Respiration in Organic Acid-Forming Molds

Cytochrome c, coenzyme Q and lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1, 1, 1, 27) in Rhizopus oryzae were studied in order to investigate the connection between the mechanism of lactate formation and terminal respiration.

Cytochrome c was extracted easily and in good yield by the addition of cetyltrimethyl ammonium bromide to mycelial suspensions. It was purified by calcium phosphate gel and Amberlite IRC-50 resin chromatography.

Coenzyme Q was extracted with ethanol, purified by chromatography on silicic acid, and, following crystallization from a mixture of ethanol and methanol, was identified as coenzyme Q₀.

Lactic dehydrogenase was partially purified and some of its properties were investigated.

Rhizopus oryzae at an early growth stage in shake culture produced almost no lactate. At this stage, the mycelia were rich in cytochrome c and FAD. On the contrary, those of later growth stages fermented a larger amount of the glucose to lactate and the contents of cytochrome c and FAD were lower than in the young mycelia.

Surface cultures produced lactate at a rate very nearly equivalent to the rate of glucose consumption. Addition of zinc to the medium resulted in decreased lactate production, but no increase was observed in the mycelial content of either cytochrome c or FAD in this case. On the other hand, increased quantities of FMN were found in mycelia from shake or surface cultures when zinc was added.     

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.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

Aerial mycelia of Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation system

Aspergillus oryzae is commonly used in solid-state fermentation (SSF) and forms abundant aerial mycelia. Previously, we have shown that aerial mycelia are extremely important for the respiration of this fungus during growth on a wheat-flour model substrate. In this paper, we show that aerial mycelia of this fungus give a strong increase in fungal biomass and α-amylase production. Cultures of A. oryzae on wheat-flour model substrate produced twice the amounts of fungal biomass and α-amylase, when aerial mycelia were formed. Utilization of these findings in commercial solid-state fermenters requires further research; results from packed beds of grain indicate that aerial mycelia are of limited importance there. Probably, substrate pre-treatment and an increase in bed voidage are required.     

Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.     

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.     

Studies on the respiratory system of Aspergillus oryzae I. Development of respiratory activity during germination in the presence and absence of antimycin A

Active growth of Aspergillus oryzae was observed when conidiawere inoculated into a medium containing antimycin A. Immediatelyafter adding antimycin A, to young mycelia germinated in itsabsence, growth stopped, but began again after several hours.This restored growth was antimycin A-insensitive. Percentagegermination was the same in the presence and absence of thisdrug. It seems that drug-resistant germination and growth donot result from selection of resistant cells but result frominduction of antimycin A-insensitive mitochondria in the wholepopulation. Endogenous respiration of cells germinated in theabsence of antimycin A was inhibited by this drug, whereas thatof cells grown in the presence of antimycin A was completelyinsensitive. Antimycin A-sensitivity of cellular respirationseems to determine the effect of this drug on mycelial growth.Mitochondria were isolated from mycelia grown in the presenceand absence of this drug. The difference in antimycin A-sensitivityin endogenous respiration was attributed to a difference inproperties of the mitochondrial respiratory systems. ¹Present address: Department of Chemistry, Institute of MedicalScience, University of Tokyo, Tokyo, Japan (Received December 21, 1969; )     

Chitosan production fromRhizopus oryzae mycelia

Summary Chitosan was successfully produced as a second valuable product fromRhizopus oryzae mycelia which had been previously used to make L(+) lactic acid. The highest yield of extractable chitosan was 700 mg/L.     

Chitosan production from hemicellulose hydrolysate of corn straw: impact of degradation products on Rhizopus oryzae growth and chitosan fermentation

Aims: To examine the potential use of hemicellulose hydrolysate (HH) for the production of chitosan by Rhizopus oryzae and investigate the influence of contents in HH on mycelia growth and chitosan synthesis. Methods and Results: Compared to xylose medium, HH enhanced mycelia growth, chitosan content and production of R. oryzae by 10·2, 64·5 and 82·1%, respectively. During sulfuric acid hydrolysis of corn straw, sugars (glucose, galactose, etc) and inhibitors (formic acid, acetic acid and furfural) were generated. Acetic acid (2·14 g l^?1) and formic acid (0·83 g l^?1) were stimulative, while furfural (0·55 g l^?1) was inhibitory. Inhibitors, at different concentrations, increased the mycelia growth and chitosan production by 24·5–37·8 and 60·1–207·1%. Conclusions: HH of corn straw is a good source for chitosan production. Inhibitors in HH, at proper concentrations, can enhance chitosan production greatly. Significance and Impact of the Study: This work for the first time reported chitosan production from HH. Chitosan production can be greatly enhanced by cheap chemicals such as inhibitors in HH.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1 capable of growing aerobically

Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1, which is able to grow aerobically, were investigated using a microbial electrode system. The respiration of strain AMB-1 was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), KCN and dicumarol. Strain AMB-1 cannot grow in the presence of these inhibitors under aerobic conditions. On the other hand, strain AMB-1 can grow and form magnetite (Fe₃O₄) particles with HQNO and KCN under anaerobic conditions. Growth and magnetite formation of strain AMB-1 were reduced by dicumarol, which also inhibited iron reduction under anaerobic conditions, whereas iron reduction was not inhibited by HQNO and KCN.     

High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccharomyces cerevisiae

Summary The expression and secretion of Rhizopus oryzae glucoamylase were studied in the yeast Saccharomyces cerevisiae. Rhizopus oryzae glucoamylase was highly expressed and efficiently secreted into a medium to a high level (above 300 mg/l) under control of a yeast promoter and the original signal sequence. Excess expression of the secretable glucoamylase with high copy number plasmid slightly decreased growth of the transformant cells in glucose medium but not in fructose medium.     

Alternative respiration and antibiotic production of Acremonium chrysogenum

The influence of potassium cyanide (KCN), dissolved O₂ concentration and medium composition on alternative respiration (AR) of Acremonium chrysogenum were investigated. The respiration of the fungus was only partially inhibited by KCN, but completely inhibited by the combination of KCN with salicylhydroxamic acid. It has been proved by in-situ measurements of the NADH-dependent fluorescence that the AR is active at low dissolved O₂ concentrations. The influence of the medium composition and the age of the fungus on the specific oxygen uptake rate is considered. Correpondence to: K. Schügerl     

Asymmetric Reduction of 2-Methyl-2-aryloxyacetic Acids by Glomerella cingulata

The mycotoxin citrinin had antifungal activity under acidic conditions. At the minimum inhibitory concentration, it completely inhibited cellular respiration and partially inhibited the incorporation of radioactive precursors into macromolecules in Saccharomyces cerevisiae. It had no effect on cell permeability. In mitochondrial preparations, it significantly inhibited succinate oxidase and NADH oxidase. Rhizopus chinensis was more sensitive than S. cerevisiae; its growth and mycelial respiration at acidic pH were completely inhibited by lower concentrations of citrinin. The pH-dependent antifungal activity of citrinin seems to be associated with its uptake by fungi. Approximately half of the citrinin taken up was found in mitochondria. The main site of the antifungal action of citrinin, therefore, appears to be the mitochondrial electron transport system.     

Changes in morphology of <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> in submerged fermentation and their effect on production of mycelium-bound lipase

In order to control suitable mycelium morphology to obtain high lipase productivity by Rhizopus chinensis in submerged fermentation, the effects of fungal morphology on the lipase production by this strain both in shake flask and fermentor were investigated. Different inoculum level and shear stress were used to develop distinctive morphologies. Analyses and investigations both on micromorphology and macromorphology were performed. Study of micromorphology reveals that micromorphologies for dispersed mycelia and aggregated mycelia are different in cell shape, biosynthetic activity. Macromorphology and broth rheology study in fermentor indicate that pellet formation results in low broth viscosity. Under this condition, the oil can disperse sufficiently in broth which is very important for lipase production. These results indicate that morphology changes affected the lipase production significantly for R. chinensis and the aggregated mycelia were suggested to achieve high lipase production.     

Chitin contents in different morphological forms ofRhizopus nigricans

Chitin (one of the major cell wall constituents ofZygomycetes) was determined in different morphological forms ofRhizopus nigricans mycelia grown at different cultivating conditions. Microscopic observation and chemical analysis has revealed that at the phase of active growth, long and sparsely branched hyphae of dispersed mycelia contain 2.3% of dry cell mass of this structural polysaccharide, which is higher than the value found in pellets of the same age. A large increase in chitin content up to 9.0% of cell dry mass was determined in older, partially autolyzed mycelia in the form of pellets.     

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.     

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.     

Optimization of physiological factors for direct saccharification of cassava starch to glucose by Rhizopus oligosporus 145f

Direct saccharification of 2.64% cassava starch by Rhizopus oligosporus 145F was attempted under various cultural conditions. Maximum glucose yield of 18.0 g/L culture filtrate was obtained with an initial pH 3.8, 2% (v/v) inoculum of R. oligosporus spores, and an incubation temperature of 45 degrees C in shake flask cultures for 48 h. This concomitantly produced 2.7 g mycelia/100g cassava starch containing 20.2% true protein. The production of glucose and mycelia was accomplished with 92.8% starch saccharification having 67.9% starch to glucose conversion efficiency.     

Studies on the Residual Respiration not Inhibited by KCN Plus Hydroxamic Acid in Tobacco Callus Cultures

A residual respiration not inhibited by KCN plus hydroxamic acid had been observed in many plant organs and tissues. The relative O2 uptake of it was 20–30% of total respiration in tobacco callus cultures. However, there is no report concerning the nature of the residual respiration and its localization in cell. The object of this study is to elucidate the characteris- tics of this residual respiration and its localization in cell. The experimental results are as follows: 1. The additions of glycolate and glyoxylate cause a marked rise in residual respiration not inhibited by KCN (or NaN3) plus m-CLAM. 2. The O2 uptake induced by glycolate and the residual respiration is inhibited by the addition of α-hydroxy ethanesulfonate. 3. The mitochondrial respiration is completely inhibited by KCN plus m-CLAM, but no effect by adding of glycolate. 4. Oxidation reactions of glycolate and glyoxylate in supernatant are observed after mitochondria are removed. Based on the above results, it is suggested that the residual respiration not inhibited by KCN plus m-CLAM in tobacco callus cultures is primarily catalyzed by glycolic acid oxidase localized within microbodies.