Penicillin production during a fermentation process using industrial strains of Penicillium chrysogenum is a research topic permanently discussed since the accidental discovery of the antibiotic. Intact cell mass spectrometry (ICMS) can be a fast and novel monitoring tool for the fermentation progress during penicillin V production in a nearly real-time fashion. This method is already used for the characterization of microorganisms and the differentiation of fungal strains; therefore, the application of ICMS to samples directly harvested from a fermenter is a promising possibility to get fast information about the progress of fungal growth. After the optimization of the ICMS method to penicillin V fermentation broth samples, the obtained ICMS data were evaluated by hierarchical cluster analysis or an in-house software solution written especially for ICMS data comparison. Growth stages of a batch and fed-batch fermentation of Penicillium chrysogenum are differentiated by one of those statistical approaches. The application of two matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instruments in the linear positive ion mode from different vendors demonstrated the universal applicability of the developed ICMS method. The base for a fast and easy-to-use method for monitoring the fermentation progress of P. chrysogenum is created with this ICMS method developed especially for fermentation broth samples.

• Klíčová slova
• Batch, Fed-batch, Fermentation, Intact cell mass spectrometry, Penicillin V, Penicillium chrysogenum,
• MeSH
• časové faktory MeSH
• fermentace * MeSH
• hmotnostní spektrometrie * MeSH
• Penicillium chrysogenum cytologie   růst a vývoj   MeSH
• techniky vsádkové kultivace přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Clostridium acetobutylicum immobilised in polyvinylalcohol, lens-shaped hydrogel capsules (LentiKats(®)) was studied for production of butanol and other products of acetone-butanol-ethanol fermentation. After optimising the immobilisation protocol for anaerobic bacteria, continuous, repeated batch, and fed-batch fermentations in repeated batch mode were performed. Using glucose as a substrate, butanol productivity of 0.41 g/L/h and solvent productivity of 0.63 g/L/h were observed at a dilution rate of 0.05 h(-1) during continuous fermentation with a concentrated substrate (60 g/L). Through the process of repeated batch fermentation, the duration of fermentation was reduced from 27.8h (free-cell fermentation) to 3.3h (immobilised cells) with a solvent productivity of 0.77 g/L/h (butanol 0.57 g/L/h). The highest butanol and solvent productivities of 1.21 and 1.91 g/L/h were observed during fed-batch fermentation operated in repeated batch mode with yields of butanol (0.15 g/g) and solvents (0.24 g/g), respectively, produced per gram of glucose.

• Klíčová slova
• Butanol, Clostridium acetobutylicum, Fermentation, Immobilisation, LentiKats(®),
• MeSH
• aceton metabolismus   MeSH
• anaerobióza MeSH
• butanoly metabolismus   MeSH
• Clostridium acetobutylicum cytologie   metabolismus   MeSH
• ethanol metabolismus   MeSH
• fermentace * MeSH
• imobilizované buňky metabolismus   MeSH
• techniky vsádkové kultivace metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aceton MeSH
• butanoly MeSH
• ethanol MeSH

The aim of this study was to evaluate the impact of short-term repeated exposure to a static magnetic field (induction 370 mT) on the Rhodococcus erythropolis cells. Specifically, it was ascertained the magnetic field's potential to influence degradation of a phenol substrate, cell growth and respiration activity (oxygen consumption) during substrate biodegradation. The experiment took place over 3 days, with R. erythropolis exposed to the magnetic field for the first day. During the experiment, different recirculation rates between the reactor and the magnetic contactor has been tested. Use of the magnetic field at higher recirculation rates (residence time in contactor was less than 7 min) stimulated substrate (phenol) oxidation by around 34%; which, in turn, promoted R. erythropolis growth by around 28% by shortening the lag- and exponential-phases and increasing bacterial respiration activity by around 10%.

• Klíčová slova
• Bacterial degradation enhanced, Fed-batch bioreactor, Phenol biodegradation, Rhodococcus erythropolis, Static magnetic field,
• MeSH
• aerobióza MeSH
• biodegradace MeSH
• bioreaktory mikrobiologie   MeSH
• fenol metabolismus   MeSH
• magnetické pole * MeSH
• počítačová simulace MeSH
• Rhodococcus růst a vývoj   metabolismus   MeSH
• techniky vsádkové kultivace přístrojové vybavení   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fenol MeSH

BACKGROUND: Inhibitors that are released from lignocellulose biomass during its treatment represent one of the major bottlenecks hindering its massive utilization in the biotechnological production of chemicals. This study demonstrates that negative effect of inhibitors can be mitigated by proper feeding strategy. Both, crude undetoxified lignocellulose hydrolysate and complex medium supplemented with corresponding inhibitors were tested in acetone-butanol-ethanol (ABE) fermentation using Clostridium beijerinckii NRRL B-598 as the producer strain. RESULTS: First, it was found that the sensitivity of C. beijerinckii to inhibitors varied with different growth stages, being the most significant during the early acidogenic phase and less pronounced during late acidogenesis and early solventogenesis. Thus, a fed-batch regime with three feeding schemes was tested for toxic hydrolysate (no growth in batch mode was observed). The best results were obtained when the feeding of an otherwise toxic hydrolysate was initiated close to the metabolic switch, resulting in stable and high ABE production. Complete utilization of glucose, and up to 88% of xylose, were obtained. The most abundant inhibitors present in the alkaline wheat straw hydrolysate were ferulic and coumaric acids; both phenolic acids were efficiently detoxified by the intrinsic metabolic activity of clostridia during the early stages of cultivation as well as during the feeding period, thus preventing their accumulation. Finally, the best feeding strategy was verified using a TYA culture medium supplemented with both inhibitors, resulting in 500% increase in butanol titer over control batch cultivation in which inhibitors were added prior to inoculation. CONCLUSION: Properly timed sequential feeding effectively prevented acid-crash and enabled utilization of otherwise toxic substrate. This study unequivocally demonstrates that an appropriate biotechnological process control strategy can fully eliminate the negative effects of lignocellulose-derived inhibitors.

• Klíčová slova
• ABE fermentation, Butanol, Clostridium, Fed batch, Ferulic and coumaric acid, Inhibitors, Lignocellulose hydrolysate, Salinity, Wheat straw,
• Publikační typ
• časopisecké články MeSH

The pharmaceutical production of recombinant proteins, such as monoclonal antibodies, is rather complex and requires proper development work. Accordingly, it is essential to develop appropriate scale-down models, which can mimic the corresponding production scale. In this work, we investigated the impact of the bioreactor scale on intracellular micro-heterogeneities of a CHO cell line producing monoclonal antibodies in fed-batch mode, using a 10 mL micro-bioreactor (ambr™) scale-down model and the corresponding 300 L pilot-scale bioreactor. For each scale, we measured the time evolution of the proteome, which enabled us to compare the impact of the bioreactor scale on the intracellular processes. Nearly absolute accordance between the scales was verified by data mining methods, such as hierarchical clustering and in-detail analysis on a single protein base. The time response of principal enzymes related to N-glycosylation was discussed, emphasizing major dissimilarities between the glycan fractions adorning the heavy chain and the corresponding protein abundance. The enzyme expression displayed mainly a constant profile, whereas the resulting glycan pattern changed over time. It is concluded that the enzymatic activity is influenced by the changing environmental conditions present in the fed-batch processes leading to the observed time-dependent variation.

• Klíčová slova
• CHO, Fed-batch, Micro-bioreactor, Monoclonal antibody, Proteomics, Scale-down model,
• MeSH
• biologické modely * MeSH
• bioreaktory * MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• glykosylace MeSH
• křečci praví MeSH
• monoklonální protilátky metabolismus   MeSH
• proliferace buněk MeSH
• proteomika metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• shluková analýza MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• monoklonální protilátky MeSH
• rekombinantní proteiny MeSH

Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for β-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for β-glucosidase production. The results showed that at 72 h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55 g/L, respectively, while the total mutant β-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60 h of cultivation, the highest total mutant β-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant β-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73 g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 gcell/gMeOH, methanol consumption rate ([Formula: see text]) 5.76 gMeOH/h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and β- glucosidase activity were produced under exponential feed rate than constant and linear feed rates.

• Klíčová slova
• Exponential feed rate, Fed-batch fermentation, Methanol feeding profile, Pichia pastoris, β-glucosidase,
• MeSH
• bioreaktory MeSH
• celulasy * metabolismus   MeSH
• fermentace MeSH
• methanol * metabolismus   MeSH
• Pichia metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• celulasy * MeSH
• methanol * MeSH
• rekombinantní proteiny MeSH

The basidiomycetous yeastPhaffia rhodozyma was grown in batch, fed-batch and continuous culture, and some parameters governing growth and total carotenoid production were determined.

• Publikační typ
• časopisecké články MeSH

Analysis of the kinetics of alpha-amylase production in a batch and a fed-batch culture of Bacillus subtilis made it possible to derive a kinetic model of the process describing mutual interactions between growth and production. The specific growth rate is limited by the concentration of both corn-steep liquor and starch. Higher concentrations of reducing sugars in the medium also inhibit growth. The overall production of alpha-amylase is a result of an equilibrium between the rate of enzyme production and its degradation due to the effect of environment. The actual specific production rate is directly proportional to the specific growth rate (characterizing the physiological state of the culture) and is inhibited by higher concentrations of corn-steep liquor in the medium.

• MeSH
• alfa-amylasy biosyntéza   MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriologické techniky MeSH
• kinetika MeSH
• kultivační média MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-amylasy MeSH
• kultivační média MeSH

Saccharomyces cerevisiae with an increased content of ergosterol or delta 5,7-sterols, growing on a molasses medium with a feed of ethanol and (NH4)2HPO4, was analyzed as to the age of cell population. The analysis was done by centrifugation in a dextran gradient and by a fluorescence-microscopic technique. In the phase of batch fermentation at a mean specific growth rate of 0.22 h-1 daughter cells contained less than 1% ergosterol while the ergosterol content of mother cells depended on the time of cultivation, a maximum level (4%) being found after two generation times. In the fed-batch phase at a mean growth rate of 0.052 h-1, both daughter and mother cells contained about the same amount of ergosterol (4.7-5.5%). Differences between daughter and mother cells are discussed in view of the relationship between the growth rate and the growth cycle.

• MeSH
• časové faktory MeSH
• ergosterol biosyntéza   MeSH
• fermentace MeSH
• kinetika MeSH
• Saccharomyces cerevisiae růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ergosterol MeSH

The physiology of a commercial strain of bakers' yeast was studied in terms of the cell composition under different growth conditions and of its response to stress. The study comprised fed-batch experiments since this is the system used in bakers' yeast industry. The classical fed-batch fermentation procedure was modified in that the yeast cells were continuously grown to a steady-state at a dilution rate of 0.1/h in order to achieve more or less the same initial starting point in terms of cell composition. This steady-state culture was then switched to fed-batch concomitantly with exposure to stress. The highest amount of trehalose accumulation was achieved when nutrient depletion and heat stress were applied concomitantly. The highest amount of trehalose, 12%, was attained in cells stressed by both nitrogen depletion and heat stress. The protein content remained constant, although with some oscillations, at a value of 30% throughout this dual stress experiment.

• MeSH
• biomasa MeSH
• dusík nedostatek   MeSH
• fosfáty nedostatek   MeSH
• fungální proteiny analýza   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• dusík MeSH
• fosfáty MeSH
• fungální proteiny MeSH