Simultaneous biosynthesis of (+)-geodin by a lovastatin-producing fungus Aspergillus terreus

The simultaneous biosynthesis of lovastatin (mevinolinic acid) and (+)-geodin by Aspergillus terreus ATCC 20542 with regard to the medium composition, i.e. the concentrations of carbon and nitrogen source, was described in this paper. A. terreus is a lovastatin producer but the formation of lovastatin was accompanied by the significant amounts of (+)-geodin, when the elevated concentration of carbon source (lactose) was still present in the medium in the idiophase and nitrogen source (yeast extract) was deficient. It was observed for runs, in which the higher (above 20 g l(-1)) initial lactose concentration was applied or when the nitrogen deficiency led to the decrease of biomass content in the system. In contrast to lovastatin, there was not optimum initial concentration of yeast extract, as its lowest tested initial concentration (2 g l(-1)) led to the highest (+)-geodin volumetric formation rates and final yield. What is more, even higher final (+)-geodin concentrations were achieved at elevated initial lactose concentration (40 g l(-1)) and in the lactose-fed fed-batch run. In the fed-batch run lovastatin concentration increased significantly too, as this metabolite formation is also carbon source dependent. Finally, (+)-geodin occurred to be a metabolite, whose formation, in contrast to lovastatin, is non-growth associated.     

Increased lovastatin formation by Aspergillus terreus using repeated fed-batch process

Lovastatin biosynthesis with Aspergillus terreus in batch fermentation reached 160 U/l in 161 h at pH 6.8 and a dissolved O tension maintained at 70%. At the end of repeated fed batch fermentations, the yield of lovastatin was increased by 37% though this took over twice as long as in the batch fermentation.     

Enhanced production of valienamine by Stenotrophomonas maltrophilia with fed-batch culture in a stirred tank bioreactor

Valienamine is an important medicinal intermediate with broad use in the synthesis of some stronger α-glucosidase inhibitors. In order to improve valienamine concentration in the fermentation broth and make the downstream treatment easy, a fed-batch process for the enhanced production of valienamine by Stenotrophomonas maltrophilia in a stirred tank bioreactor was developed. Results showed that supplementation of validamycin A in the process of cultivation could increase the valienamine concentration. One-pulse feeding was observed to be the best strategy. The maximum valienamine concentration of 2.35 g L⁻¹ was obtained at 156 h when 86.4 g of validamycin A was added to a 15-L bioreactor containing 8 L fermentation medium with one-pulse feeding. The maximum valienamine concentration had a great improvement and was increased above 100% compared to batch fermentation in the stirred tank bioreactor. The pH-controlled experiments showed that controlling the pH in the process of one-pulse feeding fermentation had not obvious effect on the production of valienamine.     

Itaconic acid production using an air-lift bioreactor in repeated batch culture of Aspergillus terreus

Repeated itaconic acid production using an air-lift bioreactor was carried out by three methods—two with cell recycling by means of centrifugation and filtration by a stainless steel filter set inside the bioreactor and one by repeated batch culture without cell recycling. In a flask culture, repeated itaconic acid production was stable for 9 cycles (45 d) and the production rate was 0.47 g/l/h. However, in the air-lift bioreactor, it was difficult to produce itaconic acid in the repeated batch culture with cell recycling for a long period due to a decrease in fluidity resulting from an increase in mycelium concentration. In the method without cell recycling, however, repeated itaconic acid production was stable for 4 cycles (21 d) and the production rate was 0.37 g/l/h.     

Comparison of air-lift and stirred tank reactors for itaconic acid production by Aspergillus terreus

In a 2-l stirred tank reactor (STR), maximum production rate ofitaconic acid was 0.48g/l.h , for an agitation rate of 400 rpm andan aeration rate of 0.5 vvm. In an air-lift reactor (ALR) themaximum production rate was 0.64 g/l.h at an O supply rate of0.41 l O /l. min. Power input per unit volume which gave themaximum production rates for STR and ALR were 1180 and 542 W/m 3,respectively. If O -enriched air was used in place of air for ALR,the corre-sponding power input per unit volume was decreased to 34W/m 3 . ALR requires less power input per unit volume in comparisonwith that of STR whether therefore air or O -enriched air is used.ALR would be a suitable bioreactor for a large production of itaconicacid.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

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.     

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of approximately 1200 microm initial diameter were reduced to a final stable size of approximately 900 microm when the agitation intensity was >/=600 rpm (u(t)>/=2.03 ms(-1)). A stable pellet diameter of approximately 2500 microm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.     

Production of the biocontrol agent Pantoea agglomerans PBC-1 in a stirred tank reactor by batch and fed-batch cultures

Concerns about food safety as well as the development of resistance to many fungicides by major postharvest pathogens have increased recently. Biological control, using microorganisms antagonistic to the fungal plant pathogens, appears to be promising as an alternative to fungicides. The microbial biocontrol agent has to be produced on an industrial scale, maintaining its biocontrol efficacy. The purpose of the current study was to optimize the conditions for microbial biomass production of the biocontrol agent Pantoea agglomerans PBC-1 in a 2-l mechanically stirred reactor (STR), defining mixing and mass transfer technological parameters and the growth kinetics for different saccharides. In the batch mode, different impellers and spargers were tested. Despite the oxygen mass transfer improvement achieved with marine propeller combined with porous sparger, the biomass did not increase, if compared with the use of a Rushton turbine and L-sparger, pointing out the relevance of a radial flux for better broth homogenization. Different carbon sources were used: sucrose, glucose and fructose; each of which led to viable populations 3.9 × 10⁹, 1.4 × 10⁹, 3.9 × 10⁹ c.f.u/ml, respectively, after 20 h of incubation. Fed-batch technology allows the maintenance of high cell viability for longer periods of time in the stationary growth phase, which can be crucial for the scale-up of biocontrol agent production process that is achieved together with a reduction of 85% on the incidence caused by the pathogens, brought about by fresh microbial biomass preparation on artificially wounded apples or oranges, stored for 7 days at 25°C against Penicillium expansum and Penicillium digitatum.     

A macrokinetic modelling of the biosynthesis of lovastatin by Aspergillus terreus

In this work a simple kinetic model to describe the biosynthesis of lovastatin by Aspergillus terreus ATCC 20542 was proposed. Several series of experiments were conducted at different media compositions. The concentrations of C- and N-sources were changed over a wide range and so were the initial biomass concentrations. From these runs the relationships ruling the substrates uptake, biomass and product formation were learnt. Lovastatin biosynthesis appeared to be partly growth associated. The inhibitive effect of organic nitrogen on lovastatin biosynthesis was found and lactose appeared to be an important limiting substrate in the formation of lovastatin. The parameters of the model were evaluated on the basis of the kinetic data obtained in the separate experiments made in triplicate at two chosen media compositions. Other results obtained at different media compositions were independent of the ones mentioned above and used for the verification of the model. The validity of the model was also examined for the lactose-fed fed-batch run. Finally, a sensitivity analysis of the model parameters was performed. The formulated model, although relatively simplified, described the experimental data quite well and could be regarded as the background for further attempts to mathematically describe the process of lovastatin biosynthesis.     

Production of lovastatin by a wild strain of Aspergillus terreus

Of 68 Aspergillus terreus, three produced lovastatin with equivalent or better yield than strain ATCC 20542 originally described for lovastatin production. Medium optimization experiments with the best isolate (TUB F-514) indicated that lactose, rapeseed meal and KNO3 were the best carbon, organic nitrogen and inorganic nitrogen sources, respectively. In shake-flasks with optimized medium containing 4 % (w/v) lactose, 400 g lovastatin/ml was produced, with a yield of 10 mg/g lactose. In solid substrate fermentation on extracted sweet sorghum pulp supplemented with cheese whey 1500 g lovastatin/g dry weight was produced with a yield of 37.5 mg/g lactose. © Rapid Science Ltd. 1998     

Lovastatin biosynthesis by Aspergillus terreus with the simultaneous use of lactose and glycerol in a discontinuous fed-batch culture

The influence of various combinations of glycerol and lactose feed on the biosynthesis of two polyketide metabolites, lovastatin and (+)-geodin, by Aspergillus terreus ATCC20542 in a discontinuous fed-batch culture was presented. In these experiments lactose and/or glycerol were also used as the initial carbon substrates in the cultivation media. The application of glycerol feed, when lactose is the initial substrate, leads to the appreciable lovastatin concentration in the broth (122.4 mg l⁻¹), nevertheless the abundant (+)-geodin level is at the same time obtained (255.5 mg l⁻¹). The cultures with glycerol as the initial substrate and fed with lactose produce less lovastatin and (+)-geodin. The application of the various combined glycerol and/or lactose feeds allows for improving lovastatin production up to 161.8 mg l⁻¹ and decreases (+)-geodin concentration to 98.7 mg l⁻¹. The analysis of product formation rates and yield coefficients indicates that lovastatin is more efficiently produced on lactose, especially in the initial stages of the cultivation. Glycerol efficiently sustains fungal activity to form these polyketides in the late idiophase but it mainly favours (+)-geodin formation, if solely used in the feed. The feeds performed both with lactose and glycerol occur to be the most desired to maximise lovastatin and minimise (+)-geodin formation.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

A cartridge oxygenator for mammalian cell culture in a stirred tank bioreactor

Summary Tubing made of membrane with high oxygen permeability is often used in supplying oxygen to animal cell culture bioreactors. We have fabricated the tubing into a cartridge configuration. Such an arrangement allows damaged tubing to be replaced conveniently and eases the maintenance of such an oxygenator in bioreactors.     

Physiological,morphological and kinetic aspects of lovastatin biosynthesis by Aspergillus terreus

This review focuses on selected aspects of lovastatin biosynthesis by Aspergillus terreus. Biochemical issues concerning this process are presented to introduce polyketide metabolites, in particular lovastatin. The formation of other than lovastatin polyketide metabolites by A. terreus is also shown, with special attention to (+)-geodin and sulochrin. The core of this review discusses the physiology of A. terreus with regard to the influence of carbon and nitrogen sources, cultivation broth aeration and pH control strategies on fungal growth and product formation. Attention is paid to the supplementation of cultivation media with various compounds, namely vitamins, methionine, butyrolactone I. Next, the analysis of fungal morphology and differentiation of A. terreus mycelium in relation to both lovastatin and to (+)-geodin formation is conferred. Finally, the kinetics of the process, in terms of associated metabolite formation with biomass growth is discussed in relation to published kinetic models. The review concludes with a list of the most important factors affecting lovastatin and (+)-geodin biosynthesis.     

In situ filtration of Anchusa officinalis culture in a cell-retention stirred tank bioreactor

Summary The effect of agitation and aeration on filtration of Anchusa officinalis culture in a stirred tank bioreactor integrated with an internal filter unit was investigated. Increases in suction head of the pump that drove the filtration process were measured at impeller speeds of 100 and 200 rpm. Surprisingly, suction head attained at 200 rpm was about 40% higher than at 100 rpm. Direct observation of the cake deposition process in the reactor using a dilute cell suspension revealed that the filter cake formed at 100 rpm was thicker, but less compact. Aeration at 0.4 vvm was shown to have little effect on the filtration rate, since the bulk fluid flow was dominated by the impeller hydrodynamics. The initial flux can be recovered by filter backwashing with compressed air at a flow rate of 0.6 vvm for a duration of 5 minutes.     

Enhanced production of (+)-terrein in fed-batch cultivation of Aspergillus terreus strain PF26 with sodium citrate

(+)-Terrein is a fungal metabolite with multiple biological activities, especially with great value in medicine. However, the mass production of single configuration terrein is still a big challenge. In this study, the effects of acetic acid, sodium acetate, citric acid and sodium citrate on the (+)-terrein production by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca were investigated. Sodium citrate was selected for fed-batch cultivation because it showed the best effect on (+)-terrein production among the four regulators tested. As a result, 5.38 g/L (+)-terrein production was achieved by feeding 10 mM sodium citrate on the 3rd day in shake flask, which was 33.8 % higher than the control and represented the highest yield of (+)-terrein. In a 7.5-L stirred bioreactor, 2.58 g/L of (+)-terrein production was achieved by the feeding of 10 mM sodium citrate on the 8th day. The results from this study lay a basis for the high-yield production of (+)-terrein by fermentation.     

Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Response surface methodology for lovastatin production by Aspergillus terreus GD13 strain

A wild type Aspergillus terreus GD13 strain, chosen after extensive screening, was optimized for lovastatin production using statistical Box-Behnken design of experiments. The interactive effect of four process parameters, i.e. lactose and soybean meal, inoculum size (spore concentration) and age of the spore culture, on the production of lovastatin was evaluated employing response surface methodology (RSM). The model highlighted the positive effect of soybean meal concentration and inoculum level for achieving maximal level of lovastatin (1342 mg/l). The optimal fermentation conditions improved the lovastatin titre by 7.0-folds when compared to the titres obtained under unoptimized conditions.