Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana

High density cultivation is essential to industrial production of biodiesel from microalgae, which involves in variations of micro‐environment around individual cells, including light intensity, nutrition distribution, other abiotic stress and so on. To figure out the main limit factor in high inoculum cultivation, a quantitative proteomic analysis (iTRAQ‐on‐line 2‐D nano‐LC/MS) in a non‐model green microalga, Chlorella sorokiniana, under different inoculum sizes was conducted. The resulting high‐quality proteomic dataset consisted of 695 proteins. Using a cutoff of P < 0.05, 241 unique proteins with differential expression levels were identified between control and different inoculum sizes. Functional analysis showed that proteins participating in photosynthesis (light reaction) and Calvin cycle (carbon reaction pathway) had highest expression levels under inoculum size of 1 × 10⁶ cells mL^?1, and lowest levels under 1 × 10⁷ cells mL^?1. Canonical correlation analysis of the photosynthesis related proteins and metabolites biomarkers showed that a good correlation existed between them (canonical coefficient was 0.987), suggesting photosynthesis process greatly affected microalgae biodiesel productivity and quality. Proteomic study of C. sorokiniana under different illuminations was also conducted to confirm light intensity as a potential limit factor of high inoculum size. Nearly two thirds of proteins showed up‐regulation under the illumination of 70–110 µmol m^?2 s^?1, compared to those of 40 µmol m^?2 s^?1. This result suggested that by elegantly adjusting light conditions, high cell density cultivation and high biodiesel production might be achieved. Biotechnol. Bioeng. 2013; 110: 773–784. © 2012 Wiley Periodicals, Inc.     

Shear rate dependence of free energy barrier height for phenyl ring rotations in polystyrene based on non-equilibrium molecular dynamics simulations

Polystyrene properties are influenced by ring motions in side groups. The main chain conformation and interaction with the surroundings dominate the ring rotations. It is known that shear flow affects linear chain conformation and molecular distribution. However, shear-induced variations in the ring rotations have yet to be studied. This study presents a shear flow system of polystyrene via non-equilibrium molecular dynamics simulations. The free energy barrier of the phenyl ring rotations was obtained from the distribution of angle χ between the ring and main chain based on the Boltzmann distribution law. The results showed that the barrier height approaches a constant value at a shear rate less than 10¹⁰ s^? 1, but decreases with an increase in shear rate higher than 10^10.5 s^? 1. Furthermore, the radial distribution function and potential energies were compared. Remarkably, the shear flow reduced the bond vibrations of the phenyl rings, but increased the separation between intermolecular particles. Hence, a smaller cavity is necessary for the rings to rotate once but more volume is occupied by the rings. The smaller volume obtained via main chain motions needed to construct the cavity lowers the energy barrier height at shear rate higher than 10^10.5 s^? 1.     

Kinetics of dimerization of the bence-jones protein Au

The dimerization reactions of complete Bence-Jones protein Au (VC-Au) and of its variable fragment (V-Au) were compared in 0.2 M (ionic strength) sodium phosphate buffer, pH 6.8 at 20° C. The dimerization constant for VC-Au (6.6 × 10⁴ M^?1) was slightly smaller than a previously published value for the fragment (1.1 × 10⁵ M^?1). The reaction enthalpies were positive for both processes. Temperature jump experiments exhibited two kinetic phases. The relaxation time of the fast phase as well as its concentration dependence and amplitude were almost identical for VC-Au and V-Au. Only small differences were observed in the slow phase. These close similarities between the reactions of the two proteins demonstrate that dimerization occurs mainly via interactions between the variable domains and that the constant domains interfere very little. From the observation of two relaxation times it follows that the dimerization mechanism for both VC-Au and V-Au must include at least three reacting species. Mechanisms with an isomerization between monomers in two conformational states and a single dimer species are excluded by the data. Alternative mechanisms with a single monomeric species but isomerization between dimers give a rather unsatisfactory fit. A good fit can be obtained if it is assumed that both monomers and dimers can exist in two states. Rate constants of the association and dissociation steps are of the order of 10⁷ M^?1 s^?1 and 10² s^?1. Isomerization rate constants are in the range of 10 s^?1.     

Effect of the cations sodium,potassium and calcium on the interaction of hyaluronate chains: a light scattering and viscometric study

Light scattering and viscometric studies have been carried out on two preparations, A and B, of rooster comb hyaluronate. Sedimentation rate studies have also been performed with A. Light scattering measurements in 0.2 m KCl for preparation A gave a molecular weight of 3.3 × 10⁶ and for B, 1.0 × 10⁶. In (0.1–0.3) M NaCl similar measurements gave a particle weight for A of (4.4–6.4 × 10⁶ and for B (1.7–2.8 × 10⁶. In 0.066 m CaCl₂ molecular weight values of 9.5 × 10⁶ for A and 1.7 × 10⁶ for B were obtained. Thus in the presence of Na⁺ and Ca²⁺ ions aggregates of chains persisted into dilute solution. Measurements by light scattering on A and B in 4 m guanidinium chloride gave values in the same range as those obtained in 0.2 m KCl. Sedimentation rate studies on A gave values of 10.3 Svedbergs in 0.2 m KCl and 12.2 Svedbergs in 0.2 m NaCl and 0.066m CaCl₂. The shear dependence of the viscosity was studied using a conicylindrical viscometer at shear rates between 0.5 and 20 s^?1. Preparation A in 0.2 m KCl and NaCl yielded values for (ν_sp/cc→0 of 5000 and 7100 ml g^?1 respectively in keeping with the tendency to aggregate. The behaviour for preparation B was similar. In 0.066 m CaCl₂ there was a marked dependence of viscosity on shear speed below 10 s^?1 for all concentrations and the value of (ν_sp/c)→0 at 0 s^?1 for preparation A was 7700 ml g^?1 while at a shear rate of 8 s^?1 (ν_sp/c)c→0 ? 5000 ml g ^?1. Similar effects were found for preparation B and the data suggest associations of chains disruptable by weak shear forces. The increase in viscosity with concentration in the presence of 0.066 m CaCl₂ was much less than in the presence of KCl or NaCl, suggesting that the Ca²⁺ had a marked effect on the ”rigidity’ of the molecules in solution. A viscometric titration experiment with Ca^2? showed that a level of 0.02 m CaCl₂ in 0.2 m NaCl was sufficient to produce the change in viscosity presented above and that significant perturbations of the viscosity were present at 0.005?0.01 m CaCl₂.     

Cytarabine-Induced Destabilization and Bending of a Model Okazaki Fragment

Abstract

The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV hyperchromicity and 2D ¹H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 °C to 42.4 °C at 1.33 × 10^?5M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20° to 42°. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.     

X-ray diffraction measurements of dipalmitoylphosphatidylcholine as a function of pressure

A kinetics of azide binding by horseradish peroxidase was studied by temperature-jump method. It was found that the reaction of the enzyme with azide is quite rapid, occuring in microsecond time range. This rate is unusually rapid in contrast to the usual hemoprotein ferric iron-ligand interactions so far reported. The resulting value for the apparent association and dissociation rate constants were k₁=6.8×10⁶ M^?1 s^?1 and k¹=3.5×10⁵ s^?1 at 23°C and pH 5.0 for the reaction. The pH dependence of the rate constants was also studied to show a strong linkage of the ligand binding with a proton uptake of a dissociable group on the enzyme.     

Process optimization of xylanase production using cheap solid substrate by Trichoderma reesei SAF3 and study on the alteration of behavioral properties of enzyme obtained from SSF and SmF

This study aimed to assess the variability in respect of titer and properties of xylanase from Trichoderma reesei SAF3 under both solid-state and submerged fermentation. SSF was initially optimized with different agro-residues and among them wheat bran was found to be the best substrate that favored maximum xylanase production of 219 U (gws)^?1 at 96 h of incubation. The mycelial stage of the fungi and intracellular accumulation of Ca⁺⁺ and Mg⁺⁺ induced maximum enzyme synthesis. Inoculum level of 10 × 10⁶ spores 5 g^?1 of dry solid substrate and water activity of 0.6 were found to be optimum for xylanase production under SSF. Further optimization was made using a Box-Behnken design under response surface methodology. The optimal cultivation conditions predicted from canonical analysis of this model were incubation time (A) = 96–99 h, inoculum concentration (B) = 10 × 10⁶ spores 5 g^?1 of dry substrate, solid substrate concentration (C) = 10–12 g flask^?1, initial moisture level (D) = 10 mL flask^?1 (equivalent to a _w  = 0.55) and the level of xylanase was 299.7 U (gws)^?1. Subsequent verification of these levels agreed (97 % similar) with model predictions. Maximum amount of xylanase was recovered with water (6:1, v/w) and under shaking condition (125 rpm). Purified xylanase from SSF showed better stability in salt and pH, was catalytically and thermodynamically more efficient over enzyme from SmF, though molecular weight of both enzymes was identical (53.8 kDa).     

EFFECTS OF SUBSTRATE RELIEF ON THE DISTRIBUTION OF PERIPHYTON IN LABORATORY STREAMS,I. HYDROLOGY1

We examined the hypothesis that the heterogeneity of epilithic algal assemblages in streams may be partly a result of hydrologic differences created when water flows over a rough substrate. A 32-day experiment was conducted in laboratory streams that contained either 22.5 × 22.5 × 4 cm or 7.5 × 22.5 × 4 cm tile blocks. Free water velocities in the streams overaged 28 cm·s^?1. Hydrologic parameters and algal assemblages associated with surfaces on top of blocks and with recessed surfaces between blocks were compared to corresponding surfaces in streams with of relief. In streams with blocks, shear velocities averaged 1.7 cm·s^?1 on the top of blocks and 0.8 cm·s^?1 in the recessed areas. Shear velocity at corresponding surfaces in the control (no relief) streams averaged 1.9 cm·s^?1 and exhibited little variation. The hydrologic differences created by the larger blocks significantly affected the distribution of algal biomass, with recessed areas having an average of 2.6 g·m^?2 AFDW more biomass than surfaces on the top of blocks. Differences in shear velocities and biomass accumulation between top and recessed areas for the smaller blocks were less than for large blocks. Successional changes on all substrates were similar with the exception that recessed surfaces had a significantly greater abundance of the filamentous chlorophyte Stigeoclonium tenue (Ag.) Kütz after day 16. The results suggest that in cobble riffle areas of natural streams, the interaction between current flow and substrate relief has the potential to create patches of algae which are different in biomass and taxonomic composition.     

Physical and functional mapping of rat-liver mitochondrial DNA

Summary Rat-liver mitochondrial DNA (mtDNA) contains 2 cleavage sites of the restriction endonuclease XbaI. The molecular sizes of restriction fragments are 6.6×10⁶ and 3.7×10⁶ D. The results of partial cleavage of mtDNA with EcoRI allow the fragment F (0.32×10⁶ D) to be localized in the sequence ABCEGFHDA. The functional map of mtDNA is constructed for two genes of the ATP-ase mRNAs from rat-liver mitochondria. Molecular hybridization shows that the ATPase genes are located in fragment B and in the GEHD area of mtDNA EcoRI cleavage.     

Refolding of Npro fusion proteins

The autoprotease N^pro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C‐terminus releasing the target protein with its authentic N‐terminus. N^pro wild‐type and its mutant EDDIE, respectively, were fused N‐terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence‐evasion‐factor, and the short 16 amino acid peptide pep6His. In comparison with the N^pro wild‐type, the tailored mutant EDDIE displayed an increased rate constant for refolding and cleavage from 1.3 × 10^?4 s^?1 to 3.5 × 10^?4 s^?1, and allowed a 15‐fold higher protein concentration of 1.1 mg/mL when studying pep6His as a fusion partner. For green fluorescent protein, the rate constant was increased from 2.4 × 10^?5 s^?1 to 1.1 × 10^?4 s^?1 when fused to EDDIE. When fused to small target peptides, refolding and cleavage yields were independent of initial protein concentration, even at high concentrations of 3.9 mg/mL, although cleavage rates were strongly influenced by the fusion partner. This behavior differed from conventional 1st order refolding kinetics, where yield strongly depends on initial protein concentration due to an aggregation reaction of higher order. Refolding and cleavage of EDDIE fusion proteins follow a monomolecular reaction for the autoproteolytic cleavage over a wide concentration range. At high protein concentrations, deviations from the model assumptions were observed and thus smaller rate constants were required to approximate the data. Biotechnol. Bioeng. 2009; 104: 774–784 © 2009 Wiley Periodicals, Inc.     

Determination of diffusion coefficients of octadecanoic acid in isolated cuticular waxes and their relationship to cuticular water permeabilities

Transport properties of cuticular waxes from 40 different plant species were investigated by measuring desorption rates of ¹⁴C-labelled octadecanoic acid from isolated and subsequently reconstituted wax. Diffusion coefficients (D) of octadecanoic acid in reconstituted waxes, calculated from the slopes of the regression lines fitted to the linearized portions of desorption kinetics, ranged from 1.2 × 10^?19 m² s^?1 (Senecio kleinia leaf) to 2.9 × 10^?17 m² s^?1 (Malus cf. domestica fruit). Cuticular water permeabilities (cuticular transpiration) measured with intact cuticular membranes isolated from 24 different species varied from 1.7 × 10^?11 m s^?1 (Vanilla planifolia leaf) up to 2.1 × 10^?9 m s^?1 (Malus cf. domestica fruit), thus covering a range of more than 2 orders of magnitude. Cuticular water permeabilities were highly correlated with diffusion coefficients of octadecanoic acid in isolated cuticular wax of the same species. It is therefore possible to estimate cuticular barrier properties of stomatous leaf surfaces or of leaves where isolation of the cuticle is impossible by measuring D of octadecanoic acid in isolated waxes of these leaves.     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10⁹ BHK-21 cells from 4 × 10⁶ cells in 13 day with 1,051 mL culture medium.     

Antigen-specific mouse lymphocyte stimulation by DNP-conjugated T-independent antigens studied by photobleaching recovery

Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 × 10^?11 cm² sec^?1 for DNP_0.4-POL at 0.15 μg/ml to 0.8 × 10^?11cm² sec^?1for DNP_3.5-POL at 30 μg/ml. For receptor-bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 × 10^?11cm²sec^?1 observed for single slg receptors. By an epitope density of 4.8, D has fallen to 2.1 × 10^?11cm²sec^?1. Peak immunogenicities for DNP-POL and DNP-DEX arc observed when antigen- receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single slg molecule.     

Media and culturing protocol using a full 25 factorial design for the production of submerged aggregates by the potential bio-herbicide Plectosporium alismatis against weed species of Alismataceae

The potential bio-herbicide Plectosporium alismatis produces drying and UV tolerant micro-sclerotia-like structures named aggregates, effective against the weed Alisma plantago-aquatica. In this study, we evaluated (i) optimal liquid culture conditions that supported the high dry weight, conidia and aggregate yields and (ii) stress-tolerance and effectiveness of aggregates. Using a full factorial 2⁵ design, we studied the impact of agitation (A), glucose concentration (B), Tween 80 concentration (C), malt extract concentration (D), and inoculum density (E), on P. alismatis growth yields. The inoculum density (E), the agitation rate (A), and 3-factor interactions involving E and/or A had significant effects on aggregate yields (average 213 aggregates mL^?1; main effect of E: +112.87; main effect of A: ?74.81), but no significant effect on conidial yields and dry weight. The agitation was maintained at 100?rpm and increasing numbers of conidia were inoculated into the culture media. Maximal aggregate yields (3.6?×?10³ aggregates mL^?1) were obtained with an initial conidial density of 4?×?10⁶ conidia mL^?1. While freshly-harvested, dried and/or UV exposed aggregates germinated at high rates (respectively: 100%; 99%; 76%; 85%), sporogenesis significantly decreased after stress exposure. In bioassays using leaf discs of A. plantago-aquatica, chlorosis and/or necrosis was observed after 8 days incubation, regardless of whether aggregates had been exposed to desiccation and/or UV-radiations prior to application on leaf discs. These data provide a cultural protocol for the production of high numbers of UV and drying resistant aggregates effective against weed species of Alismatacae.     

The kinetics of cyanide binding by human erythrocyte catalase

The kinetics of the binding reaction of cyanide by human erythrocyte catalase at 25 °C has been studied over the pH range 4.2 to 10.2 by means of temperature jump and stopped flow techniques. Catalase reacts with cyanide at a constant rate in the range pH 4.2 to 8.1 which decreases at higher pH. This is most simply explained by the reaction of catalase with unionized hydrogen cyanide molecules. The pH-independent rate constant for the formation of the catalase-cyanide complex is (1.3 ± 0.1) × 10⁶m^?1 s^?1. The association equilibrium constant and the dissociation rate constant for the catalase-cyanide complex were determined from the relaxation amplitudes of temperature jump experiments and by spectrophotometric titration and are (3.1 ± 0.2) × 10⁵m^?1 and 4.2 ± 0.6 s^?1, respectively in the pH-independent region.     

In situ measurements of bioluminescence response of Gonyaulax spinifera to various mechanical stimuli

A conspicuous bioluminescence during nighttime was reported in an aquaculture farm in the Cochin estuary due to Gonyaulax spinifera bloom on March 20, 2020. In situ measurements on bioluminescence was carried out during nighttime to quantify the response of G. spinifera to various mechanical stimuli. The bioluminescence intensity (BI) was measured using Glowtracka, an advanced single channel sensor, attached to a Conductivity–Temperature–Depth Profiler. In steady environment, without any external stimuli, the bioluminescence generated due to the movement of fishes and shrimps in the water column was not detected by the sensor. However, stimuli such as a hand splash, oar and swimming movements, and a mixer could generate measurable bioluminescence responses. An abundance of?~?2.7?×?10⁶ cells L^?1 of G. spinifera with exceptionally high chlorophyll a of 25 mg m^?3 was recorded. The BI in response to hand splash was recorded as high as 1.6?×?10¹¹ photons cm^?2 s^?1. Similarly, BI of?~?1–6?×?10¹⁰ photons cm^?2 s^?1 with a cumulative bioluminescence of?~?2.51?×?10¹² photons cm^?2 (for 35 s) was recorded when there is a mixer with a constant force of 494 N/800 rpm min^?1. The response of G. spinifera was spontaneous with no time lapse between application of stimuli and the bioluminescence response. Interestingly, in natural environment, application of stimulus for longer time periods (10 min) does not lower the bioluminescence intensity due to the replenishment of water thrusted in by the mixer from surrounding areas. We also demonstrated that the bioluminescence intensity decreases with increase in distance from the source of stimuli (mixer) (av. 1.84?×?10¹⁰ photons cm^?2 s^?1 at 0.2 m to av. 0.05?×?10¹⁰ photons cm^?2 s^?1 at 1 m). The BI was highest in the periphery of the turbulent wake generated by the stimuli (av. 3.1?×?10¹⁰ photons cm^?2 s^?1) compared to the center (av. 1.8?×?10¹⁰ photons cm^?2 s^?1). When the stimuli was applied vertically down, the BI decreased from 0.2 m (0.3?×?10¹⁰ photons cm^?2 s^?1) to 0.5 m (0.10?×?10¹⁰ photons cm^?2 s^?1). Our study demonstrates that the BI of G. spinifera increases with increase in mechanical stimuli and decreases with increase in distance from the stimuli.

     

Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor

C-reactive protein (CRP) is a serum marker highly upregulated in inflammation after bacterial infection. Robust, reliable and quick quantification of CRP would be a substitute for erythrocyte sedimentation rate (ESR) with superior diagnostic value. Quartz crystal microbalance (QCM) based sensors coated with specific antibodies and integrated into lab-on-chip systems are in development for rapid point of care quantification. In this study, we isolated three CRP specific single chain (sc)Fv antibody fragments using phage display from an antibody gene library. Their affinities ranged from 2.7 × 10^?8 to 1.0 × 10^?8 M when measured by surface plasmon resonance. ScFv antibody fragment LA13-IIE3 showed best affinity, high long-term stability and remarkable resistance to denaturation. This scFv antibody fragment was coupled to a QCM sensor. CRP quantification in up to 15 samples sequentially measured on the same sensor with intermitting regeneration by buffer was demonstrated.     

Effect of turbulence on feeding intensity and survival of Japanese flounder Paralichthys olivaceus pelagic larvae

Three‐day rearing experiments were conducted to study the effect of turbulence on the feeding intensity and survival of pelagic larvae of Japanese flounder Paralichthys olivaceus. Four levels of turbulence as control (10^?7·2 m² s^?3), low (10^?6·2 m² s^?3), mid (10^?5·6 m² s^?3) and high (10^?5·0 m² s^?3) were set by changing the flow rate of water pumped through pipes set on the bottom of the tanks. In B‐stage larvae, defined as having buds of elongated dorsal fin rays, the feeding intensity and growth were higher in the low and mid turbulence levels, while survival was highest in the control level. Most of the larvae surviving in the control level, however, were judged to be in a seriously starved condition leading to subsequent high mortality. Because the three‐day span of the rearing experiments was thought to be a little shorter than the periods before starvation‐induced, high mortality occurs. In contrast, for D‐stage larvae, their feeding and growth were optimal in the control and low levels. Feeding was more adversely affected in the high level for D‐stage larvae compared with B‐stage larvae. This is probably due to the compressed body shape and elongated dorsal fin rays of D‐stage larvae, which may be more strongly affected by turbulence and, as a consequence, the larval feeding behaviour such as pursuit and capture of prey organisms becomes less efficient than in lower turbulence. Considering the vertical distribution of B and D‐stage larvae in the oceanic water column, the optimum turbulence level range found in the present study corresponded to a wind speed of 7–10 m s^?1. Therefore, moderate weather conditions of this wind speed range are considered to potentially enhance survival of early larval stages of P. olivaceus.     

Influence of inoculum size on aflatoxin production in home-made yoghurt

In previous works we have studied the influence of different factors on the aflatoxin production in yoghurt. In the present paper we complete our investigations with the study of the influence of the inoculum size. The inoculum sizes used by us were from 4 × 10¹ to 4 × 10⁶. As can be expected, the fungal growth, expressed as dry mycelium weight, was lower in 4 × 10¹ and higher in 4 × 10⁶. The amount of aflatoxin in the mycelium was stable, or increased slightly with the inoculum size. In the substrate, the amount of aflatoxin was stable with little fluctuations, with a higher level of toxin in 4 × 10³ and lower one in 4 × 10⁶. We detected a higher aflatoxin level in the mycelium than in the substrate.