Enzymatic utilization of cotton soap stock

Enzymatic hydrolysis of neutral fat of cotton oil soap stock with a nonspecific lipase produced by Oospora lactis F-500 was designed. The culture liquid and a preparation of enzyme obtained by precipitation with isopropanol from a filtrate of the culture liquid were used. Utilization of cotton oil soap stock as the only source of carbon during cultivation of the fungus was studied. The rate of hydrolysis of soap stock fat strongly depended on the way of biological conversion of cotton oil soap stock. The most effective utilization was observed during cultivation of the fungus in the medium containing soap stock.     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Genetic instability of the short-living ascomycetous fungus <Emphasis Type="Italic">Podospora anserina</Emphasis> induced by prolonged submerged cultivation

Investigation of genetic variability of the short-living filamentous fungus Podospora anserina during its adaptation to conditions of prolonged submerged cultivation has been carried out for the first time. Cultivation of P. anserina under aeration (on a shaker) provides pronounced selective pressure, which makes it possible to obtain isolates with specific features, which are well adapted to cultivation in liquid media and have a life span several times exceeding that of the original strain. Static cultivation did not prevent the ageing of P. anserina. Repeated transfers in the shaker culture resulted in formation of mycelium deprived of the dark pigment melanin and actively producing carotenoids under illumination. The qualitative composition of P. anserina carotenoids was the same as in the closely-related species Neurospora crassa. The features obtained during the shaker cultivation (including changes in the colony morphology and decreased capacity for melanin synthesis) are inherited by their hybrids with the wild type strains, i.e., they resulted from the intragenomic rearrangements occurring during submerged cultivation of the fungus.     

Utilization of substrates during batch growth of Pseudomonas fluorescens on olive oil,lard, and mutton tallow

Summary Similar amounts of cell-associated and cell-free lipase activities were present in log phase cultures of Pseudomonas fluorescens growing on fatty substrates. The rates of triglyceride hydrolysis and fatty acid utilization were balanced as free fatty acids remained at low concentrations in the culture media at all times. The time required for growth initiation and optimal growth rates of P. fluorescens were similar on olive oil, lard and mutton tallow, provided the coalesced solid fats were mechanically dispersed during the initial stages of cultivation. Although olive oil was completely consumed at the end of cultivation, substantial amounts of tri- and diglycerides, presumably bearing saturated acyl residues, remained in lard culture and, to a lesser extent, in mutton tallow culture.     

Formation of organic acids by the fungus Cladosporium resinae in media containing n-alkanes]

The so-called fungus Cladosporium resinae that often occurs in oil fuels ane increases their acidity grows well at the expense of n-alkanes from C11 to C16. On the n-alkane containing media the fungus grows slowly and only under the stationary conditions. During the fungal cultivation on the media containing n-dodecane or kerosene the culture liquid shows acetic acid and other fatty acids, ketoacids (pyruvic and alpha-ketoglutaric) as well as citric and isocitric acids that dominate among nonvolatile acids. Upon nitrogen deficiency in the medium and comparatively good aeration the content of citric acids increases. The culture liquid of the fungus devoid from the mycelium and nonutilized n-alkanes can be used a a nutrient medium for different microorganisms.     

Use of the Fungus Panus tigrinus in the Manufacture of Pressed Materials from Cotton Plant Waste

Changes in the chemical composition of cotton plant stems used as a substrate for solid-phase cultivation of the fungus Panus tigrinus were studied, as well as the effect of these changes on properties of pressed materials made of these stems. During the first 3 days of growth, the fungus better consumed cellulose; then, the rate of cellulose consumption was comparable with that of lignin. The intensity and pattern of these changes depended on the age of the inoculum. The rate of cotton plant waste biodegradation was higher when a 3-day-old inoculum was used. Pressed materials made of the raw material treated with a 3-day-old inoculum of P. tigrinus for 2–3 days displayed better characteristics.     

The Effect of Nutrient Medium Composition on Peroxidase Biosynthesis by Bazidiomycete Pleurotus ostreatus,strain UzBI-I105

Production of extracellular peroxidase was studied during the submerged cultivation of xylotrophic bazidiomycete Pleurotus ostreatus, strain UZBI-I105 in nutrient media with lignocellulosic wastes, exhausted cottonseed oil cake, cotton stalks, rice husks, or ambary hemp. Enzyme production was enhanced 3–5 times in the presence of exhausted cottonseed oil cake extract in the nutrient medium. The dynamics of peroxidase production in various media was investigated.     

从米糠油二次皂脚中制取谷维素的研究

本课题的目的是从米糠油二次皂脚中制取谷维素。我们采用新工艺、新技术多相分步结晶,应用于实践,优选条件,谷维素的制取率为米糠油二次皂脚的1.9％,谷维素含量为98％。     

Chemical and Physical Factors Influencing Growth of Phytophthora parasitica var. nicotianae on Vegetable Oils

Factors influencing the growth of Phytophthora parasition var. nicotiona on vegetable oil liquid media were studied. This fungus grew poorly on oleic acid, triolein, and tristearin. Cholesterol and α-tocopherol reversed the toxicity of oleic acid. Growth was often stimulated by addition of mineral oil to a glucose medium, and sterols dissolved in mineral oil frequently stimulated grwoth. Shaking produced a 5-fold or more increase in growth on either glucose or cottonseed oil media. Cholesterol (20 μ/ml further increased growth on glucose meidum in shake culture by at least 100%. Growth was usually better at pH 6.7 than at 5.4 or 7.7. Growth in shake culture on a glucose medium supplemented with cholesterol and α-tocopherol approached that occuring on vegetable oil media.     

Antifungal Activity of the Essential Oil of Basil (Ocimum basilicum)

The antifungal and fungicidal effects of two chemotypes of basil (Ocimum basilicum) oil and its major individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungus Botrytis fabae was reduced significantly by both the methyl chavicol chemotype oil and the linalol chemotype oil, and the major individual components of the oils all reduced fungal growth, with methyl chavicol, linalol, eugenol and eucalyptol reducing growth significantly. Combining the pure oil components in the same proportions as found in the whole oil led to very similar reductions in fungal growth, suggesting that the antifungal effects of the whole oils were due primarily to the major components. When the fungus was exposed to the oils in liquid culture, growth was reduced by concentrations considerably smaller than those used in the Petri dish studies. Botrytis fabae and the rust fungus Uromyces fabae were also controlled in vivo, with the whole oils of both chemotypes, as well as pure methyl chavicol and linalol, reducing infection of broad bean leaves significantly. Most effective control of fungal infection was achieved if the treatments were applied 3 h postinoculation.     

Comparative Removal of Bentazon by Ganoderma lucidum in Liquid and Solid State Cultures

Bentazon removal by Ganoderma lucidum cultured in liquid and solid state conditions was compared in this work. In solid state cultures, the fungus produced both ligninolytic enzymes, namely laccase and Mn peroxidase. In liquid cultures, the main ligninolytic enzyme produced was laccase. In both types of cultures bentazon improved the production of laccase without significant alteration in the production of Mn peroxidase. In solid state cultures, where high levels of both laccase and Mn peroxidase activities were found, the fungus was more resistant to the action of the herbicide (50 mM in solid state cultures against 20 mM in liquid cultures) and more efficient in removing bentazon (90% removal against 55% in liquid cultures after 10 days of cultivation). Furthermore, the solid state culture filtrates were more efficient in the in vitro degradation of bentazon than the liquid culture filtrates. These observations suggest that both enzymes, laccase and Mn peroxidase, are involved in bentazon degradation. The results further suggest that solid state cultures of Ganoderma lucidum could be useful in strategies designed to reduce environmental contamination by bentazon.     

Hydroxamate siderophores of Scedosporium apiospermum

Scedosporium apiospermum is an emerging pathogen colonizing the airways of patients with cystic fibrosis and causing severe infections in immunocompromised hosts. In order to improve our knowledge on the pathogenic mechanisms of this fungus, we investigated the production of siderophores. Cultivation on CAS medium and specific assays for different classes of siderophores suggested the secretion of hydroxamates. A maximal production was obtained by cultivation of the fungus at alkaline pH in an iron-restricted liquid culture medium. Siderophores were then extracted from the culture filtrate by liquid/liquid extraction, and separated by reverse phase high performance liquid chromatography. Two siderophores, dimerumic acid and N ^α-methyl coprogen B, were identified by electrospray ionization-mass spectrometry and MS–MS fragmentation. Finally, comparison of various strains suggested a higher production of N ^α-methyl coprogen B by clinical isolates of respiratory origin. Studies are initiated in order to determine the potential usefulness of these siderophores as diagnostic markers of scedosporiosis.     

Antifungal antibiotic of the basidiomyceteOudemansiella mucida

A pure culture of the basidiomyceteOudemansiella mucida, isolated by the explantation technique, produces an intracellular antifungal antibiotic. The relatively simple basic growth requirements of the producing strain permit its cultivation on agar and liquid nutrient media. During submerse cultivation in medium containing glucose and cornsteep on a shaker apparatus or in a fermentor, the fungus produces 300–600 μg antibiotic/ml. The main growth characteristics of a mycelial culture of the producing strain and details of its transfer to submerse conditions are described. Dedicated to Academician Ivan Málek on the occation of his 60th birthday     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.     

The effect of acetic acid concentration on the growth and production of gamma-linolenic acid byMucor circinelloides CBS 203.28 in fed-batch culture

The effect of different initial acetic acid concentrations on the growth of and lipid and gamma-linolenic acid (GLA) production byMucor circinelloides CBS 203.28 was determined in a 14 litre stirred tank reactor operated in a fedbatch, pH-stat mode with acetic acid as carbon source and pH titrant. Increased acetic acid concentrations in the culture resulted in a significant increase in the crude oil content of the biomass. By contrast, all the other parameters such as the biomass concentration, GLA and oil yield on acetic acid, the GLA content of the biomass and oil, the growth rate and volumetric rate of GLA production decreased with an increase in acetic acid concentration. The best results were obtained with acetic acid at 2 g/1, which gave 39.8 mg GLA/g biomass and 15.6% GLA in the neutral lipid fraction, amounting to 340 mg GLA/1 culture. A decrease in the glyco- and phospho-lipid fractions during the cultivation coincided with an increase in the neutral lipid fraction. The GLA content of the biomass remained within rather narrow limits of 3.5% to 4% of the biomass, irrespective of the oil content of the biomass. The fatty acid profile was not greatly affected by the acetic acid concentration. The hyphae of the fungus were characterized by the accumulation of large intracellular oil droplets and some septa delimited the hyphae.     

Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus

Transmission of Pepino mosaic virus (PepMV) by the fungal vector Olpidium virulentus was studied in two experiments. Two characterized cultures of the fungus were used as stock cultures for the assay: culture A was from lettuce roots collected in Castellón (Spain), and culture B was from tomato roots collected in Murcia (Spain). These fungal cultures were maintained in their original host and irrigated with sterile water. The drainage water collected from irrigating these stock cultures was used for watering PepMV‐infected and non‐infected tomato plants to constitute the acquisition–source plants of the assay, which were divided into six different plots: plants containing fungal culture A (non‐infected and PepMV‐infected); plants containing fungal culture B (non‐infected and PepMV‐infected); PepMV‐infected plants without the fungus; and plants non‐infected either with PepMV and the fungus. Thirty‐six healthy plants grouped into six plots, which constituted the virus acquisition–transmission plants of the assay, were irrigated with different drainage waters obtained by watering the different plots of the acquisition–source plants. PepMV was only transmitted to plants irrigated with the drainage water collected from PepMV‐infected plants whose roots contained the fungal culture B from tomato with a transmission rate of 8%. No infection was detected in plants irrigated with the drainage water collected from plots with only a fungus or virus infection. Both the virus and fungus were detected in water samples collected from the drainage water of the acquisition–source plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.     

Effect of culture conditions on mycelial growth, antibacterial activity, and metabolite profiles of the marine-derived fungus Arthrinium c.f. saccharicola

The effects of culture conditions and competitive cultivation with bacteria on mycelial growth, metabolite profile, and antibacterial activity of the marine-derived fungus Arthrinium c.f. saccharicola were investigated. The fungus grew faster at 30°C, at pH 6.5 and in freshwater medium, while exhibited higher antibacterial activity at 25°C, at pH 4.5, 5.5, and 7.5, and in 34 ppt seawater medium. The fungus grew faster in a high-nitrogen medium that contained 0.5% peptone and/or 0.5% yeast extract, while exhibiting higher bioactivity in a high-carbon medium that contained 2% glucose. The fungal growth was inhibited when it was co-cultured with six bacterial species, particularly the bacterium Pseudoalteromonas piscida. The addition of a cell free culture broth of this bacterium significantly increased the bioactivity of the fungus. Metabolite profiles of the fungus revealed by gas chromatography (GC)-mass spectrometry showed clear difference among different treatments, and the change of relative area of three peaks in GC profile followed a similar trend with the bioactivity variation of fungal extracts. Our results showed clear differences in the optimal conditions for achieving maximal mycelial growth and bioactivity of the fungus, which is important for the further study on the mass cultivation and bioactive compounds isolation from this fungus.     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C6 to C14 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and 13C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5-87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C8 and C10 monomers as longer and smaller monomers were incorporated less efficiently.     

Adaptation of the white-rot basidiomycete Panus tigrinus for transformation of high concentrations of chlorophenols

During feed-batch cultivation of the white-rot fungus Panus tigrinus in a 5-l bioreactor on N-limited medium, 100, 200, 500, 1,000 and 2,000 mg 2,4,6-trichlorophenol (2,4,6-TCP) l^–1 were added sequentially after 90% removal of the previous portion of the toxicant. The addition of 500 mg 2,4,6-TCP l^–1 without preliminary adaptation killed the culture. The addition of 300 mg 2,4,6-TCP l^–1 without prior adaptation resulted in its slower removal than removal of 2,000 mg 2,4,6-TCP l^–1 by this adapted culture. After adaptation of P. tigrinus to 2,4,6-TCP in a 72-l bioreactor, the mixture of 2,4-dichlorophenol, 2,4,6-TCP, and pentachlorophenol, each at 500 mg l^–1, was totally removed over 3 weeks. No lignin peroxidase activity was found in the course of cultivation of the fungus. Laccase activity was suppressed by addition of 2,4,6-TCP. Mn-peroxidase was found to be responsible for transformation of the chlorophenols. As final products of the process, several newly formed aromatic polymers, both chlorinated and non-chlorinated, were found in the culture liquid. Electronic Publication