Brown tide alga, Aureococcus anophagefferens, can affect growth but not survivorship of Mercenaria mercenaria larvae

Since the collapse of populations of northern quahogs (hard clam), Mercenaria mercenaria, in Long Island bays, brown tide blooms have been proposed to pose a barrier to recovery. We tested whether the brown tide alga, Aureococcus anophagefferens, affects survivorship, development or growth in the larvae of M. mercenaria. There was no effect of A. anophagefferens (clone CCMP1708) on survivorship of hard clam larvae, even at bloom concentrations. Under most experimental conditions, larvae fed a mixed diet of Isochrysis galbana (T-Iso) and A. anophagefferens or a single species diet of A. anophagefferens, developed faster than those fed a single species diet of Isochrysis. A mixed diet of I. galbana and A. anophagefferens either had no effect on larval growth, or produced enhanced growth at moderate cell densities (8 × 10⁴ cells ml⁻¹ of A. anophagefferens). Similarly, moderate cell densities of a single food diet of A. anophagefferens (1.6 × 10⁵ cells ml⁻¹) generally had no effect on the growth of larvae. When fed bloom concentrations (10⁶ cells ml⁻¹) of A. anophagefferens, larvae developed faster, but growth was reduced, compared to those fed an equal biovolume of Isochrysis. Larvae fed slow growing or near stationary phase cultures of A. anophagefferens experienced reduced growth and slowed development. These data suggest a qualitative difference between slow or stationary phase and fast growing cultures of the brown tide alga. They also suggest that impacts of A. anophagefferens, when present, are likely to be due to the nutritional quality of this alga as a food source for hard clam larvae, which could have a lasting legacy through ontogeny. Additional studies are needed to test whether our findings apply to more recently isolated strains of A. anophagefferens.     

Effect of growth hormone and γ-aminobutyric acid on Brachionus plicatilis (Rotifera) reproduction at low food or high ammonia levels

Growth hormone (GH, 0.0025 and 0.025 I.U. ml⁻¹) and γ-aminobutyric acid (GABA, 50 μg ml⁻¹) enhance rotifer population growth in batch cultures. In order to further understand the mechanism of their actions, we conducted experiments culturing isolated females at low food and high free ammonia levels. At an optimum food level of 7×10⁶ Nannochloropsis oculata cells ml⁻¹ or at low free ammonia level of 2.4 μg ml⁻¹, the F₁ offspring of rotifers treated with GH at 0.0025 I.U. ml⁻¹ had significantly higher population growth rate (r) and net reproduction rate (Ro), and shorter generation time than untreated rotifers. At a lower food level of 7×10⁵ cells ml⁻¹ or at high free ammonia level of 3.1 μg ml⁻¹, rotifers treated with GABA at 50 μg ml⁻¹ had significantly higher r and Ro, and shorter generation time. These results indicate that GABA is effective in enhancing rotifer reproduction when rotifers are cultured under stress whereas GH enhances rotifer reproduction when culture conditions are optimal. Significant effects were also observed in F¹ and F² generations which were not treated with hormones. These data may be useful for treating rotifer mass cultures to mitigate the effects of stress caused by high population densities.     

Human fibroblast culture on a crosslinked dermal porcine collagen matrix

The use of a novel porcine-derived collagen biomaterial as a dermal tissue engineering matrix was examined. The matrix is derived from porcine dermis, and is processed to retain the native collagen (Type 1) and elastin structure. Human primary fibroblasts were cultured on the matrix to examine its potential for creating a dermal replacement. Attachment of fibroblasts on the collagen was compared to tissue culture plastic and PET membranes. Cell proliferation was assessed using the MTT assay and DAPI staining. For seeding densities of 5×10⁴ and 1×10⁵ cells cm⁻², PET and plastic demonstrated >95% attachment of seeded numbers after 3 h. The collagen matrix reached levels >80% after 3–4 h with no influence of the seeding density. Matrix samples with perforating pores of 40 μm diameter were also studied. After 216 h culture in static culture, with media replacement every 3 days, the final cell numbers reached 2.1×10⁵ (perforated) and 2.0×10⁵ cells cm⁻² (unperforated). In comparison fibroblast culture in a perfusion bioreactor, with continuous media replacement, reached 2.3×10⁵ (unperforated) and 2.5×10⁵ cells cm⁻² (perforated) after 216 h.     

Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (k_inh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and k_inh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2′-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70 °C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1–2) > BHT-CHO, BHT-OOH (0.1–0.3) > BHT-Q (0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The k_inh values declined in the order BHT-Q ((3.5–4.6)×10⁴ M⁻¹ s⁻¹) > BHT-OOH (0.7–1.9×10⁴ M⁻¹ s⁻¹) > BHT-CHO ((0.4–1.7)×10⁴ M⁻¹ s⁻¹) > BHT ((0.1–0.2)×10⁴ M⁻¹ s⁻¹). The k_inh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.     

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O₂ and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5×10⁷ cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O₂ and glucose metabolism in the scaffold (20 mm width–35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04×10⁻³ mol/s/m³ for O₂ at 0.71 mL/min and 1.91×10⁻² mol/s/m³ for glucose at 0.35 mL/min.     

Critical analysis of application of generalized distance function for optimization of important variables for esterase synthesis by Saccharomyces cerevisiae

The generalized distance function approach is employed to obtain a suitable near optimal conditions of variables. The optimal values of variables (medium constituents, microbiological parameters, and process parameters) have been evaluated separately using single responses (either specific esterase activity or cell mass) as per central-composite-design and multi-responses following generalized distance function approach. The optimal conditions (medium composition (g l⁻¹): dextrose, 13.43; peptone, 7.285; yeast extract, 2.55; and malt extract, 1.695; microbiological parameters: slant age, 39.9 h; inoculum age, 9.6 h; and number of cells, 1.49 × 10⁸ numbers ml⁻¹; process conditions: temperature, 29.9 °C; and pH, 6.2) obtained by generalized distance approach can be considered as a ‘near optimal’ solution of interactive multi-response systems of intracellular esterase synthesis by Saccharomyces cerevisiae.     

Production of cellulase by freely suspended and immobilized cells of Trichoderma reesei

Trichoderma reesei (QM 9123) was immobilized within the open porous network of reticulated polyurethane foam matrices, and the growth pattern, glucose consumption and cellulase production were compared with those of freely suspended cells. It was found that the method of immobilization was simple and had no detrimental effect on cell activity. Various production media, to be used after the cultivation of T. reesei were tried. It was found that a nitrogen source-free production medium gave the highest enzyme titers of 1.5 × 10³ FPA U l⁻¹. Similar results were obtained with both freely suspended and immobilized cells.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Plant regeneration from mesophyll protoplasts of Solanum commersonii dun

Somatic fusion of Solanum commersonii, a frost tolerant wild potato species not crossable with Solanum tuberosum, relies on the possibility to isolate and culture protoplasts. This study was conducted to determine whether protoplasts could be isolated and plants regenerated in three S. commersonii accessions. Shoot cultures for protoplast isolation were maintained on Murashige and Skoog medium. Mesophyll protoplasts were isolated and cultured using a protocol originally described for S. tuberosum with some modifications. Differences were evident among the three accessions for protoplast yield, plating efficiency and regeneration frequency. Protoplast yield ranged from 3.0 to 8.5 × 10⁶ protoplasts per g of fresh tissue. At 1–2 × 10⁴ protoplasts ml⁻¹, which was the optimal plating density, 10–20% of plated protoplasts gave multicellular colonies. Regeneration of shoots was observed in two accessions only, the maximum regeneration frequency being 66%. In one of these accessions the reduction of sucrose concentration in regeneration media improved the regeneration frequency from 14 to 35%. About three hundred plants were rooted in vitro and successfully transferred to soil.     

Kinetics of laccase-catalysed TEMPO oxidation

The oxidation of TEMPO (2,2,6,6-tetramethyl-piperidine-1-oxyl radical) has been studied in the presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL) in buffer solution pH 4.5–7.3 and at 25 °C. At pH 5.5 the oxidation constant calculated from the initial rate of TEMPO oxidation was 1.7 × 10⁴, 1.4 × 10³, 7.8 × 10² and 5.2 × 10² M⁻¹ s⁻¹ for rPpL, rRsL, rCcL and rMtL, respectively. The maximal activity of rPpL-catalysed TEMPO oxidation was at pH 5.0. The pK_a obtained in neutral pH range was 6.2. The reactivity of laccases is in a good agreement with laccases copper type I redox potential.

TEMPO oxidation rate increased 541 times in the presence of 10-(3-propylsulfonate) phenoxazine (PSPX). The model of synergistic TEMPO and PSPX oxidation was proposed. Experimentally obtained rate constants for rPpL-catalysed PSPX oxidation were in a good agreement with those calculated from the synergistic model, therefore confirming the feasibility of the model. The acceleration of TEMPO oxidation with high reactive laccase substrates opens new possibilities for TEMPO application as a mediator.     

Cultivation and preservation of vinegar bacteria

Ten strains of acetic acid bacteria were investigated for their characteristics of growth and metabolism. The strains were identified as those presently in use for industrial vinegar production in southern Germany. At the time of isolation from industrial acetators the total concentrations, i.e. acetic acid (w/v) plus ethanol (v/v), of the fermenting vinegars were 6.1–14.9%. The applicability of a previously described method for starter preparation was examined for the various isolates as well as for the type strains of species of the genera Gluconobacter and Acetobacter. Isolates from cider or wine vinegar fermentations grew readily in RAE-medium to total counts of >1×10⁹ cells ml⁻¹. For the cultivation of strains isolated from spirit vinegar fermentations AE-medium proved most suitable. Cultures of these strains exhibited lag phases of 2–5 days and grew up to total counts of <1×10⁹ cells ml⁻¹. All type strains could be grown on RAE-agar. The use of 20% malt extract as cryo-protectant was effective for the preservation of all strains. Upon revitalization the cultures were suitable as inoculum for starting fermentations in pilot acetators. 16S rRNA-targeted oligonucleotide probes were constructed which were species specific for Gluconobacter oxydans or Acetobacter aceti or group specific for Acetobacter europaeus/Acetobacter xylinum. The probes hybridized with the DNA of the respective type strains. Four isolates were allotted to A. europaeus/A. xylinum applying the group specific probe. The DNA of six of the Acetobacter sp. hybridized with none of the probes.     

A remote-query sensor for predictive indication of milk spoilage

Described is application of the remote-query (wireless, passive) magnetoelastic sensor platform for direct detection and monitoring of bacterium contamination of milk within hermetically sealed containers. Specific application is made to the quantification of Staphylococcus aureus ssp. anaerobius (S. aureus) concentrations in milk. S. aureus growth changes milk viscosity, in turn changing the resonance frequency of the liquid immersed sensor allowing S. aureus concentrations of 10³ to 10⁷ cells ml⁻¹ to be directly quantified.     

Graft copolymerization of acrylic acid onto guar gum initiated by vanadium (V)–mercaptosuccinic acid redox pair

Guar gum has been modified by graft copolymerization with acrylic acid in aqueous medium using vanadium (V)–mercaptosuccinic acid redox system. The optimum reaction conditions affording maximum grafting ratio, efficiency, add on and conversion have been determined. The grafting parameters have been found to increase with increase in vanadium (V) concentration upto 1.0 × 10⁻² mol dm⁻³, but these parameters decrease on further increasing the vanadium (V) concentration. On increasing the mercaptosuccinic acid concentration from 1.0 × 10⁻² to 4.0 × 10⁻² mol dm⁻³ grafting ratio, efficiency and add on increase up to 2.0 × 10⁻² mol dm⁻³ but decrease with further increase in mercaptosuccinic acid concentration. On varying the acrylic acid concentration from 5.0 × 10⁻² to 30.0 × 10⁻² mol dm⁻³, maximum grafting ratio, efficiency and add on have been obtained at 20.0 × 10⁻² mol dm⁻³. The grafting ratio, add on and conversion increase, on increasing the H⁺ ion concentration from 1.5 × 10⁻¹ to 6.0 × 10⁻¹ mol dm⁻³. On increasing the guar gum concentration the grafting parameters increase. The grafting ratio, add on and conversion have been found to increase with time period while efficiency started decreasing after 120 min. It has been observed that %G increases on increasing the temperature up to 35 °C. The graft copolymer has been characterized by IR spectroscopy and thermogravimetric analysis.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Synthesis of graft copolymer (k-carrageenan-g-N,N-dimethylacrylamide) and studies of metal ion uptake, swelling capacity and flocculation properties

Graft copolymer of k-carrageenan and N,N-dimethylacrylamide has been synthesized by free radical polymerization using peroxymonosulphate/glycolic acid redox pair in an inert atmosphere. The grafting parameters i.e. grafting ratio, add on and efficiency decrease with increase in concentration of k-carrageenan from 0.6 to 1.4 g dm⁻³ and hydrogen ion from 3 × 10⁻³ to 7 × 10⁻³ mol dm⁻³, but these grafting parameters increase with increase in concentration of N,N-dimethylacrylamide from 16 × 10⁻² to 32 × 10⁻² mol dm⁻³, and peroxymonosulphate from 0.8 × 10⁻² to 2.4 × 10⁻² mol dm⁻³. The metal ion sorption, swelling behaviour and flocculation properties have been studied. The intrinsic viscosity of pure and grafted samples has been measured by using Ubbelohde capillary viscometer. Flocculation capability of k-carrageenan and k-carrageenan-g-N,N-dimethylacrylamide for both coking and non-coking coals has been studied for the treatment of coal mine waste water. The graft copolymer has been characterized by Infrared (IR) spectroscopy and thermogravimetric analysis.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

Sporulation of Metarhizium anisopliae in solid-state fermentation with forced aeration

Metarhizium anisopliae was grown by solid-state fermentation using a medium based on a mixture of rice bran and rice husk (1:1 on a dry weight basis) where the initial water content was fixed at a value of 47%. The experiments were performed in glass column bioreactors of three different sizes. Moist saturated air was continuously passed through the bottom of each column at a flow rate of 0.34 l h⁻¹ g⁻¹ initial dry matter, and cultivation was performed for two weeks at 27 ± 1°C. The effect of medium packing density was studied in the columns of small size. For initial apparent densities of 0.270 and 0.357 g ml⁻¹, no significant differences were observed either in total biomass production or spore yields; however, when the initial apparent density was increased up to a value of 0.496 g ml⁻¹, both growth and sporulation were strongly affected.

Thermal gradients in the medium and larger columns packed at an apparent density of 0.357 g ml⁻¹ were observed both in the radial and axial directions. Heat build-up effects were analyzed in the larger column. In this case, there were significant differences between different segments of the column both in the water content of the fermented matter and the sporulation pattern.