Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.     

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.     

Improved exopolysaccharide production in fed-batch fermentation of Aureobasidium pullulans, with increased impeller speed

Summary Aureobasidium pullulans — when grown in fed-batch fermentation on 190 g glucose/l produced 68 g exopolysaccharide/l with a productivity of 0.42 g/l-h. When the impeller speed was increased from 280 rpm to 340 rpm, the exopolysaccharide increased to 101 g/l, the productivity was 0.90 g/l-h and the conversion ratio was 63 % (w/w).     

Culture requirements for the production of protease by Aspergillus oryzae in solid state fermentation

Summary A number of culture conditions for protease production by Aspergillus oryzae NRRL 2160 on solid substrates were investigated. The pH of the medium and the substrate markedly affected protease production. High protease yield was obtained when the fungus was cultivated for 72–96 h on rice hulls: rice bran (7:3), at an initial pH of 7.0. Maximal protease production was achieved at an initial moisture content of 35–40%, corresponding to a water activity range of 0.982–0.986. Casein and gluten were effective inducers. Polyethylene bags proved to be promising containment systems for solid state cultivation.Offprint requests to: A. M. R. Pilosof     

Pilot-and production-scale containment of cytotoxic and oncogenic fermentation processes

Proper design of fermentation facilities and equipment modification can control the risks associated with largescale production and purification of microbially produced cytotoxic agents and oncogenic viruses. The primary biohazard risks to operators and the environment are generation of aerosols and accidental spills. Fermentation and recovery facilities can be constructed to contain these agents by installing fermentation equipment within a HEPA-filter-exhausted biological barrier. Within this barrier system, large-scale processing that generates potentially hazaradous areosols (filtration, centrifugation of transformed cells or crystal slurries, and banding of viruses) should be isolated from other operations. Isolation of equipment is often required, with provision for both chemical and biological decontamination of process wastes. Failsafe fermentor over-pressure sensors, parallel exhaust gas filtration, welded transfer lines, and modified sampling systems for elimination of aerosols can be installed on most fermentation equipment. Aerosol and spill containment by proper equipment design, coupled with appropriate personnel protective equipment and medical monitoring, make possible safe production of experimental growth factors and viruses from large-scale culture of transformed mammalian cells and production of cytotoxic antitumor antibiotics.     

Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid

Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.     

Improvement of the glaB promoter expressed in solid-state fermentation (SSF) of Aspergillus oryzae

The glucoamylase-encoding gene (glaB) promoter should be very useful for recombinant protein production in solid-state fermentation (SSF) of Aspergillus oryzae. A 97-bp fragment containing the cis-element of the glaB promoter was inserted into the glaA promoter, which was little expressed in SSF. The chimeric promoter showed about a 24-fold increase in promoter activity in SSF. Eight copies of the 97-bp fragment were tandemly fused with the glaB promoter. The improved promoter showed about a 4.6-fold increase in promoter activity in SSF. The glaB gene was overexpressed under control of the improved glaB promoter in SSF. Recombinant glucoamylase production reached about 1524 mg/kg-broth for 2 d. The improved glaB promoter should be very useful for overproduction of a recombinant protein in SSF of A. oryzae.     

Pulsed feeding during fed-batch Aspergillus oryzae fermentation leads to improved oxygen mass transfer

Productivity in many fungal fermentations is detrimentally affected by high broth viscosity and consequent reduced oxygen mass transfer capacity. The goal here was to determine whether pulsed feeding of limiting carbon in a fungal fermentation could lead to reduced viscosity and improved oxygen mass transfer. As a model, an industrially relevant recombinant strain of Aspergillus oryzae was grown in carbon-limited, fed-batch mode. Maltodextrin was used as a carbon source and was added either continuously or in 1.5-min pulses, 3.5 min apart. In both feeding modes the same total amount of carbon was added, and carbon feed rate was at sufficiently low levels to ensure cultures were always carbon-limited. Compared to continuous feeding, pulsed addition of substrate led to smaller fungal elements, which resulted in a significant reduction in broth viscosity. This in turn led to higher dissolved oxygen concentrations and increased oxygen uptake rates during pulsed feeding.     

Trehalase in conidia of Aspergillus oryzae

Horikoshi, Koki (The Institute of Physical and Chemical Research, Bunkyo-ku, Tokyo, Japan), and Yonosuke Ikeda. Trehalase in conidia of Aspergillus oryzae. J. Bacteriol. 91:1883-1887. 1966.-Trehalases (soluble trehalase and coat-bound trehalase) were found in the conidia of Aspergillus oryzae, and the total activity of the trehalases increased during the germination process. The soluble trehalase was purified by diethylaminoethyl-cellulose column chromatography; its optimal pH, Michaelis constant, and heat stability were studied. In vitro, the trehalases were competitively inhibited by d-mannitol, which was also contained in the conidia. Since the trehalose content in the conidia decreased at an early stage of germination, it was assumed that trehalase might begin to hydrolyze trehalose after the inhibitory effect of d-mannitol decreased.     

Potential of Aspergillus oryzae CFTRI 1480 for producing proteinase in high titres by solid state fermentation

Aspergillus oryzae CFTRI 1480, an isolate from a spoiled moist sample of casein, produced 59,105 units of an extracellular proteinase/g dry mouldy bran (DMB) at 72 h in an arbitrarily formulated wheat bran medium in a solid state fermentation system. The enzyme production was significantly affected by mineral salt content and pH of the liquid used for moistening the wheat bran. Enzyme titres were enhanced 1.34-fold with the addition of 0.4% corn starch. Optimization of key parameters, i.e., initial moisture content, age and size of inoculum, increased the enzyme production to 191,869 units/g DMB and reduced the fermentation time to 48 h. Such high titres in a simple medium, surpassing most of the literature reports, indicate the industrial importance of the culture. The properties of acetone-precipitated enzyme, viz, the optimum pH of 10.0, more than 95% activity between pH 7.0 and 10.0, temperature optimum at 55° C and more than 90% activity between 10 and 27°C, are similar to those of commercially available fungal proteinases employed in animal feed, leather and other industries. Correspondence to: B. K. Lonsane     

Pullulan fermentation using a prototype rotational reciprocating plate impeller

A rotational reciprocating plate impeller prototype, designed to improve the mixing homogeneity of viscous non-Newtonian fermentation broth, has been tested in pullulan fermentations. With this new impeller, the operating levels of several factors were investigated to improve pullulan production with Aureobasidium pullulans ATCC 42023 in a 22-L bioreactor using experimental designs. Because both high molecular weight (MW) and high concentration of pullulan were desired; the exopolysaccharide (EPS) concentration and the broth viscosity were used as optimization objective functions to be maximized. A 6-run uniform design was used to investigate five factors. Under the best operating conditions among the six runs, 29.0 g L^?1 EPS was produced at 102 h. This condition was used as the starting point for further investigation on the two statistically significant factors, the pH and the agitation speed. An 8-run 3-level custom design that investigates up to second-order effects was used in the second stage. An optimal zone of operating conditions for large quantity of high MW pullulan production was identified. A concentration of 23.3 g L^?1 EPS was produced at 78 h. This is equivalent to an EPS productivity of 0.30 g L^?1 h^?1. The corresponding apparent viscosity of the broth was 0.38 Pa s at the shear rate of 10 s^?1.     

Effects of a casein hydrolysate prepared from Aspergillus oryzae protease on adjuvant arthritis in rats

We evaluated the effects of a casein hydrolysate (CH) prepared from Aspergillus oryzae protease on rat adjuvant arthritis, a model of human rheumatoid arthritis. CH was administered orally once a day to the animals for 22 d after the adjuvant injection. CH suppressed swelling in the adjuvant-uninjected hind paws, and a higher dose of CH suppressed the increase in arthritic score and swelling of the adjuvant-injected hind paws. A histopathological examination revealed evidence that the higher dose of CH suppressed the articular changes in the rats. In addition, CH suppressed the production of nitric oxide and prostaglandin E(2) in the plasma of the rats. These results suggest that CH had a suppressive effect on adjuvant arthritis by inhibiting the acute and chronic inflammatory reactions.     

Alcoholic chestnut fermentation in mixed culture. Compatibility criteria between Aspergillus oryzae and Saccharomyces cerevisiae strains

The main objective of the present work consisted in the transfer to the case of the chestnut of a rice fermentative process that carried out to the Japanese traditional way to lead to an alcoholic bagasse, the moromi, capable of obtaining distilled. This way, selection assays of amylolitic Aspergillus oryzae strains and studies of compatibility between microfungi and yeast were carried out. These mixed cultivations were performed operating in batch submerged culture. Later on, using solid state system (chestnut, microfungi, yeast), a fermentative fed-batch process (koji, moto, moromi) was defined. By means of this approach a yield of 70% was reached in the conversion of total carbohydrates in ethanol. Also, the time required by the traditional operation was reduced in half.     

翼形轴流桨在红霉素发酵中的应用研究

在20t工业发酵罐中,研究了翼形轴流桨搅拌对红霉素发酵过程的影响。重点考察了粘度,溶氧,效价等过程参数变化,以及搅拌功耗与发酵产量之间的关系。研究结果表明：（1）与传统的涡轮搅拌桨相比,翼形轴流桨搅拌其发酵过程参数（粘度,溶氧,效价等）随时间的变化曲线有明显的差异;（2）在相同的生产条件下,用翼形轴流桨代表涡轮桨可有效提高生产效率。