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

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

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

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

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.

• MeSH
• biomasa MeSH
• Clostridium účinky léků   růst a vývoj   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• mastné kyseliny analýza   MeSH
• membránové lipidy chemie   MeSH
• n-butanol metabolismus   farmakologie   MeSH
• nenasycené mastné kyseliny analýza   MeSH
• plasmalogeny analýza   MeSH
• techniky vsádkové kultivace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mastné kyseliny MeSH
• membránové lipidy MeSH
• n-butanol MeSH
• nenasycené mastné kyseliny MeSH
• plasmalogeny MeSH

For n-butanol production by Clostridium pasteurianum DSM 525, a modified reinforced Clostridium medium was used, where glucose was alternated with glycerol and two kinds of continuous fermentation were tested using suspended and surface immobilized cells on corn stover pieces. A steady state, with butanol productivity of 4.2g/Lh, was reached during the packed-bed continuous fermentation at a dilution rate of 0.44h(-1). The average n-butanol concentration, yield and the ratio of n-butanol/liquid by-products were 10.4g/L, 33 % and 2.5, respectively. Unexpectedly, during continuous fermentation with suspended cells, at a dilution rate of 0.01h(-1), steady-state was not achieved and regular oscillations occurred in all measured variables, i.e. concentrations of glycerol, products and the number of cells stained with the fluorescent dyes carboxy fluorescein diacetate and propidium iodide. A possible explanation for oscillatory/steady-state behavior of suspended/surface-attached cells, respectively, may be specific butanol toxicity (toxicity per cell), which was higher/lower in respective cases, and which might be caused by lower/higher cell numbers respectively in both systems.

• Klíčová slova
• Clostridium pasteurianum, Flow cytometry, Glycerol fermentation, n-Butanol,
• MeSH
• bioreaktory MeSH
• biotechnologie metody   MeSH
• Clostridium cytologie   metabolismus   ultrastruktura   MeSH
• fermentace MeSH
• imobilizované buňky cytologie   metabolismus   ultrastruktura   MeSH
• kukuřice setá chemie   MeSH
• n-butanol metabolismus   MeSH
• odpadní produkty analýza   MeSH
• techniky vsádkové kultivace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• n-butanol MeSH
• odpadní produkty 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

Nitrosomonas europaea is a chemolithoautotrophic nitrifier, a gram-negative bacterium that can obtain all energy required for growth from the oxidation of ammonia to nitrite, and this may be beneficial for various biotechnological and environmental applications. However, compared to other bacteria, growth of ammonia oxidizing bacteria is very slow. A prerequisite to produce high cell density N. europaea cultures is to minimize the concentrations of inhibitory metabolic by-products. During growth on ammonia nitrite accumulates, as a consequence, N. europaea cannot grow to high cell concentrations under conventional batch conditions. Here, we show that single-vessel dialysis membrane bioreactors can be used to obtain substantially increased N. europaea biomasses and substantially reduced nitrite levels in media initially containing high amounts of the substrate. Dialysis membrane bioreactor fermentations were run in batch as well as in continuous mode. Growth was monitored with cell concentration determinations, by assessing dry cell mass and by monitoring ammonium consumption as well as nitrite formation. In addition, metabolic activity was probed with in vivo acridine orange staining. Under continuous substrate feed, the maximal cell concentration (2.79 × 10(12)/L) and maximal dry cell mass (0.895 g/L) achieved more than doubled the highest values reported for N. europaea cultivations to date.

• MeSH
• biomasa MeSH
• bioreaktory * MeSH
• chemoautotrofní růst * MeSH
• dusitany metabolismus   MeSH
• fermentace MeSH
• kinetika MeSH
• Nitrosomonas europaea růst a vývoj   metabolismus   MeSH
• techniky vsádkové kultivace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dusitany MeSH

Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L-1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L-1 contained 42% PHB.

• Klíčová slova
• Cupriavidus necator, Grass silage, Green biorefinery, Lactic acid fermentation, Poly-3-hydroxybutyrate,
• MeSH
• bioreaktory MeSH
• Cupriavidus necator MeSH
• fermentace * MeSH
• hydroxybutyráty metabolismus   MeSH
• lipnicovité * MeSH
• polyestery metabolismus   MeSH
• siláž MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hydroxybutyráty MeSH
• poly-beta-hydroxybutyrate MeSH Prohlížeč
• polyestery MeSH