Spore Inoculum Research Articles

Sporulation and regeneration efficiency of phosphobacteria (Bacillus megaterium var phosphaticum).

Sporulation in Bacillus megaterium var phosphaticum (PB - 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB - 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10(8) cfu ml(-1)) in nutrient broth containing 5 per cent inoculum level.

Optimization of Citric Acid Production from a New Strain and Mutant of Aspergillus niger Using Solid State Fermentation

A new strain of Aspergillus niger isolated from soil and its mutant were used for citric acid production from carob under solid-state fermentation conditions. The parental strain produced 30 g/kg citric acid, while the mutant G4, selected after four rounds of gamma ray irradiation, produced 60 g/kg. Maximum citric acid production was obtained after 7 days of incubation, as the acid production was 34 and 64 g/kg for parental and mutant strains, respectively. The addition of 2% methanol increased citric acid production from the parental strain to 42 and the mutant G4 to 65 g/kg. Trace elements, namely Cu, Fe, and Zn, promoted the production of citric acid as the acid production from the parental strain increased to 46 g/kg and for mutant G4 increased to 73 g/kg after their addition. The optimum spore inoculum concentration for acid production was 107 ml−1, and the optimum pH was 5 for both parental and mutant strains.

Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using real-time PCR

The fungal pathogen Monilinia fructicola causes blossom blight and fruit brown rot of stone fruits in California. In this study, spore densities in the air were monitored in six orchard/year combinations with Burkard spore traps. A real-time PCR assay was developed to efficiently quantify the dynamics of spore density in these orchards during the growing season. Different patterns of dynamics of spore density were observed in these orchards. A linear relationship between numbers of spores counted with a compound microscope and those determined with the real-time PCR assay was obtained, using the same samples of spore traps. Spore density in five of six orchard/year combinations ranged from 0.0 to 0.05 spores l−1, except for that in orchard 4, which showed much higher values of spore density in the air, as well as higher values and wider range of incidences of blossom infection and fruit rot than those in the other orchards. The results demonstrated a potential method to quantitatively determine spore inoculum potential in orchards by using a real-time PCR assay.

Characteristics of Spore Germination in a Mouse Model of Cutaneous Anthrax

Cutaneous infection is the most common form of human anthrax, but little is known about Bacillus anthracis spore germination in these infections. We used experimental inoculations of B. anthracis Sterne spores or vegetative bacilli onto intact or abraded mouse flank skin, followed by evaluation of the infections and enumeration of germinating spores and vegetative bacilli. Bacilli developed from a spore inoculum after application onto abraded, but not intact, skin of the mice. Germination appeared to occur extracellularly at the skin surface before the development of a phagocytic response; in fact, vegetative bacilli were seen after inoculation of the spores on top of a filter that separated them from the host phagocytic cells below. Malachite green staining demonstrated that spores began germinating 1-3 h after inoculation onto abraded skin. Vegetative bacilli were found not to be capable of initiating infection in the absence of cutaneous abrasion. The results indicate that epidermal damage is required for germination of B. anthracis spores in these infections; even so, spore germination by itself is not sufficient to produce infection of undamaged skin. In contrast to events in experimental inhalational anthrax, spore germination in these cutaneous infections appears to occur extracellularly.

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO 2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO 2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO 2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.

Are field populations of arbuscular mycorrhizal fungi able to suppress the transmission of seed-borne Bipolaris sorokiniana to aerial plant parts?

The development of seed-borne Bipolaris sorokiniana in barley in the presence of arbuscular mycorrhizal fungi was studied. To exploit natural variation in their ability to control disease development, arbuscular mycorrhizal fungi from various Swedish arable soils were multiplied in trap cultures using a mixture of plant species. Six out of eight trap culture soil inocula were able to reduce transmission of B. sorokiniana from seeds to stem bases when grown together with infected barley seed. Based on this result two soil inocula, of different origin, from semi-natural grassland and barley respectively, were chosen for further greenhouse studies. Both soil inocula gave significant reductions in pathogen transmission from seeds to seedlings compared to the untreated control. In addition, treatment with spore inocula, collected from the different trap culture soils, showed disease suppression. Treatment with spores from the pure culture Glomus intraradices gave significant reduction in leaf lesion development. A treatment with the commercial inoculum Vaminoc® was included and gave some suppression of the pathogen. In conclusion this study has shown that AM soil inocula from trap cultures suppressed the transmission of seed-borne B. sorokiniana in the aerial parts of barley plants.

Inhibition of Bacillus anthracis and Potential Surrogate Bacilli Growth from Spore Inocula by Nisin and Other Antimicrobial Peptides

The ability of nisin, synthetic temporin analogs, magainins, defensins, and cecropins to inhibit Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis growth from spore inocula was determined using well diffusion assays. Nisin, magainin II amide, and defensins were inhibitory in screening against B. anthracis Sterne or B. cereus ATCC 7004, but only nisin inhibited virulent B. anthracis strains. The MICs of nisin against the 10 Bacillus strains examined were 0.70 to 13.51 μg/ml. Synthetic temporin analogs also inhibited B. anthracis but were not as potent as nisin. None of the strains examined were appropriate B. anthracis surrogates for testing sensitivity to antimicrobial peptides.

Evaluation of Chlorine, Chlorine Dioxide, and a Peroxyacetic Acid–Based Sanitizer for Effectiveness in Killing Bacillus cereus and Bacillus thuringiensis Spores in Suspensions, on the Surface of Stainless Steel, and on Apples

Chlorine (10 to 200 μg/ml), chlorine dioxide (10 to 200 μg/ml), and a peroxyacetic acid–based sanitizer (40 and 80 μg/ml) were evaluated for effectiveness in killing spores of Bacillus cereus and Bacillus thuringiensis in suspensions and on the surface of stainless steel and apples. Water and 5% horse serum were used as carriers for spore inoculum applied to the surface of stainless steel coupons, and 5% horse serum was used as a carrier for inoculum applied to apples. Inocula were dried on stainless steel for 5 h and on apples for 22 to 24 h before treating with sanitizers. At the concentrations of sanitizers tested, sensitivities of planktonic B. cereus and B. thuringiensis spores were similar. A portion of the spores surviving treatment with chlorine and, more markedly, chlorine dioxide had decreased tolerance to heat. Planktonic spores of both species were more sensitive to sanitizers than were spores on the surface of stainless steel or apples. At the same concentrations, chlorine was more effective than chlorine dioxide in killing spores in suspension and on stainless steel. The lethality of chlorine dioxide was markedly reduced when inoculum on stainless steel coupons was suspended in 5% horse serum as a carrier rather than water. Chlorine and chlorine dioxide at concentrations of 10 to 100 μg/ml were equally effective in killing spores on apples. Significant reductions of ≥3.8 to 4.5 log CFU per apple were achieved by treatment with 100 μg/ml of either of the two sanitizers. The peroxyacetic acid sanitizer (40 and 80 μg/ml) was ineffective in killing Bacillus spores in the test systems investigated. Results provide information on the effectiveness of sanitizers commonly used in the food processing industry in killing Bacillus spores in suspension, on a food-contact surface, and on a ready-to-eat food.

Inactivation of Bacillus anthracis spores in murine primary macrophages

The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.

Predictive model for growth of Clostridium perfringens in cooked cured pork

Mathematical models have been developed and used for predicting growth of foodborne pathogens in various food matrices. However, these early models either used microbiological media or other model systems to develop the predictive models. Some of these models have been shown to be inaccurate for applications in meat and specific food matrices, especially under dynamic conditions, such as constantly changing temperatures that are encountered during food processing. The objective of this investigation was to develop a model for predicting growth of Clostridium perfringens from spore inocula in cured pork ham. Isothermal growth of C. perfringens at various temperatures from 10 to 48.9 °C were evaluated using a methodology that employed a numerical technique to solve a set of differential equations. The estimated theoretical minimum and maximum growth temperatures of C. perfringens in cooked cured pork were 13.5 and 50.6 °C, respectively. The kinetic and growth parameters obtained from this study can be used in evaluating growth of C. perfringens from spore populations during dynamically changing temperature conditions such as those encountered in meat processing. Further, this model can be successfully used to design microbiologically “safe” cooling regimes for cured pork hams and similar products.

Effect of Scleroderma Spore Density and Age on Mycorrhiza Formation and Growth of Containerized Eucalyptus globulus and E. urophylla Seedlings

Greenhouse trials with containerized Eucalyptus globulus and E. urophylla seedlings were made to determine effective inoculum spore densities (Scleroderma cepa from temperate Australia, and S. citrinum from sub-tropical China) and spore storage conditions (fresh spores or spores stored for 5 years at room temperature or at 4 °C; S. albidum, S. areolatum and S. cepa from Western Australia). Inoculation with 106 or 108 spores/seedling increased eucalypt growth by up to 46% in height and 42% in dry weight compared to non-inoculated seedlings at 12 weeks after inoculation. Mycorrhizal formation was poor at 102 spores/seeding. Spores stored at 4 °C for 5 years were as effective in forming mycorrhizas as freshly collected spores when a standard density of 106 spores/seedling was applied. It is recommended that spore densities ≥104 be used for inoculation of containerised eucalypts and that spores be stored at 4 °C until use.

The effect of grapefruit extract and temperature abuse on growth of Clostridium perfringens from spore inocula in marinated, sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag, marinated chicken breast that included additional 1.0% NaCl. The packages were processed to an internal temperature of 71.1 °C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose–sulfite–cycloserine agar followed by anaerobic incubation for 48 h at 37 °C. At 19 °C, C. perfringens levels were consistently about 2.5 log 10 CFU/g until 9.5 h regardless of the presence or absence of Citricidal®. However, storage of the unsupplemented marinated chicken samples and those with 50 or 100 ppm Citricidal® samples at 25 °C for more than 6 h resulted in rapid growth of C. perfringens, exhibiting 2–3 log 10 CFU/g increase at 7 h. Citricidal® at 200 ppm significantly ( p < 0.05) reduced the growth of C. perfringens at both 19 and 25 °C. The D-values obtained at 90 °C were significantly decreased ( p < 0.05) from 14.07 (no Citricidal®) to 9.20 min (200 ppm Citricidal®). Supplementing marinated chicken products with Citricidal® and the temperature abuses had no consistent effect on color, shear force or lipid oxidation. However, the organism may grow to unsafe levels if sous-vide products are poorly handled or temperature abused for a relatively long period. An extra degree of safety may be assured in such products by supplementation with 100 or 200 ppm Citricidal®. Industrial relevance: Since temperature abuse is a common occurrence during trasportation, distribution, storage or handling in grocery stores or by consumers, an extra degree of safety may be assured in marinated chicken products by supplementation 100–200 ppm Citricidal®. In such products, the temperature abuse should not have any consistent effect on color, shear force or lipid oxidation of products.

Quantification of the fractal nature of mycelial aggregation in Aspergillus niger submerged cultures

BackgroundFractal geometry estimates have proven useful in studying the growth strategies of fungi in response to different environments on soil or on agar substrates, but their use in mycelia grown submerged is still rare. In the present study, the effects of certain important fermentation parameters, such as the spore inoculum level, phosphate and manganese concentrations in the medium, on mycelial morphology of the citric acid producer Aspergillus niger were determined by fractal geometry. The value of employing fractal geometry to describe mycelial structures was examined in comparison with information from other descriptors including classic morphological parameters derived from image analysis.ResultsFractal analysis of distinct morphological forms produced by fermentation conditions that influence fungal morphology and acid production, showed that the two fractal dimensions DBS (box surface dimension) and DBM (box mass dimension) are very sensitive indexes, capable of describing morphological differences. The two box-counting methods applied (one applied to the whole mass of the mycelial particles and the other applied to their surface only) enabled evaluation of fractal dimensions for mycelial particles in this analysis in the region of DBS = 1.20–1.70 and DBM = 1.20–2.70. The global structure of sufficiently branched mycelia was described by a single fractal dimension D, which did not exceed 1.30. Such simple structures are true mass fractals (DBS = DBM = D) and they could be young mycelia or dispersed forms of growth produced by very dense spore inocula (108–109 spores/ml) or by addition of manganese in the medium. Mycelial clumps and pellets were effectively discriminated by fractal analysis. Fractal dimension values were plotted together with classic morphological parameters derived from image analysis for comparisons. Their sensitivity to treatment was analogous to the sensitivity of classic morphological parameters suggesting that they could be equally used as morphological descriptors.ConclusionStarting from a spore, the mycelium develops as a mass fractal and, depending on culture conditions, it either turns to a surface fractal or remains a mass fractal. Since fractal dimensions give a measure of the degree of complexity and the mass filling properties of an object, it may be possible that a large number of morphological parameters which contribute to the overall complexity of the particles, could be replaced by these indexes effectively.

Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology

BackgroundAlthough the citric acid fermentation by Aspergillus niger is one of the most important industrial microbial processes and various aspects of the fermentation appear in a very large number of publications since the 1950s, the effect of the spore inoculum level on fungal morphology is a rather neglected area. The aim of the presented investigations was to quantify the effects of changing spore inoculum level on the resulting mycelial morphology and to investigate the physiology that underlines the phenomena. Batch fermentations were carried out in a stirred tank bioreactor, which were inoculated directly with spores in concentrations ranging from 104 to 109 spores per ml. Morphological features, evaluated by digital image analysis, were classified using an artificial neural network (ANN), which considered four main object types: globular and elongated pellets, clumps and free mycelial trees. The significance of the particular morphological features and their combination was determined by cluster analysis.ResultsCell volume fraction analysis for the various inoculum levels tested revealed that by rising the spore inoculum level from 104 to 109 spores per ml, a clear transition from pelleted to dispersed forms occurs. Glucosamine formation and release by the mycelium appears to be related to spore inoculum level. Maximum concentrations detected in fermentations inoculated with 104 and 105 spores/ml, where pellets predominated. At much higher inoculum levels (108, 109 spores/ml), lower dissolved oxygen levels during the early fermentation phase were associated with slower ammonium ions uptakes and significantly lower glucosamine concentrations while the mycelium developed in dispersed morphologies. A big increase in the main and total hyphal lengths and branching frequency was observed in mycelial trees as inoculum levels rise from 104 to 109 spores/ml, while in aggregated forms particle sizes and their compactness decreased.ConclusionThe methods used in this study, allowed for the detailed quantification of the transition between the two extreme morphological forms. The impact of spore inoculum level on the detailed characteristics of the particular morphological forms produced was high. Control of mycelial morphology is often regarded as a prerequisite to ensure increased productivities in industrial applications. The research described here demonstrates that adjusting the spore inoculum level controls effectively mycelial morphology.

Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves

BackgroundThe current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.ResultsThe work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.ConclusionAn early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.

Delayed Clostridium perfringens growth from a spore inocula by sodium lactate in sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag marinated chicken breast that included 0%, 1.5%, 3%, or 4.8% sodium lactate (NaL; w/w). The packages were processed to an internal temperature of 71.1 °C, ice chilled and stored at 4, 19, and 25 °C. The total C. perfringens population was determined by plating diluted samples on Tryptose–sulfite–cycloserine agar followed by anaerobic incubation for 48 h at 37 °C. At 25 °C, addition of 1.5% NaL was effective in delaying growth for 29 h. Increasing the NaL level to 4.8%, C. perfringens growth from a spore inoculum during storage at 25 °C for 480 h was not observed. At 19 °C, the growth was >6 log 10 cfu/g by 288 h in control samples. In samples with 3.0% or 4.8% NaL, the growth of C. perfringens from spores was dramatically restricted with little or no growth in 648 h at 19 °C. C. perfringens growth was not observed at 4 °C regardless of NaL concentration. The D-values at 55 °C ranged from 47.40 (no NaL) to 57.58 min (1.5% NaL). Cyclic and static temperature abuse of refrigerated products for ⩽20 h did not permit C. perfringens growth. However, temperature abuse of products for periods 24 h or longer in the absence of NaL led to growth of C. perfringens from a spore inoculum. An extra degree of safety may be assured in such products by supplementation with NaL at 1.5–4.8% NaL level.

The effect of different initial densities of nematode (Meloidogyne javanica) on the build-up of Pasteuria penetrans population

Pasteuria penetrans will build-up faster where there is a high initial nematode density and can suppress root-knot nematode populations in the roots of tomato plants. The effect of different initial densities of nematode (Meloidogyne javanica) (150, 750, 1500, 3000) and P. penetrans infected females (F1, F3) densities (F0=control and AC=absolute control without nematode or P. penetrans inoculum) on the build-up of Pasteuria population was investigated over four crop cycles. Two major points of interest were highlighted. First, that within a confined soil volume, densities of P. penetrans can increase >100 times within 2 or 3 crop cycles. Second, from a relatively small amount of spore inoculum, infection of the host is very high. There were more infected females in the higher P. penetrans doses. The root growth data confirms the greater number of females in the controls particularly at the higher inoculum densities in the third and fourth crops. P. penetrans generally caused the fresh root weights to be higher than those in the control. P. penetrans has shown greater reduction of egg masses per plant at most densities. The effects of different initial densities of M. javanica and P. penetrans on the development of the pest and parasite populations were monitored. And no attempt was made to return the P. penetrans spores to the pots after each crop so the build-up in actual numbers of infected females and spores under natural conditions may be underestimated.

Persistence of the exotic kelp Undaria pinnatifida does not depend on sea urchin grazing

We investigated mechanisms enabling persistence of the introduced Asian kelp Undaria pinnatifida on the sea urchin (Heliocidaris erythrogramma) 'barrens' on the east coast of Tasmania. Development of dense stands of U. pinnatifida requires disturbance to reduce the cover of native algae. Observations of U. pinnatifida occurring abundantly on sea urchin barrens suggests that disturbance in the form of grazing by sea urchins prevents recovery of native canopy-forming species, allowing dense stands of U. pinnatifida to persist. We examined this hypothesis over a 30 mo period in a manipulative experiment in which the response of native algae and U. pinnatifida was examined in treatments comprising all possible combinations of +/- urchins, +/- U. pinnatifida and +/- enhanced native algal spore inoculum. The results demonstrated that the sea urchin H. erythrogramma can have a significant impact on U. pinnatifida abundance. The response was most dramatic in the 2001 sporophyte growth season, when sea urchins destructively grazed U. pinnatifida sporophytes in experimental plots on the urchin barren. In other years, when there was higher recruitment of U. pinnatifida sporophytes, urchins reduced sporophyte abundance but did not prevent development of a U. pinnatifida canopy. Removal of sea urchins resulted in a slow increase in cover of understorey red algae, but only limited recovery of native canopy-forming species. In treatments where both sea urchins and U. pinnatifida were removed, cover of canopy-forming species did not exceed 6% over the duration of the study. Thus, in the absence of sea urchin grazing, there was no evidence of inhibition of U. pinnatifida by native algae. While the intensity of sea urchin grazing may directly influence the extent of the U. pinnatifida canopy, recovery of native canopyforming species was apparently influenced by a combination of factors including seaurchin grazing, depth and, most importantly, the degree of sediment accumulation on the rocky substratum. The manipulations showed that removal of the factor (i.e. sea urchin grazing) that ostensibly facilitated replacement of native canopy-forming algae by U. pinnatifida did not realise recovery of native canopy-forming species.

Optimisation on bioleaching of incinerator fly ash by Aspergillus niger – use of central composite design

Previous work has shown that heavy metals may be bioleached from municipal solid waste (MSW) incinerator fly ash. The bioleaching of heavy metals from the ash depends on physical, chemical and biological factors. Our research objective is to establish the factors that exert a greater impact on the bioleaching process, and to determine the optimal bioleaching conditions in order to achieve the maximum metal leaching efficiency for selected metals. Four factors were investigated: sucrose concentration, inoculum spore concentration, fly ash pulp density, and the time of addition of fly ash to the fungus Aspergillus niger. Using the central composite design (CCD), 31 batch bioleaching tests were run under high and low values of these four factors. Empirical models obtained through second-order (Taylor series approximation) regression provided the optimal bioleaching conditions. Results showed that sucrose concentration and pulp density were more important factors than spore concentration and the time of addition of the fly ash. The concentration of Al, Fe, Zn, citric acid, and gluconic acid attained in the leached liquor was 12.3, 12.3, 77.6 ppm, 51 and 281 mM, respectively.

The weak acid preservative sorbic acid inhibits conidial germination and mycelial growth of Aspergillus niger through intracellular acidification.

The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 10(5)/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using (31)P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH(cyt)) by more than 1 pH unit and a depression of vacuolar pH (pH(vac)) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH(cyt). NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.