Operating conditions optimization for (+)-terrein production in a stirred bioreactor by Aspergillus terreus strain PF-26 from marine sponge Phakellia fusca

Terrein is a fungal metabolite with application values in the fields of medicine, cosmetology, and agriculture. However, mass production of single configuration terrein is still a big challenge. In this study, operating factors such as inoculation, agitation speed, aeration rate, pH control, and nutrient feeding were preliminarily optimized to improve the (+)-terrein production in the 5-L stirred bioreactor from the marine sponge-derived fungus Aspergillus terreus PF-26. Spore inoculation, low agitation speed, and aeration rate were proved to be suitable for A. terreus PF-26 to produce (+)-terrein in the stirred bioreactor. At 50?rpm agitation speed and 0.33?vvm aeration rate, 2.68?g/L (+)-terrein was achieved by feeding twofold concentrated maltose and glucose medium on the sixth day and controlling pH at 4.5 from the fourth day. This study lays foundation for the mass production of (+)-terrein by the marine filamentous A. terreus strain PF-26 in the stirred bioreactor.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture. II

The main purpose of the work reported here was to establish the effectiveness of aeration and agitation, and to determine the best conditions of aeration for the growth and production of glucose oxidase of Aspergillus niger, on a semi-industrial scale. Concentration of dissolved O₂, O₂ consumption and CO₂ production were measured. It was found that the rate of growth and the activity of glucose oxidase per gram mycelium increased with the increase of speed of agitation. The concentration of dissolved oxygen of the fermentation broth, as well as the rate of respiration (O₂ consumption and CO₂ production) increased in direct proportion to the increase of speed of agitation, while assimilation of sugars was accelerated. The values of the respiratory ratio showed a fluctuation according to the presence or absence of sugar in the medium.     

Morphology engineering of Aspergillus oryzae for l-malate production

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l -malate production. Specifically, correlation between A. oryzae morphology and l -malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l -malate production was 120–130 mm³/ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l -malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l -malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.     

Production of protease using wastewater from the manufacture of shochu

The production of protease using wastewater from a shochu distillery was investigated in order to devise a process for the treatment of shochu distillery wastewater. Aspergillus usami mut. shirousami IFO 6082 was selected from among eight strains for production of protease. Production of 240 U/ml of proteolytic activity was achieved after 72 h in a jar-fermentor culture under the following conditions: initial pH, 5; 30°C; aeration, 1 vvm; and agitation, 600 rpm. The protease was purified by column chromatography on Sephadex G-150 and isoelectrofocusing. The molecular weight of the purified enzyme determined by SDS-PAGE was 56 kDa, and the isoelectric point was pH 4.0. The optimum pH for the reaction was about 4.0, so the enzyme is therefore an acid protease. The optimum temperature for proteolysis ranged between 55 and 60°C, while the enzyme was unstable at temperatures above 60°C.     

Scale‐up of citric acid fermentation by redox potential control

To obtain high citric acid productivity in Aspergillus niger fermentation on beet molasses substrate, a certain redox potential profile with two maxima (260 and 280 mV) and two minima (180 and 80 mV) must be maintained. The most effective regulation of redox potential is by regulation of aeration and agitation. It has been shown that control of redox potential by aeration and agitation is a most successful method for scale‐up from 10‐L laboratory scale to the 100‐ and 1000‐L pilot‐plant scale, even in geometrically dissimilar stirred‐tank reactors. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 552–557, 1999.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase in<Emphasis Type="Italic"> Enterobacter aerogenes</Emphasis> expressing<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin for efficient oxygen uptake

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.     

Effect of aeration and agitation on synthesis of poly (γ-glutamic acid) in batch cultures of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324

The effects of aeration and agitation on the production and molecular weight of poly (γ-glutamic acid) (PGA) were systematically investigated in batch fermentor cultures of Bacillus licheniformis NCIM 2324. A high aeration rate and agitation speed enhanced the growth of B. licheniformis NCIM 2324, but did not always lead to high PGA production. Additionally, PGA production actually decreased at very high aeration rates and agitation speeds. The maximum PGA concentration was obtained at 750 rpm and 1 vvm. Rheological studies revealed that fermentation broth during production of PGA exhibited pseudoplastic behavior. The effects of aeration and agitation on the molecular weight of PGA were also studied, and the rate and extent of the decrease in the molecular weight of PGA as a function of time were found to be much greater at high aeration than low aeration. The PGA production of 46.34 g/L with a specific productivity of 0.17 g-PGA/g-biomass/ h and a PGA yield of 0.48 with respect to total substrate observed in the present study are much higher than the values reported in previously conducted studies.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Effect of aeration and agitation on the protease production by S<Emphasis Type="Italic">taphylococcus aureus</Emphasis> mutant RC128 in a stirred tank bioreactor

The modified rotating simplex method has been successfully used to determine the best combination of agitation rate and aeration rate for maximum production of extracellular proteases by Staphylococcus aureus mutant RC128, in a stirred tank bioreactor operated in a discontinuous way. This mutant has shown altered exoprotein production, specially enhanced protease production. Maximum production of proteases (15.28 UP/ml), measured using azocasein as a substrate, was obtained at exponential growth phase when the bioreactor was operated at 300 rpm and at 2 vvm with a volumetric oxygen transfer coefficient (K _L a) of 175.75 h⁻¹. These conditions were found to be more suitable for protease production.     

Optimization and scale up of itaconic acid production from potato starch waste in stirred tank bioreactor

Present study used Aspergillus terreus strain C1 isolated from mangrove soil for itaconic acid (IA) production from potato starch waste. Fermentation parameters were optimized by classical one factor approach and statistical experimental designs, such as Plackett-Burman and response surface designs. Anionic deionization of potato waste was found to be a very effective, economic, and easy way of improving IA production. The increase in IA production by deionization was found to correlate with removal of phosphate. In our knowledge, this is the first report on application of deionization of potato waste to enhance IA production. Other parameters like inoculum development conditions, pH, presence of peptone and certain salts in the medium also significantly affected IA production. IA production by strain C1 increased 143-fold during optimization when compared with the starting condition. The optimized IA level (35.75 g/L) was very close to the maximum production predicted by RSM (38.88 g/L). Bench scale production of IA was further optimized in 3-L stirred tank reactor by varying parameters like agitation and aeration rate. The maximum IA production of 29.69 g/L was obtained under the agitation speed of 200 rpm and aeration rate of 0.25 vvm. To the best of our knowledge, it is the first report on IA production from potato starch waste at bioreactor level. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2774, 2019.     

Effects of initial maltose concentration, agitation and aeration rates on the production of glucosyltransferase byAspergillus niger CCRC 31494

Summary The microorganism Aspergillus niger CCRC 31494 can produce an extracellular glucosyltransferase (GTase, EC 2.4.1.24) with a high transglucosylating activity. The maximal enzyme production occurred at initial maltose concentration of 40 gl^-1. The microorganism was also grown in a 5-liter jar fermenter for GTase production. It was found that the optimal agitation and aeration rates were 750 rev min^-1 and 1.0 l min^-1, respectively, and the enzyme production was about 0.25–0.26 units ml^-1.     

Oxygen requirements of Aspergillus awamori fungi in the process of glucoamylase biosynthesis

The oxygen requirements of Aspergillus awamori as well as the adaptation to it and the aeration of the cultivation medium were determined in the process of glucoamylase synthesis. Under the selected agitation and aeration conditions (impeller-tip speed = 4.2 m/s; aeration 1.5 vvm) the cultivation-medium aeration was analysed by means of dissolved-oxygen-concentration measurement during the course of the process. It was demonstrated that for obtaining the glucoamylase-activity level of 800 U GA/cm³ under the selected conditions and with the fungus applied the dissolved-oxygen concentration at the level of 25% saturation should be maintained. Those findings could serve as auxiliary indexes in the scale-up process of glucoamylase synthesis.     

Fuzzy Logic Control of Bioreactor for Enhanced Biosynthesis of Alkaline Protease by an Alkalophilic Strain of Bacillus subtilis

Present studies describe the optimization of some cultural parameters such as medium pH, incubation temperature, and agitation rate for the biosynthesis of alkaline protease by Bacillus subtilis IH-72 in a bioreactor using fuzzy logic control. The process of fermentation was carried out in a 7.5-L bioreactor (New Brunswick Scientific, USA) with a working volume of 5 L. All of the parameters were automatically controlled with the help of attached software. The optimum pH, temperature, and agitation for the production of alkaline protease by B. subtilis IH-72 were found to be 9.0, 35°C, and 175 rpm, respectively. The performance of the fuzzy logic of the bioreactor was found to be encouraging for enhanced production of the enzymes. The maximum production of alkaline protease during the present study was found to be 9.6 U mL⁻¹.     

Ribonuclease production by free and immobilizedAspergillus clavatus cells

Several fungal strains ofAspergillus andPenicillium were immobilized by cryopolymerization in polyvinyl alcohol cryogel beads.Aspergillus clavatus was the best producer of extracellular ribonuclease. Enzyme productivity and growth of free and immobilized cells in shake flasks and agitated bioreactor were studied. Ribonuclease production and growth behaviour depended on concentrations of glucose, peptone and soybean in the culture medium. Enzyme production was influenced by agitation and aeration intensity. In repeated batch, shake-flask cultures, the immobilized cells showed 2 to 3.5 times higher enzyme activity than free cells. The optimal conditions in a bioreactor were at 150 rev/min agitation speed and 0.5 vol/vol.min aeration. Enzyme productivity of immobilized cells (237 units/g dry mycelium.h) was 2.1 times higher than the productivity of free cells in a bioreactor, and 2.3 times higher than that of a shake-flask culture.R.J. Manolov is with the Institute of Microbiology, Department of Enzymes, Bulgarian Academy of Sciences, Georgy Bonchev Street 26, 1113 Sofia, Bulgaria.     

Effect of agitation rate on biomass and protease production by a marine bacterium Vibrio harveyi cultured in a fermentor

A marine bacterium Vibrio harveyi was adapted to grow and produce extracellular proteases in a seawater/Zobell-based medium, supplemented with skim milk under different hydrodynamic conditions, namely agitation and aeration rates. The addition of skim milk to Zobell medium enhanced fivefold the extracellular enzyme production. Protease production seemed to take place after maximum luminescence had been produced. Specific growth rate increased as a consequence of increasing agitation rates. The maximum activity of 4.28 units mg^–1 protein were formed with 700 rev min^–1 and 0.5 v/v/m. Protease activity detected has a molecular weight of 34 kDa. Another minor band of protease activity was found at 40 kDa.