Hydrolysis of palm and olive oils by immobilised lipase using hollow fibre reactor

Lipase from Mucor miehei immobilised by adsorption on microporous, asymmetric hollow fibre membrane reactors was used to hydrolyse two different oils, namely, palm and olive oils. The hydrolysis reaction was carried out at a temperature of 40 °C, an average transmembrane pressure (TMP) of 115 mmHg and oil and aqueous flow rates of 2.5 and 3.0 ml min⁻¹, respectively. It was experimentally proven that adsorption of lipase increased with temperature and was higher on hydrophobic membranes than hydrophilic ones. The effluent concentrations of fatty acid products were measured using gas chromatograph with FID detector. Hydrolysis experimental results were fitted to a multisubstrate kinetic model derived from the Ping Pong Bi Bi mechanism. The final model expression is useful for predicting the free fatty acid profile of the enzymatic hydrolysis of palm and olive oils for different substrate flow rates and enzyme loading.     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

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.     

Urea loading from a spring storm—Knysna estuary,South Africa

Urea concentrations were tracked in the Knysna estuary, South Africa, following a strong initial storm of the austral summer rainy season in 2000. These post-storm urea concentrations are compared against a 1-year survey of these concentrations and demonstrate the initial and longer-term impacts of urea loading on a near-pristine estuary. Fifteen stations along the main channel of the estuary were sampled four times each, 2–4 weeks before the storm, providing and average urea concentration of 1.4 μmol N L⁻¹. When these same stations were sampled 12 h after the storm the average urea concentration had increased to 9.4 μmol N L⁻¹. The average urea concentration at these 15 stations remained high, averaging 10.2 μmol N L⁻¹ and 10.0 μmol N L⁻¹ 36 and 84 h post-storm, respectively. The urea concentrations in the rivers supplying fresh water to the Knysna estuary, one draining pasturelands and the other draining informal housing settlements that relied on pit toilets and open sewers are also compared before and after this storm. These data suggest that spring storms significantly influence nutrient availability, which in turn, may be related to the large dinoflagellates blooms witnessed in the Knysna estuary during the later portion of the wet summer season.     

Effects of flooding on photosynthesis and root respiration in saltcedar (Tamarix ramosissima), an invasive riparian shrub

The introduced shrub Tamarix ramosissima invades riparian zones, but loses competitiveness under flooding. Metabolic effects of flooding could be important for T. ramosissima, but have not been previously investigated. Photosynthesis rates, stomatal conductance, internal (intercellular) CO₂, transpiration, and root alcohol dehydrogenase (ADH) activity were compared in T. ramosissima across soil types and under drained and flooded conditions in a greenhouse. Photosynthesis at 1500 μmol quanta m⁻² s⁻¹ (A₁₅₀₀) in flooded plants ranged from 2.3 to 6.2 μmol CO₂ m⁻² s⁻¹ during the first week, but A₁₅₀₀ increased to 6.4–12.7 μmol CO₂ m⁻² s⁻¹ by the third week of flooding. Stomatal conductance (g_s) at 1500 μmol quanta m⁻² s⁻¹ also decreased initially during flooding, where g_s was 0.018 to 0.099 mol H₂O m⁻² s⁻¹ during the first week, but g_s increased to 0.113–0.248 mol H₂O m⁻² s⁻¹ by the third week of flooding. However, photosynthesis in flooded plants was reduced by non-stomatal limitations, and subsequent increases indicate metabolic acclimation to flooding. Root ADH activities were higher in flooded plants compared to drained plants, indicating oxygen stress. Lower photosynthesis and greater oxygen stress could account for the susceptibility of T. ramosissima at the onset of flooding. Soil type had no effect on photosynthesis or on root ADH activity. In the field, stomatal conductance, leaf water potential, transpiration, and leaf δ¹³C were compared between T. ramosissima and other flooded species. T. ramosissima had lower stomatal conductance and water potential compared to Populus deltoides and Phragmites australis. Differences in physiological responses for T. ramosissima could become important for ecological concerns.     

Sex-specific light acclimation of Chara canescens (Charophyta)

Bisexual populations of the charophyte Chara canescens (Desv. et Loisel. in Loisel., 1810) containing male and female individuals are rarely found. Two experiments were carried out to study whether male and female algae from the same site exhibit different physiological capacities, especially with respect to light acclimation.Algae from two different shore levels and from laboratory cultures acclimated to six irradiance conditions (35–500 μmol photons m⁻² s⁻¹) were compared. Field measurements showed that both female and male algae of C. canescens are able to acclimate to daily changes in solar irradiance. The quantum yield of Photosystem II (PSII) decreased with increasing irradiance in the morning and increased with decreasing irradiance in the afternoon. Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼7 mg FW) up to 500 μmol photons m⁻² s⁻¹ (∼27 mg FW) in female and male C. canescens. The irradiance saturation point for photosynthesis (E_k) was about 140 μmol m⁻² s⁻¹ for both sexes within the whole range of acclimation irradiances. The maximum photosynthesis rate at saturating irradiances (P_max) of male algae was highest at E_k, whereas P_max of female algae was highest at 500 μmol photons m⁻² s⁻¹. The photosynthetic efficiency in the light-limited range (α) increased in female C. canescens and decreased in male C. canescens. The ratio of the non-photochemical quenching parameter (NPQ) to the relative electron transport rates rETR_(MT) increased in both sexes with irradiance, but showed a steeper increase in male than in female algae. Pigment analysis showed similar acclimation pattern for male and female C. canescens. Chl a/Chl b ratios of both sexes were constant over the whole range of E_g, whereas Chl a/carotenoid ratios in male and female C. canescens decreased from 70 μmol photons m⁻² s⁻¹ upwards. Pigment analysis pointed out that the carotenes α-, β- and γ-carotene were more prominent in male than in female algae.Our results indicate that female C. canescens are more efficient in light acclimation than male algae from the same site. Nevertheless, further investigations of bisexual C. canescens populations resolving CO₂-uptake mechanisms and/or genetic differences are needed.     

Ectoine improves yield of biodiesel catalyzed by immobilized lipase

To improve the production of biodiesel by enzymatic conversion of triglycerides in cottonseed oil, compatible solutes were added to the solvent-free methanolysis system to prevent competitive methanol inhibition on the immobilized lipase (Novozym^® 435). The results indicated that the addition of ectoine increased biodiesel synthesis using a three-step methanol addition process. The concentration of methyl ester (ME) reached a maximum of 95.0% in the presence of 1.1 mmol/l ectoine, an increase of 20.9% compared to that in the absence of ectoine. On the other hand, excess ectoine decreased the ME concentration. Ectoine was also shown to enhance reuse of the immobilized lipase, significantly improving ME concentrations in each recycling test. Total concentrations of ME with added ectoine were about 1.5 times that without ectoine during five recycling tests (molar ratio of cottonseed oil to methanol, 1:4). Enzymatic reaction kinetics showed, in the concentration ranges of 0.8–1.14 mol/l and 0.03–8 mol/l for triglyceride and methanol, respectively, that ectoine had no effect on the initial reaction rates when methanol concentrations were below 0.5 mol/l. When methanol concentration exceeded 0.5 mol/l, the addition of 0.8 mmol/l ectoine increased the initial reaction rates, and the lipase exhibited a lower affinity for methanol and higher affinity for triglyceride (kinetic parameters of K_mA increase, K_mTG decrease). However, the initial reaction rates decreased significantly when 8 mmol/l ectoine was added, with the lipase having higher affinity for methanol and lower affinity for triglyceride (K_mA decrease, K_mTG increase). The supplementation of ectoine provided a new method for the purpose of improving yield of biodiesel catalyzed by enzyme.     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Salt uptake and shoot water relations in mangroves

The effects of salinity on water relations and ion concentrations were investigated in seedlings of the mangroves Avicennia alba, Bruguiera gymnorrhiza, Heritiera littoralis and Xylocarpus granatum grown at salinities of 0, 10, 20, 30, 40 and 60‰. All four species survived and grew at salinities ranging from 0 to 40‰, but none survived at a salinity of 60‰. The concentration of sodium and chloride in the xylem sap increased with increasing salinity in both A. alba and B. gymnorrhiza. Sodium and chloride concentrations in the xylem sap of A. alba grown at 40‰ salinity both reached 114 mol m⁻³, about 15% of the external concentration around the roots. The xylem sap of B. gymnorrhiza grown at 40‰ salinity, by contrast, contained only 7.0 mol m⁻³ sodium and 4.1 mol m⁻³ chloride, about 1% of their concentrations in the external solution around the roots. The results indicated that B. gymnorrhiza, which does not have salt-secreting glands, was more effective at excluding salt than A. alba, which has salt-secreting glands.Analysis of pressure–volume curves showed that the bulk modulus of elasticity increased with increasing salinity. This was accompanied by a decrease in shoot water potential, mainly associated with a reduction in shoot osmotic potentials with increasing salinity. The decrease in osmotic potential was attributed to increasing solute concentrations, particularly sodium and chloride, in the leaves of all species except H. littoralis, which had little sodium and chloride in the leaves.     

An ozone response relationship for four Phleum pratense genotypes based on modelling of the phytotoxic ozone dose (POD)

In the last decade extensive research has focused on the development of dose–response relationships based on stomatal plant ozone uptake (phytotoxic ozone dose, POD). So far most work has concentrated on crops and forest trees. This study provides a flux-based dose–response function for timothy (Phleum pratense), a widespread grassland species, which can be used in risk assessment for ground-level ozone. In 1996 and 2001 timothy was exposed in open-top chambers to ozone concentrations ranging from around 10 nmol mol⁻¹ in the charcoal filtered treatments up to 60 nmol mol⁻¹ in the fumigated treatments (08:00–20:00) in. In 1996 there was a negative effect of ozone on biomass production in the non-filtered treatment while in 2001 no such ozone effect in the non-filtered treatment could be seen. Measurements of stomatal conductance on four timothy genotypes in 2001 were used to calibrate a Jarvis-type multiplicative stomatal conductance model. The maximum conductance varied between the genotypes, from 477 to 589 mmol O₃ m⁻² s⁻¹ (projected leaf area). The model includes functions describing the reduction of stomatal conductance of senescing leaves and the direct effects on stomatal conductance by light, temperature and water vapour pressure deficit. A function describing ozone induced senescence of the leaves was included since exposure to ozone is known to cause premature senescence. The function for ozone was applied when it suggested ozone to be more limiting to stomatal conductance than phenology. To avoid overestimation of stomatal conductance in days with high VPD, a function reflecting the effect on leaf water potential on stomatal conductance was included. Comparison between modelled and measured conductance for the four timothy genotypes resulted in an r² value at 0.57 and a very small average deviation of observed from modelled values. The calibrated stomatal conductance model was used to estimate the accumulated POD, i.e. the accumulated stomatal flux of ozone, of the plants in the 1996 and 2001 experiments. The strongest relationship between ozone relative effects on biomass was obtained when POD was accumulated from 105 degree days after emergence to 1000 degree days after emergence, and integrated using an uptake rate threshold of 7 nmol m⁻² s⁻¹ (POD₇). The response relationship between biomass and POD₇ resulted in an r² value of 0.71 over all four genotypes. This r² value was somewhat higher than for the corresponding relationship based on the accumulated ozone exposure over 40 nmol mol⁻¹ (AOT40; r² = 0.66). With an uptake rate threshold at 7 nmol m⁻² s⁻¹, ozone concentrations above ∼20 nmol mol⁻¹, contribute to reduce the biomass production of timothy if meteorological conditions promote maximum stomatal conductance.     

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO₂ assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100 μmol photons m⁻² s⁻¹ light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100 μmol photons m⁻² s⁻¹ light condition. Under 15 μmol photons m⁻² s⁻¹ light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.     

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g⁻¹ dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g⁻¹ dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m⁻² s⁻¹ had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g⁻¹ dry weight in darkness to 20 mg g⁻¹ dry weight at a light intensity of 80 μmol photons m⁻² s⁻¹. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day⁻¹, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g⁻¹ dry weight was found at 65 μmol photons m⁻² s⁻¹. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.     

Synthesis of S-licarbazepine by asymmetric reduction of oxcarbazepine with Saccharomyces cerevisiae CGMCC No. 2266

S-licarbazepine was synthesized by asymmetric reduction of oxcarbazepine with CGMCC No. 2266. The optimum batch reduction conditions were found to consist of a reaction time of 36 h, temperature of 30 °C, and initial pH value of 7.0. The optimum concentration of the glucose co-substrate was found to be 0.3 mol L⁻¹. The addition of glucose contributed to in situ regeneration of NADPH in cells and improved conversion. Conversion increased with the addition of more biomass and with a decrease in the initial concentration of substrate. Within the membrane reactor, a continuous reduction process was used to improve production efficiency and reduce the inhibition of high-concentration substrate upon reduction. The optimum flux was found to be 20 ml h⁻¹. S-licarbazepine yield was 3.7678 mmol L⁻¹ d⁻¹ in continuous reduction over four days. The enantiometric excess of S-licarbazepine was 100% for both batch and continuous reduction processes.     

Thresholds for morphological response to light reduction for four tropical seagrass species

Seagrasses worldwide are highly vulnerable to, and at increasing risk from reduced light availability, and robust light thresholds are required for evaluating future impacts of changing light conditions. We tested the morphological response (shoot density and growth) of four Indo-West Pacific seagrass species (Cymodocea serrulata, Halodule uninervis, Halophila ovalis and Zostera muelleri) to six daily light levels ranging from 0 to 23 mol m⁻² d⁻¹ (0–70% surface irradiance) in cool (∼23 °C) and warm temperatures (∼28 °C) over 14 weeks. The impact of light limitation on shoot densities and growth rates was higher at warm than at cool temperatures, and for Z. muelleri and H. ovalis than for C. serrulata and H. uninervis, in terms of both the time taken for the low light treatment to take effect and the predicted time to shoot loss (e.g. 17–143 days at 0 mol m⁻² d⁻¹). Using fitted curves we estimated temperature-dependent thresholds (with estimates of uncertainty) for 50% and 80% protection of growth and shoot density, defined here as “potential light thresholds” in recognition that they were derived under experimental conditions. Potential light thresholds that maintained 50% and 80% of seagrass shoot density fell within the ranges 1.1–5.7 mol m⁻² d⁻¹ and 3.8–10.4 mol m⁻² d⁻¹, respectively, depending on temperature and species. Light thresholds calculated in separate in situ studies for two of the same species produced comparable results. We propose that the upper (rounded) values of 6 mol m⁻² d⁻¹ and 10 mol m⁻² d⁻¹ can be used as potential light thresholds for protecting 50% and 80% of shoot density for these four species over 14 weeks. As management guidelines should always be more conservative than thresholds for biological declines, we used error estimates to provide a quantitative method for converting potential light thresholds into guidelines that satisfy this criterion. The present study demonstrates a new approach to deriving potential light thresholds for acute impacts, describes how they can be applied in management guidelines and quantifies the timescales of seagrass decline in response to light limitation. This method can be used to further quantify cumulative impacts on potential light thresholds.     

Effects of irradiance and water flow on formation and growth of spongy and filamentous thalli of Codium fragile

Effects of irradiance and water flow on formation and growth of filamentous and spongy thalli of Codium fragile (Suringar) Hariot growing on vinylon threads were investigated at the laboratory culture. They showed clear differences in their irradiance and water flow requirements for their formation and growth. Spongy thalli were formed from the cultured filamentous thalli only at the high water flow velocity (10 cm s⁻¹). Number of the spongy thalli remarkably increased with increasing irradiance because those at 10, 50 and 100 μmol m⁻² s⁻¹ reached 0, 2 and 76 thalli m⁻¹, respectively, by 10 weeks of culture. In contrast, filamentous thalli were formed from the cultured spongy thalli at 0 and 3 cm s⁻¹, and difference in irradiance had no effect on their formation. Growth of the spongy thalli greatly accelerated under the combination of the high irradiance and high water velocity (200 μmol m⁻² s⁻¹ and 10 cm s⁻¹) because their relative growth rate in wet weight under the condition was two–four times higher than those at the other examined irradiances and water velocities. On the other hand, difference in water velocity had no effect on growth of the filamentous thalli under flowing water, and their growth decelerated at the high irradiance (200 μmol m⁻² s⁻¹). This demonstrates that water flow is a major factor controlling the formation of the spongy and filamentous thalli. The formation and growth of the spongy thalli surely occur under the combination of the high irradiance and fast flowing water. In contrast, the formation of the filamentous thalli occurs in the calm water, and their growth is inhibited under the high irradiance.     

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.     

Light intensity increases the susceptibility of Vallisneria natans to snail herbivory

Palatability to snail herbivory (Radix swinhoei H. Adams) and C/N ratios were assessed for Vallisneria natans (Lour.) Hara, in three different experimental light regimes (midday fluxes respectively 280 μmol m⁻² s⁻¹, 15 μmol m⁻² s⁻¹, and a variable intensity between these two). Higher light intensity as well as prolonged photoperiods increased palatability and growth, and improved C/N ratio by decreasing N content. Snail growth was slightly increased but juvenile survivorship decreased under higher light. The results suggest that the availability of light may affects intraspecific variation in palatability of V. natans.     

Effect of environmental and nutritional factors on growth and azaspiracid production of the dinoflagellate Azadinium spinosum

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota.Azadinium spinosum grew in a wide range of conditions; from 10 ̊C to 26 ̊C and salinities from 30 to 40, under irradiances ranging from 50 μmol m⁻² s⁻¹ to 250 μmol m⁻² s⁻¹, with or without aeration. Growth and maximum cell concentration were highest at a salinity of 35, at temperatures between 18 ̊C and 22 ̊C, and with aeration. Concerning AZA cell quota, the most significant effect was observed at low temperature; the AZA cell quota was more than 20 times higher at 10 ̊C (220 fg cell⁻¹) than at temperatures between 18 ̊C and 26 ̊C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of K-medium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth.These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.     

Biosynthesis of glycyrrhetic acid 3-O-mono-β-d-glucuronide catalyzed by β-d-glucuronidase with enhanced bond selectivity in an ionic liquid/buffer biphasic system

The bond selective hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) catalyzed by recombinant β-d-glucuronidase from Escherichia coli BL21 (PGUS-E) was successfully performed in an ionic liquid (IL)/buffer biphasic system. Five ILs were analyzed, however, a hydrophobic IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) showed the best biocompatibility with PGUS-E. An obvious enhancement in the initial reaction rate, substrate conversion, GAMG yield and chemical bond selectivity (S_cb) was observed using 40% (v/v) [BMIM]PF₆/buffer as the reaction medium when compared to the acetate buffer medium. Under the optimized conditions (pH 6.0, temperature 50 °C, substrate concentration 6 mM and shaking speed 200 rpm), the initial reaction rate, the GAMG yield and the S_cb reached 3.15 mM h⁻¹, 74.36% and 98.12%, respectively. The recyclability of [BMIM]PF₆ was also studied and found to be reusable for five batches with high recovery percentage (≥92%). Furthermore, the desired product and byproduct were easily separated since they were distributed in different phases. Additionally, higher V_max (3.14 versus 2.24 mM h⁻¹), lower apparent K_m (1.21 versus 1.80 mM) and E_a (25.97 versus 32.60 kJ mol⁻¹) were achieved in [BMIM]PF₆/buffer biphasic system than that in monophasic buffer system.     

The correction of reaction rates in continuous fluorometric assays of enzymes

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH₂, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10^− 4 × s^− 1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH₂, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was − 2.96 × 10^− 4 a.u μM^− 1 × s^− 1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate] ÷ (final − initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K_m and overestimation of V_max.