Liquid culture of the entomopathogenic nematode,Steinernema colombiense: evolution of the nematode concentration,particles volume fraction,rheological properties and surface tension

ABSTRACT

The rheological properties and surface tension (σ) of culture broths with suspended particles are factors that affect the performance of fermentation processes. This article presents the first report concerning the evolution of suspended particles, rheological properties and σ of culture broths during the liquid culture of Steinernema colombiense and its symbiont bacterium, in a medium containing 1.25% (w/v) egg yolk. Cultures started with 1087?infective-juveniles/mL and finished with 53,583?individuals/mL after 10 days. Culture broths were moderately concentrated suspensions with total particles volume fraction in the range 0.08–0.14. The suspended particles were nematodes of different sizes depending on their developmental stage, and egg yolk particles. Rheological properties corresponded to pseudoplastic fluids with flow behaviour index values ?0.3 (dimensionless), greatly determined by the symbiotic bacterium activity. After nematode inoculation, the apparent viscosity values (η_a; Pa s) for the whole culture broth and its supernatants were in the ranges 0.010–0.018 and 0.008–0.015, respectively. Concerning σ, their values were in the range 31.22–37.74?mN/m, probably determined by the contents of egg yolk.     

A modeling approach of the in vitro conversion of oil palm (Elaeis guineensis) somatic embryos

A mathematical model for the growth and conversion of somatic embryos was developed with the aim of monitoring the large scale production of oil palm microplants. The predicted biomass of somatic embryos obtained and subcultured (B _n ), together with the number of harvested shoots (Sh _n ) – two key parameters for production forecasts – have been modeled for seven different shoot harvesting procedures. For the four different clonal lines studied, observed differences between experimental B _n values at the end of each culture cycle and their theoretical counterpart generated by mathematical models were found to range between −30% to +14% at the end of the first 6-weeks culture cycle, then from −50% to +70% after the 6th subculturing operation (36 weeks). Concerning the predicted number of shoots harvested after conversion of somatic embryos (Sh _n ), average variations between experimental and theoretical values ranged between −45% and +41%. Predicted values for biomass (B _n ) between two culture cycles were found to vary slightly (+6% to +10%) indicating that the production of embryo biomass, as predicted by the model, was rather stable, for a given clonal line, from one 6-week cycle to another. The established model could thus be regarded as valid and the variations observed for B _n and Sh _n were found to be acceptable when compared to the those described by other models. Taken as a whole, predicted values for the two studied production parameters were in agreement with the corresponding experimental data (correlation=0.98).     

Caffeine-induced uncoupling of mitosis from DNA replication in tobacco BY-2 cells

Caffeine induced a mitosis-like state in cultured tobacco (Nicotiana tabacum L.) BY-2 cells after DNA synthesis had been arrested by aphidicolin. Cells were synchronized upon removal of aphidicolin. When aphidicolin was readded, the cell cycle was again interrupted and caffeine, when added with aphidicolin, induced the mitosis-like state in 5–10% of cells.     

Untersuchungen zum Anfangsstadium der enzymatischen Hydrolyse unterschiedlich vorbehandelter Linterscellulose

Starting from cotton linters cellulose modified in physical structure type of lattice, degree of order, state of swelling by different pretreatments, and from culture filtrates of Gliocladium spec., the initial stage of enzymatic hydrolysis of cellulose has been investigated. Especially with substrates of high degradability a considerable effect of stirring on rate of formation of soluble products was found. For linerization of yield-vs-time-curves (% residue resp. 7percnt; solubles as a criterion for yield), a 2-parameatric first order rate law was found to be suitable within a limited time interval, values of k₁ were higher and for the accessible part of the substrate were lower in the initial stage of hydrolysis than in the later one. The MICHAELIS -MENTEN -constant k_M has been determined for substrates of different physical structure after different times of reaction. Data found for k_M indicated a stronger dependence of k_M on reaction time than on physical structure of the substrate under conditions applied.     

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage λ Q − vector

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage λ system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage λ vector with a Q ⁻ mutation was used to enhance the expression of the λ system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h⁻¹ with 1.5 S ₀ of the medium supply. The maximum productivity of the total β-galactosidase was 16.3×10⁶ U l⁻¹ h⁻¹, which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.     

Initiation of an Aquaculture of Sponges for the Sustainable Production of Bioactive Metabolites in Open Systems: Example, Geodia cydonium

Among Metazoa, sponges (phylum Porifera) are the richest source for different bioactive compounds. The availability of the raw material is, however, restricted. To obtain enough of the bioactive compounds for application in human therapy, sponges have to be cultured in in vitro systems. One technique for the establishment of a long-term cell culture from sponges has recently been elaborated. Here, we present a procedure to cultivate tissue samples from sponges in an open system. The species Geodia cydonium, which produces bioactive compounds, has been selected. Tissue samples of approximately 10 g were attached to the bottoms of cultivation trays. After 2 to 3 days, the tissue samples formed a robust contact with the metal support. Subsequently, sets of trays, called tray batteries, either remained in huge aquaria at the Center for Marine Research or were transferred to the vicinity of a fish and mussel farm. The growth rates of the samples remained unchanged within the first month; however, after 3 and 6 months, they increased to 147% and 189%, respectively. In parallel, extracts were prepared from the tissue samples and tested for cytotoxicity in a mouse lymphoma cell assay system. Extracts from cultured tissue initially had a low inhibitory potency; however, after cultivation for 3 or 6 months, values comparable to those of extracts from sponges taken from the biotope were found. In addition, a molecular marker was applied to document the response (health state) of the tissue and the identity of the material in culture. The CD63 molecule was chosen because the expression of this molecule in mammalian systems changes with the age of the animals. The corresponding complementary DNA was isolated from Geodia cydonium. With this probe, the level of expression in cultured tissue samples decreased immediately after starting cultivation; after a cultivation period of 6 months, however, values were similar to those found in controls. These data show that sponge species that produce bioactive compounds can be cultivated in open systems, in which they retain their potency to produce bioactive compounds as well as their health state. Received September 16, 1998; accepted June 18, 1999     

Optimization of Clostridium thermocellum growth on cellulose and pretreated wood substrates

Summary Two strains of Clostridium thermocellum ATCC 27405 and NRCC 2688 demonstrated similar product yields and cellulase activities when grown on solka floc. A sequential culture of C. thermocellum and Zymomonas anaerobia supplemented with cellobiase could produce 1.8 mg/ml of ethanol when grwon on 1% solka floc. Different media were evaluated for their ability to enhance the product and cellulase yields of C. thermocellum grown on cellulose substrates. Ethanol and reducing sugar values of 1.5 and 3.8 mg/ml respectively and an endoglucanase activity of 3 IU/ml were obtained after growth of Clostridium thermocellum in a modified medium containing 1% solka floc. Three different pretreated wood fractions were assessed as substrates for growth. A steam exploded wood fraction gave comparable values to those obtained after growth on solka floc. Sequential cultures of C. thermocellum and Zymomonas anaerobia grown on a 1% steam exploded wood fraction could produce 1.6 mg/ml ethanol after 3 days growth.     

Relationship between leaf water spin-lattice relaxation time and water relation parameters in three wheat cultivars

Pot culture studies were conducted using two drought-tolerant and one susceptible cultivar of wheat (Triticum aestivum L.) under three different moisture regimes. Proton spin-lattice relaxation time, T₁, leaf water content, LWC, leaf water potential, Ψ, solute potential, Ψ_s and turgor potential, Ψ_p were measured from 45 to 75 d after sowing at weekly intervals. The three cultivars did not differ significantly in their values of LWC, leaf water potential, and their components in the stressed and unstressed plants; but they did differ significantly in their T₁ values both under stressed and unstressed conditions on all days of measurement, with the drought tolerant cultivars having a higher T₁ compared to the susceptible cultivar. This suggests that leaf water T₁ is a better parameter for describing plant water status than the traditional water relation indices. The relation between Ψ and T₁ was logarithmic, indicating the similarity between T₁ and water activity of the cellular water.     

Studies on the uptake of trivalent and hexavelent chromium by onion (Allium cepa)

Abstract

Experiments were conducted to examine the uptake and translocation of root-absorbed trivalent and hexavalent state of chromium in the onion plant (Allium cepa) grown in soil and sand culture. Chromium content in plant tissues increased with increasing amount of added chromium. Distribution of chromium in the plant in general, found to be in the order: root>>bulb>shoot. Higher uptake in the plants grown in sand from both the sources of chromium was observed as compared with the corresponding values for soil culture. Morphological and growth effects of the treatments of different oxidation state of chromium indicated that higher doses of Cr(VI) [150 and 300 μg mL^?1] were more toxic to the onion plants compared to equivalent doses of Cr(III).     

Production and properties of three pectinolytic activities produced byAspergillus niger in submerged and solid-state fermentation

Three extracellular pectinases were produced byAspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (K _m values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but theK _m values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number on protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced byA. niger.     

Effect of long-term in vitro shoot culture on somatic embryogenesis of quince leaves treated with different light qualities

Summary The effect of long-term in vitro shoot culture on somatic embryogenesis in quince BA 29 was investigated. Three experiments were performed on leaves explanted at about 8-mo. intervals from the same culture stock and maintained under different light qualities. Embryo production was assessed either in terms of percentage of embryogenic leaves or number of embryos per leaf. By appropriate data processing both these responses were linearly related to photoequilibrium in each experiment. Statistical comparisons among the three experiments showed significant differences both in mean (computed over light qualities) and line slope values. In particular, with increasing shoot culture age, both percentage of embryogenic leaves and number of embryos per leaf progressively increased, while mean slope values decreased. The increase in mean values suggests a positive effect on somatic embryogenesis due to possible tissue rejuvenation when mother cultures were cultivated in vitro for longer periods. Slope decrease over time indicated the interactions between age of the in vitro culture and photoequilibrium. Thus, embryo production at different culture ages was consistently found to be highest at high photoequilibrium values; in contrast, if a low level of phytochrome was activated, embryogenesis in the youngest cultures was low or absent, but increased with the progressive tissue rejuvenation arising from long-term in vitro culture.     

Variability for the in vitro culture response in tomato recombinant inbred lines

The aim of this work was to estimate genetic variability for in vitro culture response of recombinant inbred lines (RILs) of the genus Lycopersicon. The callus percentage (C), the regeneration percentage (R) and the productivity rate (PR) were evaluated 45 d after culture initiation in a set of 16 elite tomato RILs and their parents. The narrow sense heritability (h ²) values were 0.38 ± 0.04 for C, 0.46 ± 0.04 for R, and 0.28 ± 0.03 for R, while the genetic correlation (r _g ) values were −0.96 ± 0.07 between C and R, 0.81 ± 0.14 between PR and R, and −0.79 ± 0.16 between PR and C. Three AFLP markers associated to the in vitro traits were identified.     

Adventitious root induction inCorylus avellana L. cotyledon slices

Summary An experimental rooting system was developed to study in vitro adventitious root formation in hazelnut cotyledons. Experiments involveda) assay of several culture media,b) use of different developmental status of the seeds (germinated and ungerminated),c) cotyledons subjected to various light regimes, andd) different size of cotyledons slices. It was observed that higher rooting was induced in cotyledonary portions of 5- or 7-mm thickness (250 and 350 mm³, respectively) cultured on half-strength basal medium supplemented with 50 μM indole-3-butyric acid and 5 μM kinetin. Rooting was affected by light and the developmental state of seeds. Preinitiation, initiation, and root manifestation stages were defined according to specific culture periods and in relation with morphologic and histologic changes. The first histologic changes (cell division and root primordia induction) were observed after 12 days in culture. At 30 days of culture in rhizogenic medium root primodia were fully differentiated with well-developed vascular tissues.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Evaluation of the different <Emphasis Type="Italic">Alternaria</Emphasis> prediction models on a potato crop in A Limia (NW of Spain)

The main crop of the A Limia region is the potato, with an average production of 5 million kilos per year, under the protected geographic indication (IGP) Pataca de Galicia (Galician potato) label recognized by the European Community. Alternaria represents an important part of the pathogenic fungus spectra on this crop, representing an average of 1.9–3.1% of the spores collected. It represents the fourth and third most common type, respectively, in the years of study, after the Cladosporium type (which represented 80% and 64% respectively in each year of study), the Basidia type (5.5% and 1.4%), Botrytis (3.5% and 1.3%), and Fusarium (2.7% and 25.2%). A Lanzoni VPPS 2000 Hirst volumetric sampler was placed in Damil, in the region of A Limia in order to determine the airborne concentration of Alternaria conidium over a potato crop and finally to try to establish the most suitable prediction models for Alternaria attacks and how they might be controlled. We applied different models based on temperature, such as the model of propitious days (P-Days), the model of accumulation of disfavourable days (DD), and the relative humidity model of interrupted wet periods (IWP). The most efficient model was found to be the interrupted wet periods (IWP) method as it could predict several days of attack during the development of the crop. It is efficient in A Limia even when the features described in this model are fulfilled during five consecutive days. In the first year of study the efficiency of this method was lower as alternariosis did not proliferate to the same degree due to the adverse meteorological conditions registered for the development of the fungus. This study is the first step towards prediction of infection by Alternaria in potato crops. We need more years of study in order to adjust these models to the conditions of the sampling area, and thus be able to confirm the necessary values and their suitability with the aim of developing possible alternatives to reduce the negative consequences of this pathogen.     

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC₅₀ (24 h) 47.1 μM NP (primary cell culture) and 44.2 μM NP (EPC)) values and apoptotic rate (significantly increased apoptotic rate after exposure to 50 μM NP for 24 h, p < 0.001 (primary cell culture), p = 0.008 (EPC)). The explant culture was slightly less sensitive (increased apoptotic rate after exposure to 50 μM NP for 24 h, but not significant: p = 0.385), which could be due to the capabilities of a differentiated tissue, providing more protective repair mechanisms, compared with single cells. All cultures revealed a concentration–response relationship for the endpoint apoptotic rate after the application of nonylphenol for 24 h. After wastewater exposure, a significant decrease in the apoptotic rate was measured in the primary cell culture (dilution wastewater : medium 1:1:p = 0.018; dilution wastewater : medium 1:2:p = 0.003), whereas the cell line EPC did not reveal any effects. Our results show that the endpoint apoptotic rate is more sensitive than the parameter cell viability for detecting adverse effects of a wastewater sample.     

Physiological responses of marine Dendryphiella species from different geographical locations

The saprobic, cosmopolitan, marine fungi Dendryphiella arenaria and Dendryphiella salina, isolated from various plant and algal substrates from different geographical locations and climatic zones, were studied for their adaptations to the abiotic and biotic parameters commonly found in their natural marine habitats. All the tested strains of D. arenaria and D. salina grew optimally on culture media with added marine salts, at pH values between 6.5 and 8.0 and at an incubation temperature of 25°C. The D. arenaria strains had faster mean colony extension rates under all conditions of culture. All strains exhibited an increased salt optimum with increasing incubation temperature. The TLC profiles of strains of the two species were similar. The culture extracts were antimicrobial, though production of the biologically active metabolites was strain-specific. There were no significant correlations between source of origin and responses to the investigated parameters. These results demonstrate phenotypic plasticity and the ability of each isolate to adapt to diverse biotopes.     

Aeration effects on metabolic events during sporulation of Bacillus thuringiensis

The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO₂ production rates during sporulation, although amaximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.     

Effects of cultural conditions on the production of phenylalanine from a plasmid-harboring E. coli strain

Summary Phenylalanine production from E. coli KA 197/pJN6 (plasmid harboring genes for aro F, phe A^FBR, Amp^R and Tc^R) was studied under varying nutritional conditions in batch and continuous cultures. In batch culture experiments where growth was deliberately interrupted by limiting concentrations of sulphate and phosphate the phenylalanine production continued from the non-growing cells. However, the depletion of phosphate resulted in an immediate cessation of phenylalanine production but thereafter a low specific rate of phenylalanine formation resumed, while the decrease in specific rate of product formation was less after sulphate depletion. In the chemostat experiments, however, phosphate limitation was the only case where the specific rate of phenylalanine formation remained constant, while at the corresponding time in sulphate and glucose limited chemostats it was declining respectively had ceased.