Nutrition of tempe moulds

The nutritional requirements in vitro of Amylomyces rouxii NRRL 3160, Rhizopus arrhizus NRRL 1469, R. arrhizus NRRL 1526, R. oligosporus NRRL 2710, R. oligosporus NRRL 5905, R. oligosporus NRRL A-10457, R. oryzae IMI 215407, R. oryzae NRRL 3563 and R. stolonifera NRRL A-2293 were investigated. All strains grew in glucose-ammonium-salts medium without addition of vitamins. None utilized, as sole carbon and energy source, raffinose or stachyose, the main flatulence-associated oligosaccharides in soybean. All the strains, except Rhizopus oryzae IMI 215407, utilized one or more long chain fatty acids as sole sources of carbon and energy but only the R. arrhizus strains, R. oryzae NRRL 3563 and R. stolonifera , were able to use glycerol as sole carbon and energy source. Phytic acid was used neither as a source of carbon nor of phosphate by any strain.     

Physico-chemical and microbiological analyses of fermented corn cob, rice bran and cowpea husk for use in composite rabbit feed

An experiment was conducted to evaluate the effect of fermentation on the proximate composition of corn cob, rice bran and cowpea husk for use in composite rabbit feed formulations. The test ingredients were moistened with tap water and allowed to ferment naturally at room temperature. During fermentation, samples of the fermenting materials were extracted at zero, 24 and 48 h for physico-chemical and microbiological analyses using standard procedures. The microorganisms associated with the fermenting materials were identified as Rhizopus oligosporus, Aspergillus oryzae, Aspergillus niger, Rhodotorula, Geotrichum candidum, Candida albicans, and Saccharomyces cerevisiae. Two (R. oligosporus and S. cerevisiae) out of microorganisms present were used as starter cultures to ferment the test ingredients and the fermented products were then analyzed. From the results obtained S. cerevisiae enhanced the protein and fat contents while R. oligosporus was able to degrade the fiber significantly.     

Yeasts from U.S.A. soils

Summary Investigations of yeasts from 38 U.S.A. soils samples show the occurrence of 22 different species (16 sporogenous and 6 asporogenous).The most widespread species were Pichia fermentans and Hansenula anomala. The other isolated species were Saccharomyces ellipsoideus, Torulaspora delbrueckii, Pichia membranefaciens, Saccharomyces smittii, Saccharomyces carlsbergensis, Saccharomyces uvarum, Torulaspora rosei, Zygosaccharomyces rouxii, Zygosaccharomyces n. sp., Hansenula saturnus, Hansenula californica, Hansenula n. sp., Hansenula suaveolens, Debaryomyces n. sp., Torulopsis glabrata, Torulopsis n. sp. Candida tropicalis, Candida robusta, Rhodotorula glutinis, Trichosporon cutaneum and Trichosporon cutaneum var. multisporus.     

Outbreak of Rhizopus Rot Caused by Rhizopus oryzae on Seedlings of Grafted Cucumber on Pumpkin Rootstock in South Korea

In 2013, an outbreak of Rhizopus rot caused by Rhizopus oryzae occurred in cucumber grafted onto pumpkin rootstock sampled from seedling farms in Changnyeong, South Korea. A water‐soaked appearance of the affected tissue was the first symptom of this soft fungal rot in the seedling stems of grafted cucumber. Lesions at the graft sites softened and rapidly, rotted, and turned brown or dark brown. Measurements and taxonomic characteristics were most similar to R. oryzae. DNA sequencing and phylogenetic analysis of the internal transcribed spacer rRNA gene region confirmed that the isolates were indeed R. oryzae. Koch's postulates were supported by pathogenicity tests conducted on healthy plants. Based on mycological characteristics, pathogenicity test, and molecular analysis, the causal fungus was identified as R. oryzae Went & Prinsen Geerligs. To our knowledge, this is the first report of Rhizopus rot caused by R. oryzae in seedlings of grafted cucumber on pumpkin rootstock in South Korea.     

Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe)

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.     

Screening for extracellular enzymes of fungi from manufacturing wastes

Summary Three different types of manufacturing waste, dried soybean meal after oil extraction, sesame seed meal after oil extraction, and brewery spent grain from beer production, were plated for fungi with malt extract agar and potato-dextrose agar. Brewery spent grain gave the highest fungal counts and sesame seed meal the least. The more abundant fungi were:Absidia sp.,Aspergillus flavus andAsp. ochraceus in soybean meal;Asp. flavus, Rhizopus oryzae and yeast in sesame seed meal, andAsp. restrictus and yeast in brewery spent grain. Nine fungal isolates were screened for extracellular amylase, protease, cellulase and lipase activity.Aspergillus flavus andFusarium oxysporum were good producers of these enzymes, butAbsidia sp.,Aspergillus ochraceus and the yeasts showed a limited range of enzyme activities.Aspergillus restrictus, an osmophilic fungus, had no activity for the four enzymes.

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Resumen En placas de agar de patata y dextrosa (PDA) y de extracto de malta se sembraron tres tipos de residuos industriales: pulpa de soja, una vez realizada la extracción de aceite, pulpa de semillas de sésamo, después del mismo proceso, y residuos de cereales de fábrica de cerveza. El mayor número de hongos se obtuvo a partir de los residuos de cereales y el menor a partir de la pulpa de semillas de sésamo. Los hongos más abundantes fueron:Absidia sp.,Aspergillus flavus yAsp. ochraceus en pulpa de soja,Asp. flavus, Rhizopus oryzae y levaduras en pulpa de semillas de sésamo, y,Asp. restrictus y levaduras en residuos de fábrica de cerveza. Se estudiaron las actividades extracelulares de amilasa, proteasa, celulasa y lipasa.Aspergillus flavus y Fusarium oxysporum eran buenos productores de estos enzimas, sin embargo,Absidia sp.,Asp. ochraceus y las levaduras mostraron una limitada variedad de actividades enzimáticas.Aspergillus restrictus, hongo de tipo osmofílico, no mostró actividad para niguno de los cuatro enzimas estudiados.

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Résumé Les champignons présents dans trois sortes de déchets industriels (tourteaux délipidés de soja et de sésame, et graines résiduelles de brasserie) ont été cultivés sur milieux gélosés à l'extrait de malt et à la dextrose de pomme de terre. Les graines résiduelles de brasserie ont donné les numérations de champignons les plus élevées, et le tourteau de sésame les plus faibles. Les champignons les plus abondants sont: dans le tourteau de soja,Absidia sp.,Aspergillus flavus etAsp. ochraceus; dans le tourteau de sésame,Asp. flavus, Rhizopus oryzae et la levure; et dans les graines résiduelles de brasserie,Asp. restrictus. Neuf souches de champignons ont été isolées et examinées en ce qui concerne la production extracellulaire des activités amylase, protéase, cellulase et lipase.Aspergillus flavus etFusarium oxysporum sont de bons producteurs de ces enzymes. Par contre,Absidia sp.,Aspergillus ochraceus et les levures ont des activités enzymatiques faibles. Le champignon osmophileAspergillus restrictus ne produit aucun des quatres enzymes.

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Paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–18 August 1985     

Cellulose-decomposing fungi of salt marshes in Egypt

Seventy-five species and three varieties which belong to thirty-four genera were identified from 74 soil samples collected from salt marshes in Egypt. The most frequent fungi wereAspergillus fumigatus, Aspergillus niger, Cladosporium herbarum andAlternatia alternata, followed byAspergillus terreus,Curvularia spicifera andPenicillium notatum. Six genera were of moderate occurrence:Penicillium, Futarium, Curvularia, Rhizopus, Stachybotrys, andChaetomium. Five genera were of low occurrence:Paecilomyces, Oephalosporium, Epicoccum, Mucor andMyrothecium.     

Synthesis of ethyl phenylacetate by lyophilized mycelium of Aspergillus oryzae

Lyophilized mycelia of Aspergillus oryzae CBS 102.07, Aspergillus oryzae MIM, Rhizopus oryzae CBS 112.07, Rhizopus oryzae CBS 391.34, Rhizopus oryzae CBS 260.28 and Rhizopus oryzae CBS 328.47 were tested in this study to select the best biocatalysts for ethanol acylation with phenylacetic acid. The mycelium-bound carboxylesterase activity of A. oryzae MIM, which exhibited the best performances, was initially investigated at 50°C, either in 0.1 M phosphate buffer or in n-heptane to catalyse the hydrolysis or the synthesis, respectively, of ethyl phenylacetate. The results in terms of product and substrate concentrations versus time were used to estimate the maximum molar conversions at equilibrium, the equilibrium constants, and the times needed to reach half maximum conversions, thus providing sufficient information about this biotransformation. The values of the apparent equilibrium constants, estimated at 20°C<T<50°C, were finally used to estimate the thermodynamic parameters of ethanol acylation by this biocatalyst.     

Use of mono- and diacylglycerol lipase as immobilized fungal whole cells to convert residual partial glycerides enzymatically into fatty acid methyl esters

The accumulation of partial glycerides such as monoglyceride (MG) and diglyceride (DG) is one of the rate-limiting steps in plant oil methanolysis catalyzed by Rhizopus oryzae producing triacylglycerol lipase. To convert partial glycerides efficiently into their corresponding methyl esters (MEs), we attempted to use a mono- and diacylglycerol lipase (mdlB) derived from Aspergillus oryzae. By considering cost efficiency, R. oryzae and recombinant mdlB-producing A. oryzae were immobilized independently within polyurethane foam biomass support particles and directly utilized as a whole-cell biocatalyst. The mdlB-producing A. oryzae effectively exhibited substrate specificity toward MG and DG and was then used for the methanolysis of intermediate products (approximately 82% ME), which were produced using R. oryzae. In the presence of 5% water, the use of mdlB-producing A. oryzae resulted in less than 0.1% of MG and DG, whereas a considerable amount of triglyceride was present in the final reaction mixture. On the basis of these results, we developed a packed-bed reactor (PBR) system, which consists of the first column with R. oryzae and the second column containing both R. oryzae and mdlB-producing A. oryzae. Ten repeated-batch methanolysis cycles in the PBR maintained a high ME content of over 90% with MG and DG at 0.08–0.69 and 0.22–1.45%, respectively, indicating that the PBR system can be used for long-term repeated-batch methanolysis with partial glycerides at low levels. The proposed method is therefore effective for improving enzymatic biodiesel production.     

Microbial catalyzed esterification of primary and secondary alcohols in organic solvent

Summary Microbial esterification of primary and secondary short chain alcohols with butyric acid in organic solvent has been studied. A screening for 2-octylbutyrate hydrolysis between microorganisms belonging to different genera allowed the selection of 12 microbial strains able to hydrolyze this substrate. The potential of these microorganisms in catalyzing ester formation was checked for various 1- and 2-alkylbutyrate derivatives:Rhizopus delemar,Rhizopus oryzae andSarcina lutea promoted both 1- and 2-alkylbutyrate synthesis with almost complete molar conversion of the primary alcohols, whileAspergillus niger andYarrowia lipolytica only catalyzed 1-alkanol esterification.     

Efficient and Direct Fermentation of Starch to Ethanol by Sake Yeast Strains Displaying Fungal Glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.     

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg     

Examination of several selected fungi by scanning electron microscope

Scanning electron microscopy with high resolution and great depth of focus has been known to be able to show the fine features of surface ornamentation of fungi, and with this technique we could obtain the interesting results about the surface structures of the selected fungi,Penicillium chrysogenum, Aspergillus niger, Aspergillus oryzae andCandida utilis.     

Solid state fermentation for the production of lipolytic fungal enzymes

The production of lypolitic moulds in solid state fermentation was studied.Aspergillus oryzae andRhizopus oryzae were grown on rice hulls using different media. Tween 80 and olive oil were employed as main carbon sources; the whole solid cultures were lyophilised and employed in heptane for catalysing the formation of various geranyl esters with molar conversions ranging from 40 to 95%, under optimised conditions, starting from 50 mM geraniol and equimolar amount of the acid.     

Some new hosts to leptosphaerulina McAlp from India-II

Summary In this paper seven new and economically important hosts to the genusLeptosphaerulina are reported and the morphology of the fungi described.Leptosphaerulina ricini onRicinus communis, L. brassicae onBrassica nigra, B. oleracea var.capitata andB. oleracea var.botrytis; Leptosphaerulina australis McAlp. onCyamopsis tetragonoloba, Phaseolus sp., andLeptosphaerulina oryzae comb. nov. onOryza sativa.     

Three new species of Eupenicillium from soil

Three new species ofEupenicillium are described from soils from the western United States. They areE. idahoense sp. nov.,E. tularense sp. nov., andE. lasseni sp. nov. The conidial stages are new species ofPenicillium.

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Zusammenfassung Drei neue Arten vonEupenicillium vom Erdboden der westlichen Vereinigten Staaten sind beschrieben worden. Diese sind:E. idahoense, sp. nov.,E. tularense, sp. nov. undE. lasseni sp. nov. Die Konidialformen sind neue Arten vonPenicillium.

     

Agar plate growth studies of Rhizopus oligosporus and Aspergillus oryzae to determine their suitability for solid-state fermentation

Summary Colony radial growth rates of Rhizopus oligosporus and Aspergillus oryzae were compared under various conditions on agar plates containing cassava starch. Both organisms grew well on cassava starch as their sole source of carbon and energy, although growth was stimulated by the addition of yeast extract and peptone. Neither organism utilized ungelatinized starch effectively. The optimum initial pH for R. oligosporus was 7, although good growth was obtained at pH 5 when ammonium sulfate was partially replaced by urea. A. oryzae grew well over a range of initial pH values from 5 to 8. Growth of R. oligosporus was inhibited by NaCl concentrations above 0.5% (w/v) while A. oryzae was unaffected up to 4% NaCl. The best colony radial growth rate obtained for R. oligosporus was 1.01 mm/h, which was far superior to that obtained for A. oryzae (0.29 mm/h). R. oligosporus was chosen as the more suitable organism for future studies of the protein enrichment of cassava by solid-state fermentation.     

Experimental cerebral zygomycosis in alloxan-diabetic rabbits: variation in virulence among zygomycetes

We investigated the potential of 33 different zygomycete isolates to cause cerebral disease following the intranasal instillation of their spores into ketotic rabbits with alloxan induced diabetes. The isolates represented six thermotolerant species of Rhizopus (R. arrhizus, R. chinensis, R. microsporus, R. oligosporus, R. oryzae, and R. rhizopodiformis), Absidia corymbifera, Cunninghamella bertholletiae, and Rhizomucor pusillus. All 13 isolates of the thermotolerant Rhizopus species proved to be cerebral pathogens as confirmed by culture and histopathology. One isolate of R. oligosporus and one isolate of R. rhizopodiformis, however, were less pathogenic than isolates of other Rhizopus species tested. Cerebral pathogenicity was noted with 2 of 5 isolates of Rh. pusillus and only 1 of 13 A. corymbifera isolates. Two thermotolerant C. bertholletiae cultures, recovered from human lesions, did not cause either cerebral or pulmonary disease in ketotic rabbits. The incidence of pulmonary zygomycosis caused by the isolates of the species of the four genera under study was as follows: Rhizomucor 24%, Rhizopus 22%, Absidia 9%, and Cunninghamella 0%. This study confirms the pathogenic potential of the thermotolerant species of Rhizopus to cause cerebral zygomycosis in ketotic diabetic rabbits and also revealed the potential of Rh. pusillus and A. corymbifera occasionally to cause the same disease in animals and humans.     

New anamorphic yeast species: Candida infanticola sp. nov., Candida polysorbophila sp. nov., Candida transvaalensis sp. nov. and Trigonopsis californica sp. nov

Three new species of Candida and a new species of Trigonopsis are described based on their recognition from phylogenetic analysis of gene sequences from large subunit ribosomal RNA, ITS1/ITS2 rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II. Candida infanticola sp. nov. (type strain NRRL Y-17858, CBS 7922) was isolated from the ear of an infant in Germany and is closely related to Candida sorbophila. Candida polysorbophila sp. nov. (type strain NRRL Y-27161, CBS 7317) is a member of the Zygoascus clade and was isolated in South Africa as a contaminant from an emulsion of white oil and polysorbate. Candida transvaalensis sp. nov. (type strain NRRL Y-27140, CBS 6663) was obtained from forest litter, the Transvaal, South Africa, and forms an isolated clade with Candida santjacobensis. Trigonopsis californica sp. nov. (type strain NRRL Y-27307, CBS 10351) represents a contaminant from wine in California, and forms a well-supported clade with Trigonopsis cantarellii, Trigonopsis variabilis and Trigonopsis vinaria.     

Five new filamentous,glucose-fermenting Candida isolated from decayed wood in the evergreen rainy Valdivian forest of southern Chile

Five novel representatives of the yeast genus Candida isolated from advanced stages of wood degradation from fallen trunks of Eucryphia cordifolia Cav., Laurelia sempervirens Weim., Laurelia philippiana Weim., Nothofagus dombeyii (Mirb.) Blume, and Nothofagus obliqua (Mirb.) Blume, in the evergreen rainy Valdivian forest of southern Chile, are described and illustrated. They clearly differ from all accepted Candida species to warrant their establishment as five new species of yeasts: Candida coipomensis sp. nov., Candida ralunensis sp. nov., Candida laureliae sp. nov., Candida osornensis sp. nov., and Candida llanquihuensis sp. nov.