Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production (p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.     

Manganese and iron interactions on their uptake and distribution in soybean (Glycine max (L.) Merr.)

Summary The uptake and distribution of iron and manganese were studied in a manganese-sensitive soybean cultivar (‘Bragg’) grown over a range of supply levels of these nutrients in solution culture. At high (90 and 275 μM) manganese levels, increasing the iron concentration in solution from 2 to 100 μM partially overcame the effects of manganese toxicity. Interactions between manganese and iron occurred for dry matter yields, rate of Mn absorption by the roots, and the proportions of manganese and iron transported to the tops. No interaction was observed for the rate of root absorption of iron. The percentage distribution of manganese in the plant top increased with increasing iron, despite a reduced rate of Mn uptake. On the other hand, iron uptake was independent of solution Mn concentration and increased with increasing solution Fe. Also more iron was retained in the roots at high Mn and/or Fe levels in solution. Concentrations of manganese and iron in roots, stems and individual leaves were affected independently by the manganese and iron supplyi.e. without any interaction occurring between the two elements. In general, the concentration in a plant part was related directly to the solution concentration. Symptoms resembling iron deficiency correlated poorly with leaf Fe concentrations whereas high levels of manganese were found in leaves displaying Mn toxicity symptoms.     

Influence of potassium and manganese on growth and uptake of magnesium by soybeans (Glycine max (L.) Merr. cv. Bragg)

Summary Soybean (Glycine max (L) Merr. cv. Bragg) seedlings were grown in nutrient solutions to evaluate the response to manganese nutrition as affected by potassium supply. In solutions containing 275 M manganese, increasing the solution concentration of potassium from 1 mM to 10 mM alleviated symptoms of manganese toxicity, decreased manganese concentrations in the leaves and increased dry matter yields of the plants. The reduction in manganese toxicity was brought about by a reduced rate of root absorption of manganese at high potassium supply levels.Increasing the supply of either potassium or manganese decreased the leaf concentration of magnesium although there were no apparent symptoms of magnesium deficiency in any treatment. The reduced concentration of magnesium in the leaves was due to effects of potassium and manganese on the rate of root absorption of magnesium.Under manganese deficiency conditions, growth was reduced and manganese concentrations in plant parts were very low; there was no effect of potassium supply when manganese was absent from the nutrient solution.     

The effects of lime and superphosphate on manganese toxicity in steam-sterilized soil

Summary The effects of liming and superphosphate application on the manganese nutrition of lettuce were studied in factorial experiments. A fairly acid silt loam of the Hamble series, which on steam-sterilization released considerable amounts of manganese in readily available forms, was used in the investigation.Manganese toxicity in lettuce was prevented by liming to increase the pH of the soil. The amounts of water-soluble, exchangeable and total active manganese present in the soil, and the manganese content of lettuce plants, decreased with increasing soil pH; easily reducible soil manganese increased with increasing soil pH.The effect of added superphosphate depended on the pH of the untreated soil. When the soil had a high pH, application of phosphate invariably decreased the pH and increased the manganese uptake. In acid soil supplying excessive amounts of manganese, application of phosphate reduced the manganese content of the plants; the pH of the soil was either unchanged or slightly increased by the treatment.Correlations were calculated between soil pH, various fractions of soil manganese, and the manganese content and yield of lettuce.From a comparison of plants grown in soil and sand culture it was concluded that the presence of a further toxic factor in the soil was probable.The results are discussed in relation to those obtained by other investigators.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.     

Influence of culture conditions on mycelial growth and bioluminescence of Gerronema viridilucens

Herein we describe a procedure for measuring the total light emission of the naturally bioluminescent tropical fungus Gerronema viridilucens and the optimization of culture conditions using multivariate factorial anova. Cultures growing on an agar surface in 35 mm Petri dishes at 90% humidity show optimal bioluminescence emission at 25 degrees C in the presence of 1.0% sugar cane molasses, 0.10% yeast extract and pH 6.0 (nonbuffered). Temperature and pH are the most important factors for both mycelial growth and bioluminescence.     

Influence of culture media and environmental factors on mycelial growth and pycnidial production of Sphaeropsis pyriputrescens

Sphaeropsis pyriputrescens, the causal agent of Sphaeropsis rot of pears and apples, is a recently described species. In this study the effects of culture media, temperature, water potential, pH and light on mycelial growth and pycnidial production of S. pyriputrescens were evaluated. Apple juice agar and pear juice agar were most suitable for mycelial growth of all six isolates tested. Cornmeal agar was not suitable for either mycelial growth or pycnidial production. The fungus grew from -3 to 25 C, with optimum growth at 20 C and no growth at 30 C. The fungus grew at water potential as low as -5.6 MPa on potassium chloride-amended potato-dextrose agar (PDA). Hyphal extension was not observed at -7.3 MPa after 10 d incubation, but growth resumed when the inoculum plugs were placed on PDA. The fungus grew at pH 3.3-6.3 and optimum growth was at pH 3.3-4.2. No mycelial growth was observed at pH above 7.2 after 10 d incubation, but growth resumed when the inoculum plugs were transferred onto PDA. Regardless of medium tested, few pycnidia formed at 20 C in the dark. Pycnidial production was enhanced significantly by fluorescent light, but continuous light appeared to reduce pycnidial production, depending on the medium. Oatmeal agar (OMA) was most suitable for production of pycnidia and conidia. Pycnidia that formed on 3 wk old OMA cultures at 20 C under 12 h light/12 h dark produced abundant conidia, and the technique is recommended for inoculum production.     

Unfolding and pH studies on manganese peroxidase: role of heme and calcium on secondary structure stability

The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding-unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.     

Survival-promoting activity of nimodipine and nifedipine in rat motoneurons: implications of an intrinsic calcium toxicity in motoneurons

L-type calcium channel antagonists, nimodipine and nifedipine, were tested for effects on the survival of purified rat motoneurons in culture. They showed significant activity, with maximum survival at 30 microm after 3 days in culture as high as 75%, which was comparable to the maximum effect obtained with brain-derived neurotrophic factor, a potent neurotrophic factor for rat motoneurons. It was also found that depolarizing conditions with a high potassium concentration (30 mm) were toxic to motoneurons. This toxicity was blocked by co-treatment with nimodipine. These results implicate a pre-existing calcium burden through calcium channels in motoneurons; they may offer further insights into understanding the selective death of motoneurons and have therapeutic implications in amyotrophic lateral screlosis.     

Effect of outdoor conditions on growth rate and chemical composition of <Emphasis Type="Italic">Gelidium crinale</Emphasis> in culture

A comprehensive study of the major environmental factors affecting growth and chemical composition of Gelidium crinale is presented here for the first time. The purpose of this study was to analyze the significance of the major growth factors and to formulate them into regression models. For this purpose summer and winter experiments were conducted in small aerated seawater tanks under various light and fertilizer conditions. The effects of carbon source, pH control and microelements were also tested. Finally, an annual growth experiment was conducted in order to support the two-season experiment. The major limiting seasonal factor was found to be temperature, which positively affected weekly growth rate, dry weight and gel strength, while negatively affecting the agar percentage. Ammonium was also revealed as a major limiting factor, positively affecting chlorophyll and protein concentrations, and negatively affecting carbohydrate concentration. Other growth factors, such as light intensity, pH, carbon source and microelements, were less- or non-effective, being already almost optimal under ambient conditions.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

The Mobility of Manganese in the Wheat Plant: II. Redistribution in Relation to Concentration and Chemical State

Wheat plants were grown in solution culture at two levels ofmanganese supply until the second leaves were fully expanded.The supply of manganese was then curtailed and symptoms of deficiencyappeared in subsequent growth. Although redistribution of manganeseat rates dependent upon initial content occurred in both high-and low-manganese plants, first and second leaves always retainedsufficient for normal function, and remained healthy. In a secondexperiment no redistribution from the second leaves was observedunder similar conditions of supply and demand. When second leaves were killed and placed in water, up to 80per cent, of the manganese diffused out, but extraction of similarleaves with 80 per cent, methanol removed only 15—20 percent, of that present. These results suggest that the lack ofmobility shown by the element is not due to precipitation orcomplex formation within the tissue, although at high levelsof manganese supply the solubility of manganese silicate mightbe exceeded, and some precipitation may occur.     

Optimising sporulation and virulence in Drechslera avenacea

Studies were conducted on agar media to optimise sporulation of Drechslera avenacea, a fungal pathogen being evaluated as a biological control agent for Avena species (wild oats). Conidium production was affected by nutrition, pH, temperature and light conditions. Of the agar media tested, Czapek Dox agar (CZA) and half-strength oatmeal agar (½OMA) were the only media where sporulation occurred at all temperatures tested under a 12-h light:12-h dark photoperiod (L/D). The optimum temperature for conidium production was 20°C on ½OMA, whereas there was no optimum temperature on CZA. Under a 12-h near-ultraviolet (NUV):12-h dark photoperiod (NUV/D), similar numbers of conidia were produced on CZA at 6.66, 14.56, and 22.78 W m^?2, whereas on ½OMA conidium production was the highest at 14.56 W m^?2. When NUV/D and L/D conditions were compared, similar numbers of conidia where produced on CZA, whereas ½OMA conidium production was superior under the NUV/D photoperiod. Considerable variation in sporulation and degree of virulence of D. avenacea was detected among isolates from different geographic areas. The most virulent conidia were obtained on ½OMA at 20°C incubated under continuous illumination NUV light. Therefore, the most suitable conditions for conidium production of D. avenacea were growth for 1 week on ½OMA at 20°C under continuous NUV at an intensity of 14.56 W m^?2. Under these conditions, 1.1×10⁵ conidia mL^?1 were produced which is the highest sporulation yet reported for any Drechslera spp., which are traditionally poor sporulators.     

Decreasing expression of alpha1C calcium L-type channel subunit mRNA in rat ventricular myocytes upon manganese exposure

Manganese is an essential trace element found in many enzymes. As it is the case of many essential trace elements, excessive level of manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to understand the mechanism of manganese toxicity in the heart, the effects of manganese on isolated rat ventricular myocytes were studied. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50 microM Mn2+ and 100 microM Mn2+ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover, Mn2+ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. Investigation by RT-PCR showed that the mRNA expression of alpha1C/Cav1.2 treated with manganese was decreased depending on its concentration, while the mRNA expression of alpha1D/Cav1.3 was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM), and the results showed that Mn2+ could elicit a slow and continuous increase of [Ca2+]i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, downregulate the mRNA expression of alpha1C/Cav1.2, and thus causing long-lasting molecular changes of L-type calcium channel which have probably been triggered by overloading of calcium in myocytes.     

The influence of culture conditions on mitotic activity in protoplasts derived fromPisum root cortical explants

Summary Protoplast cultures were prepared from explants of the roots of seedling peas. In defined, synthetic media these cultures were mitotically active. A variety of culture conditions were investigated and the influences of these conditions on the mitotic activity of the protoplasts were observed. Marked inhibition of mitoses were observed after exposure to high light intensities, and in the absence of a proper exogenous supply of hormones. The protoplasts showed extreme sensitivity to the nature and concentration of the exogenous hormone supply. The protoplasts were mitotically active at low population densities (6,000–8,000 protoplasts/ ml of medium). They did not divide in cultures in which glucose was supplied as a carbohydrate source, but divided actively in cultures in which sucrose (2%) was supplied. The responses to temperature and pH were similar to most protoplast systems which have been reported. The definition of a wide variety of optimal culture conditions resulted in high mitotic activity in protoplast cultures with low population densities.     

Optimization of Culture Medium for Sporulation and Biomass Production of a Nematophagous Fungus: Consideration of Nutritional and Environmental Conditions

This study reports the effects of various nutritional and environmental factors on sporulation and biomass of Paecilomyces lilacinus IPC‐P. These factors included carbon and nitrogen sources, carbon‐to‐nitrogen ratios, mineral elements and vitamins together with water potentials, temperatures, dark/light cycles and pH. On the basis of these results, together with a ‘two‐step’ cultivation and orthogonal method, the culture conditions for sporulation of this fungus were optimized. The spore suspension was inoculated on a basal medium (sucrose 19.00 g/l, soy peptone 4.06 g/l, K₂HPO₄ 1.00 g/l, KCl 0.50 g/l, MgSO₄ 0.50 g/l, FeSO₄ 0.01 g/l, agar 13.00 g/l) for 4 days, before being transferred to a sporulation medium (dextrin 2.27 g/l, urea 2.13 g/l, CaCl₂ 3.00 g/l, ZnSO₄·7H₂O 0.01 g/l, agar 13.00 g/l) for a further 4 days under the following environmental conditions: ?3.9 MPa/pH 7/light 24 h/temperature 29°C; these conditions were altered to ?0.3 MPa/pH 6/light 24 h/temperature 23°C in order to obtain better biomass yields. The data presented provide information on the nutrient and environmental requirements of this fungus, which will be essential for its commercial production.     

On the importance of calcium and magnesium ions in yeast sporulation

Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.     

Effects of Physical Factors and Antibiotics on the Growth of Higher Plant Cells in Suspension Culture

To study the influence of culture conditions on higher plant cells in suspension culture, the effects of antibiotics and physical factors, i.e. pH, temperature, shaking conditions and light, on the growth of suspended cells were investigated using three different callus cells; Populus hybrids (P. maximowiczii × P. nigra), N. glutinosa and N. tabacum var. xanthi ova. Of the conditions tested, red light as the light source, 32°C as the temperature, 7.5 as the initial pH value of the medium and 90 reci/min as the reciprocations of the shaker were the most favourable conditions for cell growth. In addition, the influence of various antibiotics was investigated. Of the antibiotics tested, penicillin, oleandomycin, cephaloridin and oxytetracycline did not inhibit cell growth and blasticidin-S was most toxic to all cultures.     

Separation of pH,dilution, lonic strength and chemical matrix effects for biological monitoring of urines with the Microtox® test using nicotine,cotinine and reference urines

The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox® test to interpret bioassay results for urine. Four reference urines were assessed as reference materials for the bioassay. Nicotine and cotinine were investigated as urinary markers for tobacco exposure. The optimum luminescence conditions were: 1.85%–3.25% NaCl, 0.33–0.58 mol/L ionic strength, and pH 5.8–6.7. Low pH values and high concentration of toxic trace metals were important factors in this study. Unexpacted toxicity for a Standard Reference Material was attributed to zinc contamination. Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity. Thus practical urinary biological monitoring with the Microtox® test necessitates excretion of metabolites and compounds that are much more toxic than the urine components. Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned. This is the first report of Microtox® EC₅₀ values for nicotine and cotinine. The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution.