Influence of a pulsed electric field on the spores and oxygen consumption of Aspergillus niger and its citric acid production

Spores of Aspergillus niger were exposed to a pulsed electric field. After treatment by the electric field, the viability of the conidia of A. niger varied depending on the field strength, pulse width and frequency. In all cases, these parameters reduced the viability rate of the conidia from 2.0 × 10⁷ to a range from 6.2 × 10⁶ to 8.5 × 10⁶ spores/ml (3.1 to 42.6%). After pulse treatment, the conidia were used as the inoculum for citric acid fermentation in shake flasks. The highest increase in citric acid yield (about 1.4-fold) was reached at a field strength of 2.85 kV/cm, a frequency of 1 Hz and a pulse width of 1 ms. When the parameters of the electric field increased there were important changes in the respiration rate of the Aspergillus niger mycelium (48-h-old) after electric shock treatment. The highest consumption of dissolved oxygen (22.9%) in the medium by Aspergillus niger mycelium was observed at an electric field strength of 2.85 kV/cm, a 1 Hz frequency, a pulse width of 1 ms and a 1-min exposure period. It seems that an electric-field stimulation of the conidia prior to inoculation may offer an important method of improving the efficiency of citric acid. The treatment of the conidia is both simple from the technical point of view and extremely rapid.     

Some factors affecting the formation of protoplasts inAspergillus niger

The highest yield of protoplasts in the strainAspergillus niger K 10 was obtained from young, freshly harvested hyphae, grown on simple medium of sucrose-asparagine type on a rotary shaker. The residual cultivation medium has to be washed from the mycelial suspension with a solution of high osmotic pressure. Lyophilized snail digestive juice in concentration of 1 %, temperature 31° C, and incubation in Erlenmayer flasks on reciprocal shaker were optimal for the release of protoplasts. Good stabilizers of released protoplasts (in combination with CaCl₂) were for example galactose, mannitol, inositol and mixture of NaCl with glucose, sucrose or lactose.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Adaptation of Aspergillus niger to short-term salt strees

Adaptation of filamentous fungi to short-term salt stress has been analysed by a continuous measurement system. Spores of Aspergillus niger were immobilized on the polylysine-coated glass bottom of a culture vessel, which enabled the exchange of a medium containing salt (NaCl) without disturbing continuous observation. Repeated contacts with 0.75% NaCl produced hypha insensitive to this concentration of NaCl. When the NaCl concentration was increased stepwise, the tolerated concentration increased up to 1.25%. The acquisition of such a tolerance to 0.75% NaCl required about 10 min prior contact with 0.5% NaCl. Based on these results, the adaptation mechanism is discussed. Correspondence to: H. Matsuoka     

A preliminary study of the applicability of sweet potatoes for mycological fat production

Summary For mycological fat production,Aspergillus nidulans andPenicillium lilacinum were grown on media made up of crushed and boiled sweet potatoes. Mycelial mats containing a fair amount of fat were obtained, namely, 15 % in case ofAspergillus and 24 % in case ofPenicillium. Media containing an external supply of glucose gave rise to increase in percentage fat in mycelium, but the amount of mycelium and consequently the total fat content decreased.Addition of sodium nitrate gave heavier mycelial mats but the total fat content could not be raised.Addition of potassium phosphate or magnesium sulphate or both to the sweet potato medium gave rise to heavier mycelial mats but with lower fat content.     

Protease-conidia relationships of Aspergillus niger grown in solid state fermentation

Protease activity of Aspergillus niger growing on solid substrate correlated well with conidia formation (R: 0.91–0.96) for initial moisture contents of 38–48% (wet basis), initial pH 5.4 and 6 and temperature (29–37 °C ). However, conidia/protease ratio varied with most of these conditions and by NaCl addition indicating only a partial association between them.     

Production of mycelial protein and cellulolytic enzymes from food wastes

Summary Extracted grape waster material and pressed apple pulp were tested as carbon sources forPenicillium funiculosum 515,Myrothecium verrucaria 9095 andAspergillus niger TMF-15. They were good growth substrates, especially forA. niger. When cultivated on mixed substrate in optimized nutrient medium,A. niger accumulated a product of 35% crude protein with a maximum productivity of 0.117 g protein/1/h and cellulose consumption of 90.92%.A. niger also produced the highest levels of cellulase activity. Maximum carboxymethyl cellulase and activity against filter paper were 494 units/l and 97 units/l, respectively.     

Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

EFFECT OF SALINITY ON POLLEN I. POLLEN VIABILITY AS ALTERED BY INCREASING OSMOTIC PRESSURE WITH NaCl,MgCl2, and CaCl2

Petunia (Petunia hybrida Vilm. cv. ‘Snowstorm') plants were grown in saline solution (NaCl, MgCl₂, and/or CaCl₂) of 0, 1, 2, and 3 bars osmotic pressures. Pollen viability was tested by tetrazolium chloride staining and by germination (by the hanging drop method, using 15 % sucrose and 0.01 % boric acid as the nutrient medium, at 27 ± 1 C). Pollen viability decreased with increased salinity. Pollen from plants grown in single salt solutions of NaCl, MgCl₂, and CaCl₂ (each at 0, 1, 2, or 3 bars osmotic pressure) was germinated in base culture medium. Pollen viability decreased more with NaCl than with MgCl₂ or CaCl₂. In vitro studies of the effects of three salts, viz., NaCl, MgCl₂, and CaCl₂, on pollen germination and tube growth showed that NaCl inhibited germination and pollen tube growth more than did MgCl₂ or CaCl₂. MgCl₂ was least injurious, and even promoted tube growth at 0.5 and 0.75 bars osmotic pressure. Adding low concentrations of MgCl₂ reduced the toxic effect of NaCl and increased the percentage of germination. CaCl₂ reduced the effect of NaCl less than did MgCl₂. We conclude that specific ion effects were more important than osmotic pressure.     

PROTEIN AND AMINO ACID COMPOSITION OF THE OBLIGATE HALOPHILE APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Total protein was determined for cells of Aphanothece halophytica Fremy harvested during early log, mid-log and linear growth phases in media containing 1, 2, and 3 M NaCl. Cells grown in medium containing 1 M NaCl showed a progressive increase in protein content up to a maximum of 76% of dry weight (linear phase). Total protein also increased in cells grown in 2 M NaCl. medium (56.5–72.0%). Cells grown in 3 M NaCl medium showed a progressive decrease in total protein (59.9–43%). Although amounts of protein varied, the percentages of the respective amino acids of hydrolyzed bulk protein were consistent to within 1% for linear phase cells grown in 1, 2, and 3 M NaCl cultures. Percentages of acidic amino acids were 2.3–2.6 times greater than those of the basic amino acids. The amino acid composition of phycocyanin was similar to that of bulk protein. Free amino acids varied with both age of the culture and the concentration of NaCl. The high quantity and quality of the protein observed suggest that A. halophytica might be a useful food organism.     

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K_m (15.6260 g/L) and the rate constant of deactivation, K_de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V_max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.     

Effects of Nutrients at Different Salinities on Growth of the Freshwater Green Alga Scenedesmus bijugatus in Water of Narendra Pond,Puri, Orissa

The growth and biochemical composition of Scenedesmus bijugatus grown in normal and nutrient enriched Narendra Pond water was evaluated at different levels of salinity. In normal pond water (NPW) the growth was enhanced when the initial salinity of the water was doubled (to 7.2 mmol/l) but further salinity increase caused growth retardation. In nutrient enriched pond water (NEW), however, the maximum growth occurred in the culture containing 29 mmol/l of NaCl. At growth retarding salt concentrations the chlorophyll, carbohydrate, protein and lipid contents per cell decreased in both NPW and NEW while the proline content first increased and was then reduced. All nutrients, taken separately or in combination, caused growth enhancement of the alga at the three selected salinity levels. The alga showed a better halotolerance when enriched with nitrogen or phosphorus.     

Fatty acid composition of lipids in vegetative organs of the halophyte Suaeda altissima under different levels of salinity

Qualitative and quantitative composition of fatty acids (FA) in the lipids of vegetative organs of the halophyte Suaeda altissima (L.) Pall. grown at different NaCl concentrations in nutrient solution was studied. Along with this, the biomass of these organs, the content of water and Na⁺, Cl^?, and K⁺ ions in them, and the ultrastructure of root and leaf cells were determined. At both low (1 mM) and high (750 mM) NaCl concentrations in nutrient solution, plants could maintain growth and water content in organs, demonstrating a noticeable increase in the dry weight and a slight increase in the water content at 250 mM NaCl. At all NaCl concentrations in nutrient solution, S. altissima tissues contained a relatively high K⁺ amount. Under salinity, Na⁺ and Cl^? ions contributed substantially into the increase in the cell osmotic pressure, i.e., a decrease in their water potential; in the absence of salinity, K⁺ fulfilled this function. In the cells of both roots and leaves, NaCl stimulated endo- and exocytosis, supposedly involved in the vesicular compound transport. 750 mM NaCl induced plasmolysis and changes in the membrane structure, which can be interpreted as degradation processes. Under optimal NaCl concentration in medium (250 mM), the content of lipids in plant aboveground organs per fresh weight was more than 2.5-fold higher than under 1 or 750 mM NaCl, whereas in the roots opposite patten was observed. When plants were grown under non-optimal conditions, substantial changes occurred in the qualitative and quantitative FA composition in lipids of both aboveground organs and roots. Observed changes are discussed in relation to processes underlying S. altissima salt tolerance and those of disintegration occurring at the high external NaCl concentration (750 mM).     

Production of15N-labelled hen egg white lysozyme usingAspergillus niger

Summary Aspergillus niger has been used as a host organism for the production of¹⁵N-labelled hen egg white lysozyme (HEWL). In order to achieve maximum incorporation of label, strains expressing the HEWL gene were grown in medium containing ammonium¹⁵N chloride as sole nitrogen source. Yields of HEWL protein were reduced relative to those obtained on more complex media. Gains in yield using complex media were offset by reduction in¹⁵N incorporation. No differences in either yield or kinetics of production were observed when ammonium¹⁵N chloride was replaced by unlabelled ammonium chloride as sole nitrogen source. Yields of¹⁵N-HEWL produced in this way are adequate for, and offer considerable advantages to, NMR studies of structure and folding of mutant and wild-type lysozymes.     

Enhancement of Intact Bean Leaf Senescence by NaCl Salinity

Red kidney bean (Phaseolus vulgaris L.) plants were grown in nutrient solution and in nutrient solution plus four bars of added NaCl. Chlorophyll and protein decay occurred much more rapidly in intact leaves from plants subjected to four bars of added NaCl in the growth medium than in intact leaves from plants without added NaCl. Ribonucleic acid (RNA) content in intact leaves of salt treated plants was higher than in intact leaves from plants grown in nutrient solution alone. However, the tendency for RNA content variation in leaves during the experimental period was the same for both control and salt treated plants. The results support the idea that salinity enhances senescence and suggests that hormone imbalance plays an important role in this process.     

Influence of manganese on morphology and cell wall composition of Aspergillus niger during citric acid fermentation

Morphology and cell wall composition of Aspergillus niger were studied under conditions of manganese sufficient or deficient cultivation in an otherwise citric acid producing medium. Omission of Mn²⁺ (less than 10^-7 M) from the nutrient medium of Aspergillus niger results in abnormal morphological development which is characterized by increased spore swelling, and squat, bulbeous hyphae. Fractionation and analysis of manganese deficient cell walls revealed increased chitin and reduced -glucan contents as well as reduction of galactose containing polymers, as compared to cell walls from manganese sufficient grown hyphae. Addition of copper induced the same effect as manganese deficiency, both on morphology and cell wall composition. Addition of cycloheximide also produced a very similar type of morphology with increased chitin and reduced -glucan contents of the cell wall but its effect on galactose was less pronounced.Dedicated to emer. Prof. Dr. J. Kisser on the occasion of his 80th birthday     

Minimal nutrient requirements for induction of germination of Aspergillus niger conidia

Aspergillus niger reproduces asexually by forming conidia. Here, the minimal nutrient requirements were studied that activate germination of A. niger conidia. To this end, germination was monitored in time using an oCelloScope imager. Data was used as input in an asymmetric model to describe the process of swelling and germ tube formation. The maximum number of spores (P_max) that were activated to swell and to form germ tubes was 32.54% and 20.51%, respectively, in minimal medium with 50 mM glucose. In contrast, P_max of swelling and germ tube formation was <1% in water or 50 mM glucose. Combining 50 mM glucose with either NaNO₃, KH₂PO4, or MgSO4 increased P_max of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these P_max values up to 25.85% and 10.99%. Next, 10 mM amino acid was combined with a phosphate buffer and MgSO₄. High (e.g. proline), intermediate and low (e.g. cysteine) inducing amino acids were distinguished. Together, a combination of an inducing carbon source with either inorganic phosphate, inorganic nitrogen or magnesium sulphate is the minimum requirement for A. niger conidia to germinate.     

Effects of osmotic shock and calcium on growth and solute composition of Phaseolus vulgaris plants

Identification of the primary response to salinity is of great importance in order to develop salt tolerant species. In this work the effect of a NaCl osmotic shock on leaf elongation of Phaseolus vulgaris L. cv. Contender has been studied. After establishing an osmotic shock by adding NaCl to the root medium, three consecutive events could be distinguished. First, there was a sudden interruption of leaf growth, followed by a period of retardation of growth, and then by a restoration of growth to reach the steady-state growth rate. High calcium (5 mM) in the growth medium diminished the initial loss of water in the roots and kept the water content higher upon restoration of leaf growth, as compared to low calcium levels (0.5 mM). During the low calcium treatment, leaf malate started to increase already 3 h after the start of the osmotic treatment, showing a maximum concentration at around 9 h, then decreasing and approaching the value of control plants. At high calcium, malate concentration remained unchanged with time. Plants grown in the presence of low calcium showed an increase in the concentration of total amino acids upon NaCl shock. It is estimated that organic metabolites, if confined to the cell cytoplasm, contribute significantly to the osmotic adjustment, together with inorganic ions. Our results support the hypothesis that water shortage in the roots is responsible for the initial inhibition of leaf elongation. The beneficial effect of calcium on restoration of growth after the NaCl shock is a consequence of short-term, energetically expensive osmotic adjustment, in which mainly organic metabolites are involved. Under steady state conditions, high calcium treatment results in a faster growth rate than low calcium. In a process of osmotic adjustment, in which inorganic ions are principally involved, this is the result of a decrease in leaf Na⁺ concentration in expanding leaves, together with an increase in K⁺ and Ca²⁺.     

Leaf gas exchange,water relations,nutrient content and growth in citrus and olive seedlings under salinity

The effects of salinity on growth, leaf nutrient content, water relations, gas exchange parameters and chlorophyll fluorescence were studied in six-month-old seedlings of citrus (Citrus limonia Osbeck) and rooted cuttings of olive (Olea europaea L. cv. Arbequina). Citrus and olive were grown in a greenhouse and watered with half strength Hoagland’s solution plus 0 or 50 mM NaCl for citrus, or plus 0 or 100 mM NaCl for olive. Salinity increased Cl⁻ and Na⁺ content in leaves and roots in both species and reduced total plant dry mass, net photosynthetic rate and stomatal conductance. Decreased growth and gas exchange was apparently due to a toxic effect of Cl⁻ and/or Na⁺ and not due to osmotic stress since both species were able to osmotically adjust to maintain pressure potential higher than in non-salinized leaves. Internal CO₂ concentration in the mesophyll was not reduced in either species. Salinity decreased leaf chlorophyll a content only in citrus.