Improved glucoamylase production by less unstable strains ofAspergillus awamori

Summary The NRRL 3112 strain ofAspergillus awamori showed spontaneous segregation and sector formation tentatively attributed to a chromosomal instability. Spontaneous segregants showing stablepro1 (proline) requirement were easily isolated and stablepro+ strains were not isolated at all. Derivatives showing lower frequency ofpro1 conidia produced significantly more glucoamylase than the parental strain.     

Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation

The feasibility of using grape pomace for the production of xylanase and exo-polygalacturonase by Aspergillus awamori in solid state fermentation has been evaluated. Solid state fermentation experiments indicated that the particle size did not influence the enzyme production. The addition of extra carbon sources and the initial moisture content of the grape pomace were found to have a marked influence on the enzymes yields. Xylanase and exo-PG activities were high at 65% (w/w) initial moisture content and glucose supplementation.     

Transformation ofAspergillus awamori andA. niger by electroporation

A method is described which allows transformation ofAspergillus awamori andA. niger mediated by electroporation. This procedure gave transformation frequencies similar to those obtained with polyethylene glycol. ForA. niger no differences were observed between the two procedures with respect to the number of integrated plasmid copies or the frequency of homologous integration.     

Cellulase production by a solid state culture system

Production of cellulase using solid culture systems of Trichoderma reesei QM9414 and Sporotrichum cellulophilum on wheat bran was studied. By using moisture-controlled solid culture equipment, the effect of water content of wheat bran on cell growth and cellulase production was investigated. Cellular biomass grown on solid substrate was estimated by measuring oxygen consumption rate and glucosamine content of the cells. These parameters were shown to have a good linear correlation with the specific growth rate. This reliable method of estimating the cell growth rate enabled us to simulate the enzyme production in a solid culture system by means of multiple linear regression analysis which takes into account of the water content, cell mass, and the oxygen consumption rate as variables. The cell growth and cellulase production were maximized at different water content of the medium. A high water content, 57% for T. reesei and 70% for S. cellulophilum, favored mycelial growth, while the maximum cellulase activity was obtained at a lower water content such as 50% for both fungi. It was observed that cellulase production by T. reesei depended on the culture conditions that support the optimal growth rate for the maximum enzyme production.     

Isolation of a transglucosidase-nonproducing mutant ofAspergillus awamori yielding improved quality and production of amyloglucosidase preparations

Summary The fermentative production of amyloglucosidase (AMG) by differentAspergillus species simultaneously yields transglucosidase (TG), which is undersirable in the conversion of starch to dextrose, as it catalyses the reversion of dextrose and maltose to maltosaccharides, in turn providing disproportionately lower yields of dextrose (DX). To overcome this problem, using UV-irradiation, a novel mutant (ND-1-283) has been isolated fromAspergillus awamori, which has lost the ability to produce TG and which secretes 45% more AMG than its parent strain, giving the mutant a dual operational advantage. The inability of this mutant to produce TG was demonstrated by thin layer chromatography (TLC) of starch hydrolysate; this was substantiated by obtaining 96.0% DX (w/w) at the end of saccharification.     

Influence of carbon, nitrogen and phosphorous sources on glucoamylase production by Aspergillus awamori in solid state fermentation

It was the objective of the present study to increase the production of glucoamylase by Aspergillus awamori through solid state fermentation, using wheat bran as the main carbon source and (NH4)2SO4, urea, KH2PO4, glucose, maltose and starch as additional nitrogen, phosphorus, and carbon sources. The production of glucoamylase is strongly influenced by N and C sources. A 100% increase was observed when the (NH4)2SO4 was replaced by urea, with C/N = 4.8, using maltose as the additional carbon source. C/P ratios in a range of 5.1 to 28.7 did not induce glucoamylase production under the studied conditions.     

Citric acid production in submerged and solid state culture of Aspergillus niger

In this work, the citric acid production in solid state culture was performed, evaluating the isolated effect and interactions of particle size and liquid phase employed, by means of the factorial design of first order. The results indicate that the particle size is the most determinant variable. An analysis comparing submerged and solid state in optimal conditions was performed. When solid state culture was used, the productivity of citric acid was doubled, reducing the fermentation time from 14 to 6 days, compared to the submerged culture, obtaining a maximum citric acid concentration of 21.24 g/l.List of Symbols B _s , B _v main effects - B _sv crossed effects - s cm particle size - S coded particle size - v ml liquid phase volume - V coded liquid phase volume     

Mutation and screening to increase chymosin yield in a genetically-engineered strain ofAspergillus awamori

Summary Through the course of five rounds of mutagenesis of a genetically-engineered strain ofAspergillus awamori, the yield of a heterologous protein (the acid protease, calf chymosin) increased four-fold. This was accomplished through the use of an agar plate screen incorporating the colony restrictor 2,6-dichloro-4-nitroaniline (dichloran) and the acid protease inhibitor diazoacetyl-norleucine methyl ester (DAN) to reduce high background concentrations of the native acid protease. A miniaturized liquid culture growth method using 24-well culture plates was an intermediate screen between agar plate and shake flask cultures. Analysis of broth samples for active calf chymosin was accomplished with a highly specific, 96-well microtiter plate turbidimetric assay.     

Potential of fed-batch culture in solid state fermentation for production of gibberellic acid

Summary Fed-batch culture technique, applied to the solid state fermentation process for the production of gibberellic acid, improves the yield by 18.2% as compared to a conventional batch solid state fermentation.     

A novel application of solid state culture: production of lipases by Yarrowia lipolytica

An extracellular lipase was produced in solid state cultures of Yarrowia lipolytica CECT 1240 using nylon sponge and several food and agroindustrial wastes (barley bran and triturated nut) as, respectively, inert support and support substrate. The highest activity was obtained with triturated nut (23 kU l^–1) followed by sunflower oil-soaked barley bran (21 kU l^–1). The activities were 5 fold greater those obtained in the control cultures with just inert support.     

Penicillin production by solid state fermentation

Summary Penicillin was produced by a non-sterile solid state fermentation (SSF) on bagasse impregnated with culture medium. The use of concentrated media greatly enhanced the antibiotic production in this system. It was observed that adequate initial moisture content (70%) of the impregnated solid medium results in higher production. A comparison between solid and liquid fermentation showed superior yield and productivity.     

Effect of culture conditions on lipolytic enzyme production by Penicillium candidum in a solid state fermentation

Lipolytic enzymes were produced using wheat bran as substrate in a solid state fermentation with Penicillium candidum. The best production of lipolytic activity occurred at 29 degrees C. One hundred micromoles of free butyric acid (FBA) was released from tributyrin by 1 mL of cell free supernatant in the absence of control of environmental relative humidity. When a closed chamber saturated with water vapour was used the lipolytic activity increased to 320 micromoles of free butyric acid. The best initial reaction pH was 7.0. The highest activity, 480 micromoles of FBA, was obtained at a moisture content of 67.5 % of saturation.     

Fungal solid state culture of palm kernel cake

Palm kernel cake (PKC), an agro-industrial by-product used extensively in the animal feed industry, has limited use in fish feeds due to its high fiber and low protein contents. In this study, PKC was processed under solid state culture conditions with five fungal strains and the effect of this fungal culturing on the amino acid, fatty acid, cellulose and hemicellulose fractions was evaluated. Fungal strains used were Sclerotium rolfsii, Trichoderma harzianum, Trichoderma longiobrachiatum, Trichoderma koninggi and Aspergillus niger. Fungal growth was carried out at 50% moisture level and 1% inoculum level for 7 days. A significant increase in protein content from 18.76% to 32.79% was obtained by growing T. longibrachiatum on PKC. Cellulose level decreased significantly from 28.31% to 12.11% for PKC cultured with T. longibrachiatum, and hemicellulose from 37.03% to 19.01% for PKC cultured with A. niger. Fungal culturing of PKC brought about an increase in the level of unsaturated- and a decrease in the level of the saturated-fatty acids.     

Repeated batch production of citric acid from sugarcane molasses using recycled solid-state surface culture ofAspergillus niger

Summary The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Zymomonas mobilis

The production of ethanol from starch by a coimmobilized mixed culture system of aerobic and anaerobic microorganisms in Ca-alginate gel beads was investigated. The mold Aspergillus awamori was used as an aerobic amylolytic microorganism and an anaerobic bacterium, Zymomonas mobilis, as an ethanol producer. By controlling the mixing ratio of the microorganisms in the inoculum size, a desirable coimmobilized mixed culture system, in which the aerobic mycelia grew on and near the oxygen-rich surface of the gel beads while the anaerobic bacterial cells mainly grew in the oxygen-deficient central part of the gel beads, was naturally established under the aerobic culture conditions, and ethanol could be directly produced from starch by the system. The ethanol productivity by the system in flask culture was particularly affected by the shear stress (dependent on the shaking speed) which controlled the mycelial growth on the surface of the gel beads. Under optimum culture conditions in the flask culture, the glucose produced was instantly consumed, and was not observed in the culture broth; the final concentration of ethanol produced from 100 g/L starch was 25 g/L and the yield coefficient for ethanol, Y(pls), was 0.38. The ethanol productivity by the coimmobilized mixed culture system was compared with those by other various culture systems and the advantages of the system were clarified.     

Variability in amylase production among morphological mutants ofAspergillus wentii

Mutational experiments were performed to produce morphological mutants fromAspergillus wentii Wehmer (IMI 17295) by UV and X-ray irradiation. Among the morphological mutants, five representative types were recognized. Marked variation existed in amylase activity between the morphological mutants and the wild type.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Conversion of cellulose into fungal cell mass in solid state culture

Summary To satisfy the demand for simple production technology (simple and cheap reactor, cheap recovery and finishing), solid state cultivations were carried out with pretreated straw in a simple fixed bed reactor under nonsterile conditions.The results of these investigations were compared with those evaluated in a stirred tank reactor. The same cell mass fractions were obtained in both reactors. However, about double the cultivation time is necessary for a solid state cultivation as compared to a submerse cultivation.Symbols N₂ nitrogen content of dry biomass (%) - P productivity on cell protein (%/h) - T temperature (°C) - t_F cultivation time (h) - X fungal cell mass fraction (%)     

Production of protein and cellulase byAspergillus niger in solid state culture

Summary Single-cell protein (SCP) and cellulase production byAspergillus niger AS-101, grown on alkalitreated corn cobs, was studied under various cultural conditions. The maximum yields of SCP and cellulase, under solid-state fermentation were obtained when the culture was incubated at pH 5.5 for 18 d at 30°C with 12% substrate.

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Effect des conditions de culture sur la production de protéines et de cellulases par Aspergillus niger en milieu solide

Résumé La production de protéine d'origine unicellulaire (POU) et de cellulase parAspergillus niger AS-101, cultivé sur des épis de maïs traités à l'alcali, est étudiée sous diverses conditions de culture. Les rendements maximum en POU et en cellulase par fermentation en milieu solide sont obtenus lorsque la culture est incubée à pH 5.5 pendant 18 jours à 30°C et avec une concentration en substrat de 12%.