25 g L⁻¹ monosodium glutamate. Using a PHB/Ect co-production system with growing and non-growing cell phases prevents the inhibition of PHB synthesis by high concentration of NaCl and significantly reduces ectoine degradation. PHB and ectoine concentrations as high as 35.3 g L⁻¹ and 8.6 g L⁻¹, respectively, were achieved. The efficient co-production of PHB and ectoine at a low NaCl concentration has been realised.     

Continuous succinic acid production by Actinobacillus succinogenes in a biofilm reactor: Steady-state metabolic flux variation

Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO₂ as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g⁻¹ at a glucose consumption of 10 g L⁻¹, to 5.7 g g⁻¹ at a glucose consumption of 50 g L⁻¹. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g⁻¹) at a glucose consumption of 10 g L⁻¹ to a value close to zero at 50 g L⁻¹. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g⁻¹ and 48.5 g L⁻¹ respectively. Metabolic flux analysis based on the established C₃ and C₄ metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway.     

Oxygen enhances the recovery of Potamogeton maackianus from prolonged exposure to very low irradiance

Recovery ability in relation to carbohydrate content of Potamogeton maackianus growing in two dissolved oxygen concentrations (8 and 2 mg L⁻¹) was investigated during 28 days exposure to very low irradiance (about 0.06 μmol m⁻² s⁻¹). Plant weight remained relatively constant (0.19 g dry wt plant⁻¹) within the initial 21 days in the high oxygen treatment, but decreased to 0.14 g dry wt plant⁻¹ at the end of the experiment. In low oxygen environments, plant weight was similar within the initial 14 days, but decreased to 0.08 g dry wt plant⁻¹ at 21 day. During the experimental period, both soluble sugar and starch contents in shoots decreased with time. Compared to high oxygen treatment, plants in the low oxygen treatment depleted starch more quickly (25 versus 18 mg g⁻¹ at 28 day) but remained a relatively high soluble sugar content (0.9 versus 1.8 mg g⁻¹ at 28 day). After recovery in high light and high dissolved oxygen conditions for 1 week, plant growth rate, new branch number, stem elongation rate and leaf recruitment number were significantly higher in high oxygen than in the low oxygen treatments. These data suggest that the ability of the plant to recover from prolonged exposure to very low irradiance is related to the depletion level of carbohydrate stored in plant tissues, which is regulated by oxygen availability in the water.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.     

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.     

Treatment of acidic wastewater arising from the refining of vegetable oil by crossflow microfiltration at very low transmembrane pressure

The refining process of vegetable oils generates acidic wastewater with the following characteristics: pH (1–1.5), COD (10–30 g O₂ L⁻¹), suspended solids (7–12 g L⁻¹) and fats (2–4 g L⁻¹). In order to reduce the effluent load and recover a fraction of the fats without using any additives, a microfiltration (0.2–1.4 μm) process involving ceramic membranes at very low transmembrane pressure values (0.1–1 bar) was assessed. Four batches of acidic wastewater from different manufacturing runs were tested. Trials with a constant volumetric reduction ratio of 30 were carried out for periods of more than 5 h. With a 0.5 μm membrane it was possible to maintain a permeate flux of 100 L h⁻¹ m⁻² for 24 h and achieve a 91% reduction in SS, a 96% reduction in fat and a COD reduction of more than 60%. In addition, the retentate thus extracted separated spontaneously into two phases, both of which could be exploited: the upper phase mainly consisting of fats as a by-product and the lower clarified phase which could be mixed into the permeate.     

A new bioprocess for the production of prebiotic lactosucrose by an immobilized β-galactosidase

A new bioprocess for the synthesis of lactosucrose was studied using a covalently immobilized β-galactosidase on macrospheres of chitosan. The effects of temperature and pH on the production of lactosucrose and other oligosaccharides were evaluated. At 30 °C and pH 7.0, the maximum concentration of lactosucrose reached to 79 g L⁻¹. The change of the reaction conditions allowed to modify the qualitative profile of the final products without quantitative change in the total of oligosaccharides produced. At pH 7 and 30 °C, products profile was 79 g L⁻¹ of lactosucrose, 37 g L⁻¹ of galactooligosaccharides and 250 g L⁻¹ of total oligosaccharides, while at pH 5 and 64 °C the concentrations for the same compounds were 40, 62 and 250 g L⁻¹, respectively. The immobilization increased the thermal stability up to 260-fold. Using 300 g L⁻¹ of sucrose and 300 g L⁻¹ of lactose, and 8.5 mg of chitosan mL⁻¹, 30 cycles of reuse were performed and the biocatalyst kept the maximal lactosucrose synthesis. These results fulfill some important aspects for the enzyme immobilization and oligosaccharides synthesis: the simplicity of the protocols, the high operational stability of the enzyme and the possibility of driving the final products.     

MTs in Palaemonetes argentinus as potential biomarkers of zinc contamination in freshwaters

Aquatic invertebrates take up and accumulate essential and non-essential trace metals even when both are likely to be poisonous. In order to study the potential of the metallothioneins (MTs) as biomarkers of metal contamination in native shrimp Palaemonetes argentinus, organisms have been exposed at 0, 5, 50 and 500 μg L⁻¹ of zinc for 96 h. Moreover, accumulation and subcellular distribution of this essential metal were evaluated. A significant Zn accumulation was observed in different body sections. Higher Zn levels occurred in cephalothorax compared to abdomen, especially at the highest exposure concentration (500 μg Zn L⁻¹). A clear differential subcellular metal distribution between cephalothorax and abdomen was also observed. In cephalothorax Zn was similarly distributed between the soluble and insoluble fractions; while in abdomen, when total Zn increased, insoluble metal augmented more markedly than the soluble one. Cytosolic Zn levels increased more in cephalothorax than in abdomen of shrimps exposed to 500 μg Zn L⁻¹ when compared to control. Finally, a significant induction of MTs was observed in cephalothorax at 500 μg Zn L⁻¹. A potential role for MTs as biomarkers in P. argentinus should be further studied to enhance the sensitivity of the response, although it is likely that MTs play a key role in metal detoxification since the increase of these proteins is linked to metal challenge.     

Evaluation of the effects of light intensity on growth of the benthic dinoflagellate Ostreopsis sp. 1 using a newly developed photoirradiation-culture system and a novel regression analytical method

Benthic dinoflagellates of the genus Ostreopsis are found all over the world in temperate, subtropical, and tropical coastal regions. Our recent studies revealed that a putative “cryptic” species of Ostreopsis ovata is present widely along Japanese coasts. This organism, Ostreopsis sp. 1, possesses palytoxin analogs and thus its toxic blooms may be responsible for potential toxification of marine organisms. To evaluate the bloom dynamics of Ostreopsis sp. 1, the present study examined the growth responses of Ostreopsis sp. 1 strain s0716 to various light intensities (photon flux densities: μmol photons m⁻² s⁻¹) using a newly devised photoirradiation-culture system. This novel system has white light-emitting diodes (LEDs) capable of more closely simulating the wavelength spectrum of light entering the oceanic water column than do fluorescent tubes and halogen lamps. In this system, the light intensity of the white LEDs was reduced through two polarizing filters by varying the rotation angles of the filters. Thereby, the new system was capable of culturing microalgae under well-controlled light intensity conditions. Ostreopsis sp. 1 grew proportionally when light intensity was increased from 49.5 to 199 μmol photons m⁻² s⁻¹, but its growth appeared to be inhibited slightly at ≥263 μmol photons m⁻² s⁻¹. The relationship between observed growth rates and light intensity was calculated at R > 0.99 (P < 0.01) using a regression analysis with a modified equation of the photosynthesis-light intensity (P-L) model. The equation determined the critical light intensities for growth of Ostreopsis sp. 1 and the organism's growth potential as follows: (1) the threshold light intensity for growth: 29.8 μmol photons m⁻² s⁻¹; (2) the optimum light intensity (L_m) giving the maximum growth rate (μ_max = 0.659 divisions day⁻¹): 196 μmol photons m⁻² s⁻¹; (3) the optimum light intensity range (L_opt) giving ≥95% μ_max: 130–330 μmol photons m⁻² s⁻¹; (4) the semi-optimum range (L_sopt) giving ≥80% μ_max: 90 to over 460 μmol photons m⁻² s⁻¹. The L_sopt represents 4.5–23% ambient light intensity present in surface waters off of a temperate region of the Japanese coast, Tosa Bay; putatively, this semi-optimum range of light intensity appears at depth of 12.9–27.8 m. Considering these issues, our data indicate that Ostreopsis sp. 1 in coastal environments may form blooms at ca. ∼28 m depth in regions along Japanese coasts.     

Signal transducer and oxidative stress mediated modulation of phenylpropanoid pathway to enhance rosmarinic acid biosynthesis in fungi elicited whole plant culture of Solenostemon scutellarioides

This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation with phytopathogenic fungi. Amongst selected fungi, Aternaria alternata caused significant elevation (p < 0.05–0.01) in RA accumulation (∼1.3–1.6-fold) between 25 and 100 μg l⁻¹. However, elicitation at the dose of 50 μg l⁻¹ has been found to be most effective and intracellular RA content reached almost ∼1.6-fold (p < 0.01) higher in day 7. Therefore, A. alternata (50 μg l⁻¹) was selected for mechanism evaluation. A significant elevation of intercellular jasmonic acid was observed up to day 6 after elicitation with A. alternata (50 μg l⁻¹). A significant increase in tissue H₂O₂ and lipid peroxidation coupled with depletion of antioxidant enzymes superoxide dismutase and catalase indicated augmented oxidative stress associated with biotic interaction. Preceding the elicitor-induced RA accumulation, a notable alteration in the specific activities of biosynthetic enzymes namely PAL and TAT was recorded, while, no significant change in the activities of RAS was observed. HPPR activity was slightly improved in elicited plant. Therefore, it could be concluded that A. alternata elicited the biosynthesis of rosmarinic acid via signal transduction through jasmonic acid coupled with elicitor induced oxidative stress and associated mechanism.     

Expansion of harmful brown tides caused by the pelagophyte,Aureoumbra lagunensis DeYoe et Stockwell,to the US east coast

Brown tides caused by the pelagophyte Aureoumbra lagunensis DeYoe et Stockwell have formed ecosystem disruptive algal blooms in shallow lagoons of Texas (TX), USA, for more than two decades but have never been reported elsewhere. During the summer of 2012, a dense brown tide occurred in the Mosquito Lagoon and northern Indian River Lagoon along the east coast of Florida (FL), USA. While chlorophyll a levels in this system have averaged 5 μg L⁻¹ during the past two decades, concentrations during this brown tide reached ∼200 μg L⁻¹. Concurrently, levels of nitrate were significantly lower than average and levels of dissolved organic nitrogen were significantly higher than average (p < 0.001 for both). Sequences of the 18S rRNA gene of the bloom community and of single cell isolates were identical to those of Aureoumbra lagunensis DeYoe et Stockwell from TX. The A. lagunensis brown tide in FL bloomed to densities exceeding 10⁶ cells mL⁻¹ (quantified with a species-specific immuno-label) from July through September, began to dissipate in October, but maintained densities exceeding 10⁵ cells mL⁻¹ in some regions through December of 2012. The decline of the bloom was associated with near-hypoxic conditions and more than 30 fish kills reported within the Mosquito Lagoon in September 2012, a number far exceeding all prior monthly reports in this system dating to 1996. Wild northern quahog populations (a.k.a. hard clam, Mercenaria mercenaria) suffered mass die offs during the brown tide and eastern oysters (Crassostrea virginica) that settled during 2012 were significantly smaller than prior years. Clearance rates of hard clams and eastern oyster were significantly reduced in the presence of Mosquito Lagoon bloom water and A. lagunensis monocultures isolated from the Mosquito Lagoon at densities of ∼10⁶ cells L⁻¹. The expansion of harmful brown tides caused by A. lagunensis to these estuaries represents a new threat to the US southeast coast.