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.     

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.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml⁻¹, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml⁻¹ was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.     

Production of geranyl acetate and other acetates by direct esterification catalyzed by mycelium of Rhizopus delemar in organic solvent

Dry mycelium of Rhizopus delemar MIM catalyzed the formation of geranyl acetate using 110 mM geraniol and acetic acid at 55°C in heptane to give 11.9 g/l (55% molar conversion). Geranyl acetate was produced at 72.5-75 g/l after 10 days by semi-continuous addition of the substrates. Rhizopus delemar also catalyzed the direct acetylation of different primary alcohols with molar conversions ranging from 65 to 98%.     

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).     

Extracellular alkaline protease of newly isolatedRhizopus oryzae

Summary A fungal isolate identified asRhizopus oryzae, produces an extracellular alkaline serine protease. Maximum protease formation was after six days in shake flask culture at two different conditions of pH and temperature optimum (pH 5 at 30°C and pH 10 at 37°C). AgNO₃ and Tween 80 increased protease synthesis. The enzyme is stable between pH 3 and pH 11 and has a temperature optimum of 60°C.     

Microbiological studies of Indonesian fermented foodstuffs

Microbiological studies were made of certain Indonesian foodstuffs obtained from Malang, Surakarta, and Djakarta.Saccharomyces cerevisiae andCandida solani were isolated from ragi-roti (a baker's yeast preparation). From ragi-tempe and tempe were isolatedRhizopus oryzae, R. arrhizus, R. oligosporus, R. stolonifer, Mucor Rouxii, M. javanicus andTrichosporon pullulans. The microbiological flora of ragi-tape was found to includeCandida parapsilosis, C. melinii, C. lactosa sp. nov.,Hansenula subpelliculosa, H. anomala, H. malanga sp. nov.,Chlamydomucor oryzae andAspergillus oryzae. From ragi-ketjap, used to prepare soysauce, were isolatedRhizopus oligosporus, R. arrhizus, Aspergillus oryzae, andA. flavus, the latter species probably being an accidental contaminant. Two new species are described:Candida lactosa andHansenula malanga, from ragi-tape from Surakarta and Malang, respectively.From a thesis presented to the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the Ph.D. degree, August, 1969.Financial support of Dakimah Dwidjoseputro by the Ford Foundation is grate-fully acknowledged.     

Production of flavour esters by Rhizopus oryzae

Microbial production of different alipathic esters with flavour characteristic has been studied. Lyophilized whole cells of Rhizopus oryzae CBS 112-07 were found to be particularly suitable to catalyse the synthesis of different flavour esters (hexyl acetate, propionate, butyrate, caprylate; geranyl acetate, propionate, butyrate and 2- and 3-methylbutyl acetate, butyrate) in n-heptane. This strain was therefore utilized for the semipreparative production of geranyl butyrate by semicontinous and continous addition of the substrates with satisfactory yields (144 g l^–1 in 264 h and 142 g l^–1 in 48 h respectively).     

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.     

Susceptibility of Sitophilus granarius and S. Oryzae (Coleoptera: Curculionidae) to high temperature after exposure to supra-optimal temperature

S. granarius and S. oryzae were exposed to 32° and 35° for 2, 7 and 14 days and 1, 2 and 7 days, respectively, and mortality tested at 40°. Under these conditions maximum acclimation of S. granarius was achieved after 14 days at 32° and 2 days at 35°, while S. oryzae maximum acclimation occurred after 2 days at 32° and 1 day at 35°. Longer exposure at 32° for S. oryzae and for both species at 35°, appeared to reverse the process. Rearing S. granarius for three and five generations at 30°, raised its tolerance to the test temperature. Rearing S. oryzae for three generations at 30° did not have any significant effect on that species.

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Zusammenfassung Imagines von S. granarius und S. oryzae wurden physiologisch bei 32° und 35° für 2, 7 und 14 Tage, oder für 1, 2 und 7 Tage akklimatisiert und ihre Sterblichkeit bei 40° geprüft. Unter diesen Bedingungen wurde die maximale Akklimatisierung bei S. granarius nach 14 Tagen bei 32° und nach 2 Tagen bei 35° erreicht, während bei S. oryzae die maximale Akklimatisierung nach 2 Tagen bei 32° und 1 Tag bei 35° erreicht wurde. Längere Akklimatisierungs-Perioden bei 32° für S. oryzae und für beide Arten bei 35° schienen den Prozess umzukehren. Die Züchtung von S. granarius für drei und fünf Generationen, und von S. oryzae für drei Generationen bei 30° vergrösserte die Toleranz von S. granarius gegen die experimentelle Temperatur, hatte aber keinen bedeutsamen Effekt bei S. oryzae.Die Daten beweisen die Fähigkeit von S. granarius und S. oryzae, sich an höhere Temperaturen zu akklimatisieren, wobei der Grad der vergrösserten Toleranz bei den zwei Arten verschieden war. Es zeigte sich auch, dass in diesen zwei Arten der physiologische Akklimatisierungsprozess bei stufenweisem Wechsel zu einer höheren Temperatur effektiver war als der Prozess der Akklimatisierung bei Dauerzucht in einer erhöhten Temperatur. Die gegenwärtigen Daten beziehen sich nur auf die veränderte Empfindlichkeit hinsichtlich der Mortalität bei den akklimatisierten Insekten in hoher Temperatur.

     

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.     

Effects of temperature, pH and additives on the activity of tannase produced by a co-culture of Rhizopus oryzae and Aspergillus foetidus

Summary Tannase was produced by modified solid-state fermentation (MSSF) of tannin rich substrates by a co-culture of the two filamentous fungi, Rhizopus oryzae and Aspergillus foetidus. The enzyme thus produced was then partially purified by solvent precipitation and DEAE-Sephadex column chromatography. A study on the effects of temperature and pH was made on the activity of tannase so purified. The optimum values of incubation time, reaction temperature and pH for tannase activity were 5 min, 40 °C and 5.0 respectively. The half-life period thermal stability and kinetic constants (K _m 0.21 mM, V _max 4.9×10⁻² M min^-1 at 40 °C) of this tannase were determined and the effects of different metal ions, surfactants, chelators, denaturants and inhibitors on the enzyme activity were also studied.     

The effect of urea on growth of moulds and biomass yield in solid-state cultivation

Urea, added at 2 to 20 mg/g in solid bran medium supporting growth of Aspergillus oryzae and Rhizopus oligosporus, led to a higher concentration of NH₄ ⁺ and pH but a decrease in biomass production.The authors are with the Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia.     

Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-<Emphasis Type="Italic">epi</Emphasis>-5-<Emphasis Type="Italic">epi</Emphasis>-valiolone synthase gene in <Emphasis Type="Italic">Streptomyces albus</Emphasis> ATCC 21838

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.     

Composition of the fungal flora of four cereal grains in Saudi Arabia

Using the grain-plate method and on glucose-Czapek's agar at 28°C, fifty-eight species belonging to 26 genera were collected from barley (42 species and 19 genera), maize (29 species and 16 genera), sorghum (32 species and 17 genera) and wheat grains (42 species and 18 genera).The most frequent genera were Aspergillus, Penicillium, Rhizopus, Fusarium, and Mucor followed by Alternaria, Drechslera, and Curvularia. From the preceding genera Aspergillus flavus, A. niger, Penicillium notatum, Rhizopus stolonifer, Fusarium moniliforme, Mucor racemosus, Alternaria alternata, Drechslera spicifera, and Curvularia lunata were the most prevalent species in the four types of grains tested.     

Temperature effects in ethanol fermentation high corn media

Summary A relationship between temperature and high ethanol yields has been found using whole corn mashes saccharified with Aspergillus oryzae wheat bran koji. Decreased ethanol yields were obtained at 34.5°C with high concentration corn mashes in contrast to high ethanol yields with the same medium at lower temperatures. The decreased yields appear to be related to mass and/or heat transfer problems rather than primary ethanol toxicity. Scale-up of the high corn medium will require a re-evaluation of alcohol fermentation technology.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Amylolytic enzyme production byRhizopus oryzae grown on agricultural commodities

The amylolytic enzyme production byRhizopus oryzae NRRL 395 grown on different agricultural commodities was datermined. The mould produced much higher enzyme activity from barley, corn, bats, and rice than from cassava. The optimal temperature for enzyme production was 30°C. Neutralization with CaCO₃ greatly enhanced the rate of enzyme production. Nitrogen supplementation of cassava resulted in higher enzyme yields.

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Résumé On a déterminé la production d'enzymes amyloytiques parRhizopus oryzae NRRL 395. cultivé sur différents produits agricoies. La moisissure produit une activité enzymatique plus élevée à partir du malt. du maïs, de l'avoine et du riz qu'á partir du manioc. La température optimale pour la production d'enzyme est de 30°C. La neutralisation par al CaCO₂ augmente fortement la vitesse de production de l'enzyme. L'ajout d'azote au manioc résulte en un accroissement du rendement enzymatique.