Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

Internal substrate concentrations during biotransformation of octanoic acid into 2-heptanone by spores ofPenicillium roquefortii

Internal substrate concentrations were monitored during biotransformation of octanoic acid into 2-heptanone by spores ofPenicillium roquefortii. The fatty acid rapidly enters the spores in its undissociated form, and a Collander-type relation shows that it strongly accumulates in the spore wall and membrane; this accumulation is reversible. The reaction takes place with cytoplasmic substrate concentrations that quickly fall to zero, and the process is limited by octanoic acid penetration into the cells. This entry is accompanied by proton efflux and involves an active transport process with a H⁺-ATPase system that exhibits Michaelian behavior. The driving force is postulated to be pH, which takes a value set by the initial substrate concentration through the stoichiometry of the H⁺/octanoic acid exchange.     

Screening ofPenicillium species for the production of extracellular glucose oxidase

During screening ofPenicillium species for extracellular glucose oxidase production, a strain ofPenicillium variabile (P16) was selected which released high activities of enzyme into the culture medium. Maximum activity (5.49 U/ml) was after 96 h cultivation in shake-flask culture.M. Petruccioli is with the Dipartimento di Biologia, Difesa e Biotecnologie Agro-Alimentari, University of Basilicata, Via N. Sauro 85, 1-85100 Potenza, Italy. M. Ceccarelli and F. Federici are with the Dipartimento Agrobiologia e Agrochimica, University of Tuscia, Via S.C. de Lellis, 1-01100 Viterbo, Italy.     

De novo production of (+)-aristolochene by sporulated surface cultures of Penicillium roqueforti

The de novo production of the fungal metabolite, (+)-aristolochene by sporulated surface cultures of Penicillium roqueforti is reported for the first time. The biosynthesis of fungal volatiles by various sporulated surface cultures was monitored by solid phase micro-extraction (SPME). When comparing malt extract agar with sabouraud dextrose agar, the highest yield of the fungal metabolite (0.04 mg/ml of culture) was obtained with the latter medium. The biosynthesis of (+)-aristolochene showed a maximum during the fourth day after inoculation.     

Mycotoxin production profiles ofPenicillium vulpinum (Cooke & Massee) Seifert & Samson strains

Mycotoxins produced by seven strains ofPenicillium vulpinum (formerlyPenicillium claviforme) isolated from different sources were studied. The strains were characterized by specific profiles of secondary metabolites and produced mycotoxins of different structural types. In addition to toxins already known for this fungal species (patulin, roquefortine, 3,12-dihydroroquefortine, oxalin, viridicatin, cyclopenin, and α-cyclopiazonic acid), the strains studied also produced indolyl-3-acetic acid, griseofulvin, meleagrin, and cyclopeptin.     

Comparison of growth characteristics and roquefortin C production of<Emphasis Type="Italic">Penicillium roqueforti</Emphasis> from blue-veined cheese

The properties of 21 isolates ofPenicillium roqueforti from just as many commercial blue-veined cheeses, purchased from the Argentinean market (domestic and imported products) were comparatively examined. Isolates were investigated for their ability to grow at different temperatures, pH values and concentration of NaCl, as well as for their proteolytic and lipolytic activities, respectively. The potential of these strains to produce roquefortin in vitro, and the actual levels of roquefortin in 10 of these cheeses were analysed by TLC. All strains showed similar growth properties in aspects of salt concentration and pH-value of the medium, and all grew well at 10 °C. Only four strains showed proteolytic activity on casein agar, while all strains were lipolytic on trybutirin agar. After incubation at 25 °C for 16 days, all strains produced roquefortin in Yeast Extract Sucrose (25.6–426.7 μg/g) and in reconstituted (10%) sterile skim milk (26.9–488 μg/g). Roquefortin at >0.1 μg/g was also found in 9 out of 10 analysed samples of blue-veined cheeses (8 from Argentine, 1 from Spain), with a maximum value 3.6 μg/g. During the ripening process of blueveined cheese, production of roquefortin seems to be unavoidable. Care should be taken to select strains with low toxin production characteristics, to minimize potential health risks. Roquefortin C production byP. roqueforti in vitro was not correlated with roquefortin C levels found in cheese. Financial support: Research grants from the National University of Quilmes, Argentina     

A chemically-defined medium for production of compactin by Penicillium citrinum

A mutant strain of Penicillium citrinum grown in a chemically-defined production medium, yielded 145 mg compactin l^–1. The medium also facilitated spectrophotometric analysis of compactin. Addition of KH₂PO₄in the production medium did not increase the compactin production, while addition of a surfactant, Tween 80, increased compactin to 175 mg l^–1. Inoculation with 10⁷ spores ml^–1 and initial pH of 6.5–7 were the most suitable for compactin production.     

Identification ofPenicillium sp. NR 6564 and taxonomic notes onP. janthinellum

The fungal strain NR 6564, which produces a new series of antifungal antibiotics, Ro 09-1470 and its congeners, was identified asPenicillium janthinellum from its cultural and morphological properties. The identification was confirmed by analysis of the ubiquinone system and DNA-DNA hybridization.     

Phosphatase activity ofPenicillium citrinum submerged batch cultures and its relationship to fungal activity

Acid and alkaline phosphatase activities were evaluated using batch fermenter cultues ofPenicillium citrinum, an organism used in studies of fungal functioning in soil. Fungal activity was assessed by monitoring rates of O₂ utilization, glucose utilization, dry weight changes over time, and lengths of FDA-stained hyphae. At low growth rates (7 g dry wt increases·h^–1·ml^–1) and low culture activity, phosphatase activity at both pH 8.5 and 5.5 tended to decrease with culture age, with the exception that phosphatase activity at pH 8.5 peaked during early stationary phase. At higher growth rates (25 g dry wt increase·h^–1·ml^–1) and high culture activity, phosphatase activity tended to remain constant throughout the course of the experiment. The relationship between phosphatase activity and other measures of fungal activity was consistent only at low growth rates for acid phosphatase. These results suggest that phosphatase measurements will be of limited utility in assessing activity, except at low growth rates.     

Taxonomy ofPenicillium nalgiovense isolates from mould-fermented sausages

A large number ofPenicillium nalgiovense isolates from mould fermented sausages and the ex type culture were examined for characters of morphology, physiology and production of secondary metabolites. To separate biotypes within theP. nalgiovense species, the data obtained were evaluated using multivariate statistical methods. The macromorphological characters of the ex type culture and isolates from meat products appeared to be distinctive. The ex type culture is characterized by a brown reverse on both Czapek yeast extract and malt extract agar while the isolates from meat products have a yellow to orange reverse. Proteolytic and/or lipolytic activity was demonstrated by 75% of the examined cultures and all of them demonstrated ability to utilize lactate as sole carbon source. Growth on creatine sucrose agar was very inhibited and acid production was absent or very weak. TLC analysis showed production of three unknown secondary metabolites that constituted the characteristic profile. HPLC analysis showed production of only three known secondary metabolites; chrysogine (96%), nalgiolaxin and nalgiovensin (9%). The ex type culture produced nalgiolaxin and nalgiovensin but not chrysogine. The chemometric evaluation showed thatP. nalgiovense isolates from meat products from a homogenous species, which can not be divided into biotypes. The only indication of grouping, beside a separation of the ex type culture, was related to the conidium colour (white, turquoise or grey green). The examinedP. nalgiovense isolates showed some resemblance (morphologically and chemically) toP. chrysogenum.     

Pectinase production by Penicillium frequentans

High activities of extracellular pectinase with viscosity-diminishing and reducing groups-releasing activities were produced by Penicillium frequentans after 48 h at 35°C, in agitated cultures supplemented with 0.5% citrus pectin and initial pH of 2.5. Under these conditions the fungus also produced high activity of pectinesterase. At an initial pH of 7.0 or 8.0, pectin lyase activity was also detected. Enzyme activity releasing reducing sugars was more stable at 50°C than viscosity-diminishing activity. Both activities were maximal at pH 2.5 to 5.2 and at 55°C.The authors are from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café, s/n^o, Bairro Monte Alegre, 14.049 Ribeirão Preto, S.P., Brazil.     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

A new minimal synthetic medium for germ-tube production in Candida albicans

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.     

Isolation and characterization ofPenicillium funiculosum mutants with enhanced glucose oxidase production

After the mutagenesis ofPenicillium funiculosum with UV light andN-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5–153% higher (in a medium with glucose) and 4–83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutantP. funiculosum NMU95-132 was chosen for further selection work.     

Aroma compounds produced by Kluyveromyces marxianus in solid state fermentation on a packed bed column bioreactor

Studies were carried out for the production of aroma compounds by Kluyveromyces marxianus grown on cassava bagasse in solid state fermentation using packed bed reactors, testing two different aeration rates. Respirometric analysis was used to follow the growth of the culture. Headspace analysis of the culture by gas chromatography showed the production of 11 compounds, out of which nine were identified. Ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Lower aeration rate (0.06l h^–1 g^–1 of initial dry matter) increased total volatile (TV) production and the rate of production was also increased at this aeration rate. Using an aeration rate of 0.06l h^–1 g^–1 maximum TV concentrations were reached at 24 h and at 40 h with 0.12l h^–1 g^–1.     

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L^–1; Ca-carbonate concentration, 15 g L^–1; temperature, 28°C and aeration rate, 4 VV^–1 min^–1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.     

Mass production of Glomus mosseae spores

Five crops inoculated with Glomus mosseae were grown for 10 weeks and the development of mycorrhizal infection and sporulation were assessed. Infected roots from pot cultures of different ages were used to examine the host effect on the development of mycorrhizae. The effectiveness of each host was assessed by measuring spore numbers. For all hosts, the percentage of root length infected increased rapidly up to 10 weeks after sowing. Infectivity of root inocula increased with increasing percentage of root length infected with the inoculum for all crops, except where large numbers of mature spores (1755) had been produced on barley. The highest spore numbers were achieved in the rhizosphere of barley plants, followed by chickpea and beans. The lowest spore numbers were found in the rhizosphere of corn and okra plants. The type of the crop as well as the harvest date greatly influenced the size of the spore population and the extent of root colonization of G. mosseae.     

Cellulose production by Gluconacetobacter hansenii in a medium containing ethanol

The addition of 1% (v/v) ethanol to the basal medium inhibited growth of Gluconacetobacter hansenii but decreased the numbers of non-cellulose producing cells. Cellulose production increased 1.7 times to approx. 2.5 g l(-1) and showed a pattern of mixed growth-associated production. Microbial cells produced rigid pellicle-type bacterial cellulose as the shell of a large lump of bacterial cellulose like a static culture. The inoculum cultivated for 3 d maintained cellulose production by the fifth batch.     

Riboflavin excretion byPachysolen tannophilus grown in synthetic medium supplemented withd-xylose

Pachysolen tannophilus excreted riboflavin (12.7 m g/ml) into a synthetic medium withd-xylose as carbon source, when the pH was below 2.0. The addition of glucose enhanced the quantity of riboflavin excreted. The greatest riboflavin production was at pH 1.6 after 5 days at 30°C.