Comparing phosphorus mobilization strategies using Aspergillus niger for the mineral dissolution of three phosphate rocks

Phosphorus deficiencies are limiting crop production in agricultural soils worldwide. Locally available sources of raw phosphate rock (PR) are being recognized for their potential role in soil fertility improvement. Phosphorus bioavailability is essential for the efficiency of PRs and can be increased by acid treatments. The utilization of organic acid producing micro-organisms, notably Aspergillus niger , presents a sustainable alternative to the use of strong inorganic acids, but acid production of A. niger strongly depends on the mineral content of the growth media. This study compared the phosphorus mobilization efficiency of two biological treatments, namely addition of acidic cell-free supernatants from A. niger cultivations to PRs and the direct cultivation of A. niger with PRs. The results show that addition of PR to cultivations leads to significant differences in the profile of organic acids produced by A. niger . Additions of PR, especially igneous rocks containing high amounts of iron and manganese, lead to reduced citric acid concentrations. In spite of these differences, phosphorus mobilization was similar between treatments, suggesting that the simpler direct cultivation method was not inferior. In addition to citric acid, it is suggested that oxalic acid contributes to PR solubilization in direct cultivations with A. niger , which would benefit farmers in developing countries where conventional fertilizers are not adequately accessible.     

Comparison of Suppressiveness of Vermicomposts Produced from Animal Manures and Sewage Sludge against Phytophthora nicotianae Breda de Haan var. nicotianae

The degrees of suppression produced by vermicomposts produced from cattle manure, sheep manure or horse manure and by vermicomposts produced from sewage sludge were compared in greenhouse experiments. The effect of these vermicomposts on the growth and infection of tomato seedlings by Phytophthora nicotianae var. nicotianae was studied. The density of the pathogen and the number of micro-organisms in container media amended with vermicomposts were also analysed. The vermicomposts produced from animal manure significantly reduced the infection of tomato seedlings by the pathogen. The density of P. nicotianae in media which included these vermicomposts was similar to that in infested peat substrate (control treatment). The vermicomposts from sewage sludge did not protect tomato seedlings against P. nicotianae . They also significantly inhibited growth of the plants as well as decreasing the density of the pathogen in container media. In general the vermicomposts had no effect on total number of micro-organisms in potting media compared with control. They only had higher levels of actinomycetes but this did not appear to correspond with their ability to suppress the pathogen.     

Enzymatic activity of micro-organisms isolated from cork wine stoppers

The production of enzymes by micro-organisms which are found on vegetal substrates is important due to their ability to decompose cellulose, lignin and other components, which guarantee the integrity of the vegetal cell. The objective of this study was to determine the enzymatic activity of filamentous fungi, yeasts and bacteria, isolated from natural cork stoppers for bottles of still and sparkling wines. Suspensions of fungal conidia, yeasts and bacterial cells of micro-organisms were established in concentrations of 10(6) CFU/ml. The enzymatic activity of these micro-organisms was evaluated by means of the API ZYM system, with which it was possible to determine and semi-quantify nineteen enzymatic activities simultaneously. The enzymes produced by all of the species were esterase (C1), esterase lipase and naphthol-AS-BI-phosphohydrolase. The micro-organisms with the greatest enzymatic activity were Monilia sitophila, Alternaria alternata, Aspergillus niger and Aeromonas sp.     

A new screening method for investigating microbial interactions

Interactions among Candida albicans, Staphylococcus aureus and Escherichia coli were investigated using a screening system in which test micro-organisms were incorporated in agar discs and effector micro-organisms in fluid growth media. Total as well as partial inhibition of test micro-organisms was observed in agar discs when these were incubated in broths containing effector micro-organisms. The ratio of numbers of test to effector micro-organisms was found to be of importance in the inhibition effect. The technique was found to be cheap, simple and versatile.     

Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.     

Death and Lysis of Leptospirae When Cultured in Asbestos-Filtered Growth Media

Death and lysis of leptospirae, when cultured in asbestos-filtered bovine albumin polysorbate 80 media, was quantitated. The pathogens (virulent and avirulent) required 2 x 10(6) cells/ml to initiate growth in such media, whereas inocula of 2 to 20 cells/ml grew in control medium. Saprophytic leptospirae initiated growth from 2 cells/ml in asbestos-filtered medium as well as control medium. The adverse action of asbestos-filtered medium was not removed by storage of medium for 2 years at 25 C and was not diminished when such medium was frozen at -80 C. Washing with water, HCl and NaHCO(3)-NaCl, citric acid, and medium components did not remove the lytic activity associated with asbestos-filtered culture medium. Continuous subculture in asbestos-filtered medium was possible from large inocula; however, upon subsequent dilution and reinoculation into asbestos-filtered media, there was no evidence of acquired resistance, and all pathogens failed to grow.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin-a

Cyanobacterial hepatotoxins and anatoxin-a, a neurotoxin, were shown to be degraded when crude extracts of lysed toxic laboratory strains of cyanobacteria were exposed to natural populations of micro-organisms from lakes. While anatoxin-a decayed equally fast with all the inocula from lake sediment and water, the degradation rate of hepatotoxins was higher with inocula from places at which cyanobacterial water blooms had occurred than with inocula from places with no known mass occurrences of cyanobacteria. Degradation was slowest when an inoculum from a humic lake was used. A part of the loss of the toxins was shown to be due to adsorption on lake sediments.     

Evidence that the mechanisms of fungitoxicity of 8-quinolinol and its bischelate with copper(II) are different

The copper(II) contents of the growth media, Sabouraud dextrose and Czapek-Dox broths, and of the spore inocula of Aspergillus niger (ATCC 1004), Aspergillus oryzae (ATCC 1011), Trichoderma viride (ATCC 8678), and Myrothecium verrucaria (ATCC 9095) were determined by atomic absorption spectrophotometry in a graphite furnace. The test systems composed of Sabouraud dextrose broth and spore inocula of the four fungi contained only a little over 3% of the copper(II) required to form a minimal inhibitory concentration of bis(8-quinolinolato)copper(II). The test system of Czapek-Dox broth and A. oryzae contained slightly less than 65% of the copper(II) required to form a minimal inhibitory concentration of the bischelate of 8-quinolinol with copper(II). When the minimal inhibitory concentrations of 8-quinolinol and bis(8-quinolinolato)copper(II) were added simultaneously to the test system of Czapek-Dox broth and A. oryzae, 10% of the combined mixture of toxicants caused complete inhibition of growth indicating synergism between the toxicants. These results together with the observation that alpha-lipoic acid as well as small aliphatic thiol-containing compounds (cysteine, glutathione, dithioerythritol, and dithiothreitol) reversed the toxicity of 8-quinolinol but not the toxicity of bis(8-quinolinolato)copper(II) led to the conclusion that the mechanisms of fungitoxicity of both toxicants are different.     

The effects of glucosinolates and their hydrolysis products on microbial growth

Rapemeal, which contains potentially toxic compounds such as glucosinolates, was assessed as a substrate for the growth of micro-organisms. The effects of glucosinolates and their degradation products were tested on a range of industrially important microbial species. Sinigrin (2-propenyl glucosinolate) was found to be relatively innocuous to all of the organisms tested but its hydrolysis to yield isothiocyanates, thiocyanates and nitriles resulted in inhibition of growth. The initial inhibitory sinigrin concentration before its hydrolysis was found to be species-dependent with Bacillus subtilis being the most resistant (80 μg ml^-1) and Saccharomyces cerevisiae (40 μg ml^-1) the most sensitive. Three Gram-positive organisms tested were found to be more resistant to hydrolysis products than other micro-organisms. Similar results were observed with phenylisothiocyanate for which inhibition was found to be inhibitor and cell concentration-dependent. Addition of thioglucoside glucohydrolase during active growth of Escherichia coli in a sinigrin-containing liquid medium reduced the number of viable cells. Similar effects were also observed in rapemeal media in which growth inhibition was dependent on the glucosinolate content of the rapemeal.     

Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients.

Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis.     

A fully defined, clear and protein-free liquid medium permitting dense growth of Neisseria gonorrhoeae from very low inocula

Neisseria gonorrhoeae is difficult to cultivate in liquid medium. Currently there are no liquid media, defined or undefined, that reliably permit growth of this bacterium from low inocula. Standard clinical laboratory broths may allow multiplication of some strains of gonococci from large inocula, but such media incorporate infusates, extracts or digests and are therefore undefined. In this study, 20 gonococci of ten auxotypes were tested in various experimental media in the development of an easily prepared chemically defined, clear and protein-free liquid medium. The final medium - GW medium - allowed the growth of three clinical isolates of gonococci from inocula of <10(3) CFU mL(-1) to >10(8) CFU mL(-1) by 24 h. None of four commercially-available broths (nutrient broth, brain heart infusion, tryptone soya broth, and Mueller-Hinton broth) tested in parallel reliably supported growth of these isolates to the same extent. GW medium should be useful for studies of the growth of gonococci under different conditions and, as the medium is clear and colorless, this can be monitored turbidometrically. GW medium may be suitable as a basal medium for biochemical identification tests, antimicrobial susceptibility determinations and antimicrobial synergy studies.     

The determination of threshold concentrations of biocides for museum exhibits by a photometric method

The efficiency and threshold concentration of biocides has been examined by photometric analysis of the growth of micro-organisms cultured in the laboratory. Both fungi and algae responsible for attacking museum exhibits inside and in the open air were used. The organisms were cultured on a nutrient media containing serial dilutions of biocide, and growth was assessed by measuring optical density with a multichannel photometer. The efficacy of the biocides was measured by means of an inhibition parameter α which is the degree of deviation of the growth curve determined from that of the control. Both AB catamin, which is already used in museums, and catapol were examined. The data suggest that the efficiency of catapol is very close to that calculated theoretically. The advantage of the method over traditional ones has been demonstrated.     

Rapid serotyping of campylobacters based on heat-stable antigens, using a combined passive haemagglutination/co-agglutination technique

Tryptic soy broth/agar, brain heart infusion agar and Columbia broth/agar, all widely used in the bacteriological laboratory, were radiation-sterilized at a dose of 10–15 kGy. The media were tested for the growth of 12 different micro-organisms, including fastidious pathogens such as Streptococcus pyogenes, Strep, pneumonias, Haemophilus influenzas and Neisseria meningitidis. Solid and fluid media supplemented with catalase before irradiation performed well in comparison with heat-sterilized media.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Sterilization efficiency of a cascaded dielectric barrier discharge

AIMS: To investigate the microbial inactivation efficiency of a newly developed cascaded dielectric barrier discharge (CDBD) set-up against various micro-organisms on polyethylene terephthalate (PET) foils. METHODS AND RESULTS: Inactivation kinetics in dependency of time were produced with air as process gas and test strains like Salmonella serotype Mons, Staphylococcus aureus and Escherichia coli and spores of Bacillus atrophaeus, Aspergillus niger and Clostridium botulinum, which were homogeneously distributed on the sample surface by a spray method. Highest count reduction was observed for the vegetative cells with at least 6.6 log(10) within 1 s. Aspergillus niger was the most resistant test strain with an inactivation rate of about 5 log(10) in 5 s. CONCLUSIONS: For industrial applications it is necessary to evaluate new sterilization methods against a broad range of different micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: CDBD plasma is a fast and effective technology for decontamination of heat sensitive materials in few seconds.     

Laboratory study of several enrichment broths for the detection of Salmonella spp. particularly in relation to water samples

The selectivity and efficiency of several enrichment broths used for the detection of salmonellas were comparatively evaluated under laboratory and environmental conditions. Media with selenite were less efficient in their inhibition of the growth of Gram-positive micro-organisms. Salmonellas grew slowly in tetrathionate broth and in media containing brilliant green. These media inhibited the growth of Salmonella typhi, which grew only in media containing selenite. The results obtained in the experiments with stressed salmonellas indicate that the media selenite F, selenite F with novobiocin, selenite cystine and Rappaport-Vassiliadis (RV/43), in conjunction with the double agar layer technique, showed an optimal efficiency for the detection of stressed salmonellas. When natural samples (freshwater and seawater) were used to evaluate the media, however, those containing malachite green, whether or not supplemented with sodium novobiocin, enhanced the recovery of salmonellas.     

Stainer and Scholte's pertussis medium with an alternative buffer

A comparative study of the Stainer and Scholte chemically defined growth medium (SS) for Bordetella pertussis, and a modification of it (MSS), was made. The Tris buffer which is reported to have adverse effects was replaced in MSS with sodium glycerophosphate. By using generation time as a measure of the growth promoting properties of the media, we found that the MSS was superior to SS for B. pertussis strain CN5612/67, which was used as a vaccine strain in Norway. Additionally by employing the 'dilution to extinction' method we showed that MSS supported B. pertussis growth from smaller inocula than the original SS medium. No significant differences in the quality of vaccines made from organisms grown in the two alternative media were observed in animal tests for potency and safety. The difference in buffers in SS and MSS had no influence on the formation of the agglutinogens, as tested by agglutination with specific factor sera. It was confirmed that liquid media gave a better total expression of the agglutinogens than solid media.     

Characterization of phe B gene encoding catechol 2,3-dioxygenase

A shaken thermal gradient device providing temperatures between 8.3 and 33.5° C was used to investigate the effects of silver ion on the duration of the lag phase and on minimum apparent growth temperatures of Hyphomicrobium spp. grown at 29 and 9°C. With 29°C-grown inocula, at lower temperatures, an increased time was required for growth initiation in the presence of silver ion added at 5 ng ml^-1. With silver ion added at 10 or 100 ng ml^-1, growth initiation was not observed at lower temperatures. With 100 ng ml^-1 added silver ion, this effect also was observed with 9°C-grown inocula. This increased sensitivity to silver ion could limit the ability of Hyphomicrobium spp., and possibly other microbes, to initiate growth and to contribute to microbial functioning in silver-impacted low temperature environments.     

A Rapid Method for the Detection of Non-ionic Surfactant-degrading Micro-organisms

A rapid colorimetric test, involving the use of Dragendorff's reagent, has been developed for the detection of alcohol ethoxylate-utilizing micro-organisms on agar media. A number of putative surfactant-utilizing bacteria were detected using this screening test and their ability to utilize surfactants was confirmed by more detailed growth studies. Suggestions are made concerning the use of this method in enumerating surfactant-degrading micro-organisms in natural environments.