Department of Fermentation Chemistry and Bioengineering Institute of Chemical Technology Prague Technicka 5 166 28 Prague 6 Czech Republic
tomas.branyik@vscht.cz
1989-1994 MSc degree (Ing.), Institute of Chemical Technology (ICT) Prague. 1994-1999 Postgraduate study (PhD), ICT Prague and Intitute of Chemical Process Fundamentals ASCR. 2000-2004 Post-doc, University of Minho, Braga, Portugal. 2004-2008 Assistant Professor at the Department of Fermentation Chemistry and Bioengineering, ICT Prague. since 2008 Associate Professor in the field of Biotechnology, ICT Prague.
Current Research Topics: Surface interactions of microorganisms (adhesion, immobilization, biofilms, flocculation), biotechnology of microalgae, continuous bioreactors, formation and decay of beer foam.
Abstract: The increasing interest of consumers in health and alcohol abuse issues motivates breweries to expand the assortment of products with low alcohol content. The goal of producing beers with low alcohol content can be achieved by two main strategies: namely by gentle removal of alcohol from regular beer and by limited ethanol formation during the beer fermentation. Within these two basic strategies, there are a number of techniques that vary in performance, efficiency and usability. This paper presents an overview and comparison of these techniques and provides an evaluation of sensorial properties of low-alcohol and an alcohol-free beer produced as well as suggests possibilities for their additional improvement. (C) 2011 Elsevier Ltd. All rights reserved.
Abstract: The aim of this study is to demonstrate the influence of production strains (bottom-fermenting Saccharomyces pastorianus and S. cerevisiae with disruption in the KGD2 gene), carrier materials (spent grains and corncobs), reactor arrangements (packed-bed and gas-lift reactors), and mixing regimes (ideally mixed and plug flow) on the formation of flavor-active compounds during alcohol-free beer production. In addition, the composition of alcohol-free beer produced on a laboratory scale was compared with those of commercial products. The results confirmed the influence of each component of the production system (yeast strain, reactor, and carrier) on flavor formation, but their individual importance was case specific. The results indicate that the interplay between the appropriate production strain, carrier material, and bioreactor design is very important in continuous immobilized cell reactors and that suitable combinations could be used to improve both system performance and product quality.
Abstract: J. Inst. Brew. 117(3), 368-376, 2011 In order to understand how xanthohumol affects a brewing yeastâs metabolism, yeast viability and vitality were studied during the production of a xanthohumol enriched beer (10 mg/L xanthohumol) on a 50 L pilot plant scale. The results showed that yeast viability was not significantly affected, but yeast vitality in the xanthohumol enriched brewing trials was slightly better. The content of higher alcohols, esters and organic acids was similar to the control in all the xanthohumol enriched brewing trials, however the content of sulphur dioxide, acetaldehyde and saturated fatty acids was lower in the xanthohumol enriched brewing trials. To the authorsâ knowledge, this is the first time brewing trials have been carried out showing that xanthohumol has a positive effect on a yeastâs physiological condition.
Abstract: One of the industrially important qualities of yeast is their ability to provide the cellâcell and cellâsupport interactions. This feature of yeast is responsible for technologically significant phenomena such as flocculation (brewing) and yeast biofilm formation (immobilization to supports), whereas these phenomena are time, environment, and strain dependent. Therefore, the goal of this work was to verify the possibility to predict and subsequently select yeast strains capable to colonize solid supports by using physicochemical adhesion models. Three different industrial yeast strains (Saccharomyces cerevisiae) were tested for their adhesion onto spent grain particles in the continuous gas-lift reactor. The cell adhesion energies were calculated, based on physicochemical characteristics of surfaces involved, according to three adhesion models (DLVO theory, thermodynamic approach, and extended DLVO theory). The role of physicochemical surface properties in the cellâcell and cellâsupport interactions was evaluated by comparing the computed predictions with experimental results. The best agreement between forecast and observation of the yeast adhesion to spent grains was achieved with the extended DLVO (XDLVO) theory, the most complex adhesion model applied in this study. Despite its relative comprehensiveness, the XDLVO theory does not take into account specific biochemical interactions. Consequently, additional understanding of the yeast adhesion mechanism was obtained by means of quantifying the expression of selected FLO genes. The presented approach provides tools to select the appropriately adhesive yeast strains and match them with solid supports of convenient surface properties in order to design immobilized biocatalysts exploitable in biotechnological processes.
Abstract: Iso-alpha-acids or isohumulones are hop-derived beer constituents formed in the brewing process. Iso-alpha-acids are light-sensitive; they play a key role in the origin of the well-known light-struck off-flavour of beer. To prevent the negative effect of light, beers are generally stored in light-proof cans or dark bottles. Alternatively, reduced iso-alpha-acids, resistant to light-induced degradation, can be added to beer. These substances also show a positive effect on foam stability. This paper describes the impact of iso-alpha-acids, reduced iso-alpha-acids, tetrahydro-iso-alpha-acids, and hexahydro-iso-alpha-acids on brewing; their food legislation frame is also mentioned. Chemical and physical methods of analysis of the substances are also reviewed.
Abstract: An expeditious method of yeast age estimation was developed based on selective bud scar staining (Alexa Fluor 488-labelled wheat-germ agglutinin) and subsequent fluorescence intensity measurement by flow cytometry. The calibration Curve resulting from the cytometric determination of average bud scar fluorescence intensities vs. microscopically counted average bud scar numbers of the same cell populations showed a good correlation and allowed routine cell age estimation by flow cytometry. The developed method was applied for yeast age control in traditional batch and continuous beer fermentations. At the pitching rates used in industrial beer fermentations, our results support former findings by locating a gradient of increasing yeast age from the top to the bottom zone of the fermenter cone. The results also indicate that in continuous beer fermentation, the increasing bud scar fluorescence of immobilized Cells Could help to schedule the replacement of aged biomass, prior to loss of viability or deterioration of process performance and product quality.
Abstract: This work presents a yeast-cell vitality-assessment method based on on-line intracellular fluorescence measurement. The intracellular NAD(P)H fluorescence of a cell suspension is recorded during transition from aerobic to anaerobic conditions and the output signal is evaluated as a measure of yeast vitality (quality). This fluorescence method showed a highly satisfactory correlation with even low dead cell numbers where the acidification power test could not be applied.
Abstract: In order to study the formation and conversion of the most important flavour compounds, the real wort used in alcohol-free beer fermentation was mimicked by a complex model medium containing glucose, yeast extract, and selected aldehydes. The fermentation experiments were carried out in a continuously operating gas-lift reactor with brewing yeast immobilised on spent grains (brewing by-product). During the continuous experiment, parameters such as oxygen supply, residence time (Rt), and temperature (T) were varied to find the optimal conditions for the alcohol-free beer production. The formation of ethanol, higher alcohols (HA), esters (ES), as well as the reduction of aldehydes and consumption of glucose were observed. The results suggest that the process parameters represent a powerful tool in controlling the degree of fermentation and flavour formation brought about by immobilised biocatalyst. Subsequently, the optimised process parameters were used to produce real alcohol-free beer during continuous fermentation. The final product was compared with batch fermented alcohol-free beers using the methods of instrumental and sensorial analysis.
Abstract: This study deals with two innovative brewing processes, high gravity batch and complete continuous beer fermentation systems. The results show a significant influence of the variables such as concentration and temperature on the yield factor of the substrate into ethanol and consequently on the productivity of the high gravity batch process. The technological feasibility of continuous production of beer based on yeast immobilization on cheap alternative carriers was also demonstrated. The influence of process parameters on fermentation performance and quality of the obtained beers was studied by sensorial analysis. No significant difference in the degree of acceptance between the obtained products and some traditional market brands was found. (c) 2008 Institute of Chemistry, Slovak Academy of Sciences.
Abstract: The influence of oxygen supply oil the formation and conversion of the most important flavor compounds during Continuous, alcohol-free beer production was studied in a complex model medium. The medium contained pro-organic salts, nutrients, and aldehydes (hexanal, 2-methyl propanal, 3-methyl butanal, and furfural) and mimicked real brewery wort, with the advantage of a constant composition. Fermentation experiments were carried out in it continuously operating gas-lift reactor, with brewing yeast immobilized oil spent grains. The formation (ethanol, higher alcohols, esters, vicinal diketones, and acetaldehyde) and reduction (aldehydes) of flavor-active compounds at different aeration rates were observed. The results suggest that the oxygen supply represents all influential tool for controlling the degree of fermentation and flavor formation carried out by an immobilized biocatalyst. Under optimal oxygen Supply conditions in the continuously operating gas-lift reactor, it was possible to obtain a fermented model medium with a composition approaching that of commercial alcohol-free beers produced by batch process.
Abstract: The attractive prospect of a continuous beer fermentation system consists mostly of the accelerated transformation of wort into beer. Although continuous beer fermentation has been studied as a promising technology for several decades, the number of industrial applications is still limited. The major obstacle hindering the extensive industrial exploitation of this technology is the difficulty in achieving the correct balance of sensory compounds in the short time typical for continuous systems. This paper offers an integral view on the particularities of continuous systems, which may impart beer a sensorial character distinct from conventionally fermented counterparts. The main groups of flavour active compounds are discussed from the perspective of possible control strategies by means of process parameters and strain selection.
Abstract: Despite extensive research carried out in the last few decades, continuous beer fermentation has not yet managed to. outperform the traditional batch technology. An industrial breakthrough in favour of continuous brewing using immobilized yeast could be expected only on achievement of the following process characteristics: simple design, low investment costs, flexible operation, effective process control and good product quality. The application of cheap carrier materials of by-product origin could significantly lower the investment costs of continuous fermentation systems. This work deals with a complete continuous beer fermentation system consisting of a main fermentation reactor (gas-lift) and a maturation reactor (packedbed) containing yeast immobilized on spent grains and corncobs, respectively. The suitability of cheap carrier materials for long-term continuous brewing was proved. It was found that by fine tuning of process parameters (residence time, aeration) it was possible to adjust the flavour profile of the final product. Consumers considered the continuously fermented beer to be of a regular quality. Analytical and sensorial profiles of both continuously and batch fermented beers were compared.
Abstract: A one stage continuous primary beer fermentation consisting of brewing yeast immobilized on spent grain particles in a gas lift reactor was studied. The goal of this work was to adjust the flavor of the continuously produced green beer to the desired character by sparging an adequate amount of air and by controlling the fermentation temperature in the immobilized yeast reactor as well as to predict the rate of the brewing yeast immobilization using a kinetic model adapted to the conditions of beer fermentation. The volumetric productivity of the continuous system was approximately 5 times higher than of the batch fermentation. The aroma profile of green beer from the continuous immobilized fermentation, run at zero air flow and temperatures 13 16°C, was fully comparable to that produced by industrial batch technology. Generally, the diacetyl concentration in green beer from the continuous fermentation was higher than in batch process, however, its re-assimilation was enhanced by high total biomass concentration in the system.
Abstract: A packed bed reactor (PBR) design was tested for the purpose of continuous pectinase production with yeasts, as a possible alternative to the traditional batch process using fungal cultures. Two different carriers - a porous glass (Siran) and a cellulosic carrier obtained from spent grains (barley) - were used to immobilize Kluyveromyces marxianus CCT 3172, a yeast strain secreting endopolygalacturonase. To improve cell distribution throughout the column, part of the outflow was recycled. Cell loads of 0.204 and 0.247 g(biomass)/g(carrier) were obtained at the top and bottom of the PBR with spent grains, respectively. Using the PBR with Siran as the immobilization support, 0.071 g(biomass)/g(carrier) was the biomass load at the top of the column while at the bottom a value of 0.147 g(biomass)/g(carrier) was found. The highest value for pectinase volumetric productivity (Pv = 1.68 U/ml h) was achieved in the PBR with Siran for a D = 0.260 h(-1) and a glucose concentration on the inlet of S-in = 40 g/l. Both carriers were suitable for pectinase production. The best results were obtained with a high and uniform biomass concentration in the column, together with high dilution rates and total glucose consumption. (c) 2004 Elsevier Ltd. All rights reserved.
Abstract: Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO2 removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.
Abstract: This review surveys rapid bioluminescent detection techniques applied in food industry and discusses the historical development of the rapid methods. These techniques are divided into two groups: methods based on bioluminescent adenosine triphosphate (ATP) assay, and on bacterial bioluminescence. The advantages and disadvantages of these methods are described. The article provides the bibliography of fluorescent method applications in food samples.
Abstract: In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate (q(im)) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble-column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (X-im(act)) of the total immobilized biomass (Xi,,) was subsequently estimated at approximately m 0.12 g(IB) g(C)(-1) (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell-to-carrier adhesion and cell-to-cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (X-im(act)) and the maximum attainable biomass load (X-im(act)) was included into the model. Since the average biofilm thickness was estimated at ca. 10 mum, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble-column reactor. Good agreement between model simulations and experimental data was achieved.
Abstract: Immobilization of brewing yeast onto a cellulose-based carrier obtained from spent grains, a brewing byproduct, by acid/base treatment has been studied in a continuously operating bubble-column reactor. The aim of this work was to study the mechanisms of brewing yeast immobilization onto spent grain particles through the information on physicochemical surface properties of brewing yeast and spent grain particles. Three mechanisms of brewing yeast immobilization onto spent grains carrier were proposed: cell-carrier adhesion, cell-cell attachment, and cell adsorption (accumulation) inside natural shelters (carrier's surface roughness). The possibility of stable cell-carrier adhesion regarding the free energy of interaction was proved and the relative importance of long-range forces (Derjaguin-Landau-Verwey-Overbeek theory) and interfacial free energies was discussed. As for the cell-cell attachment leading to a multilayer yeast immobilization, a physicochemical interaction through localized hydrophobic regions on cell surface was hypothesized. However, neither flocculation nor chain formation mechanism can be excluded so far. The adsorption of brewing yeast inside sufficiently large crevices (pores) was documented with photomicrographs. A positive effect of higher dilution rate and increased hydrophobicity of base-treated spent grains on the yeast immobilization rate has also been found. (C) 2004 Wiley Periodicals, Inc.
Abstract: A one-stage continuous primary beer fermentation with immobilized brewing yeast was studied. The objective of the work was to optimize the operational conditions (aeration and temperature) in terms of volumetric productivity and organoleptic quality of green beer. The system consisted of an internal-loop airlift reactor and a carrier material prepared from spent grains (a brewing by-product). An industrial wort and yeast strain were used. The immobilized biomass (in amounts from two to sevenfold greater than free biomass) contributed 45-75% to the total fermentation. The volumetric productivity of the continuous system was as much as five times higher than that of the batch fermentation. An optimum higher-alcohols-to-esters ratio in green beer was found at approximately 2 mg/L of oxygen dissolved in wort, mixing induced by pure CO2, and temperatures at 13-16degreesC. At high total biomass concentration, the diacetyl formation was low, and the volumetric productivity of the system was high. Therefore, the amount of immobilized biomass in the reactor has to be kept at high concentration by regular replacement of the carrier losses.
Abstract: A yeast strain secreting endopolygalacturonase was used in this work to study the possibility of continuous production of this enzyme. It is a feasible and interesting alternative to fungal batch production essentially due to the specificity of the type of pectinase excreted by Kluyveromyces marxianus CCT 3172, to the lower broth viscosity and to the easier downstream operations. In order to increase the reactors' productivity, a cellulosic carrier obtained from barley spent grains was tested as an immobilization support. Two types of reactors were studied for pectinase production using glucose as a carbon and energy source--a continuous stirred tank reactor (CSTR) and a packed bed reactor (PBR) with recycled flow. The highest value for pectinase volumetric productivity (P(V)=0.98 U ml(-1) h(-1)) was achieved in the PBR for D=0.40 h(-1), a glucose concentration on the inlet of S(in)=20 g l(-1), and a biomass load in the support of X(i)=0.225 g g(-1). The results demonstrate the attractiveness of the packed bed system for pectinase production.
Abstract: This work demonstrated the technological feasibility of the three-phase airlift bioreactor (ALR) with brewing yeast immobilized on spent grains (a brewing by-product) for continuous beer production. The optimum fermentation performance of the one stage immobilized cell bioreactor was achieved at residence times between 18-25 h (dilution rate 0.04-0.055 h(-1)) and was characterized by an apparent degree of attenuation in the range of 70-80%. The productivity of the system in terms of ethanol concentration in green beer (ca. 4.2%) was satisfactory. Although the diacetyl concentration in the young beer was high (0.32 mg L-1 at D = 0.04 h(-1)) it is speculated that the level could be reduced by cell growth control, aeration and temperature optimisation. The immobilized yeast fermentation in the ALR was shown to be robust in recovery after process upsets.
Abstract: A novel carrier obtained from spent grains, a brewing by-product, was used for brewing yeast immobilisation in a continuous bubble-column reactor. The multiple-layer cell adhesion to the carrier particles resulted in a maximum cell load of 430 mg dry cell g(-1) dry carrier (d.c.). After 120 h of reactor operation, the cell load of DEAE-modified carrier was below 40 mg dry cell g(-1) d.c. while the values for non-modified carrier reached at least 100 mg dry cell g(-1) d.c. The changes in substrate composition on the rate of yeast attachment and on its stability were also studied.
Abstract: A mixed microbial culture was entrapped into porous silica gel prepared by two different sol-gel methods. The immobilization of cells into prepolymerized tetraethoxysilane was more stressful to living microbial cells than the entrapment into colloidal SiO2. Our experimental equipment operating in a sensor mode was able to detect 0.5 mg phenol l(-1) and had a linear response in the range from 2 to 10 mg phenol l(-1).
Abstract: A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (q(s)max) decreased in the order: PBRC (598 mg l(-1) h(-1)) > PBR (PU, 471 mg l(-1)h(-1)) > PBR(SG, 394 mg l(-1) h(-1)) > FBR (PU, 161 mg l(-1) h(-1)) > FBR (SG, 91 mg l(-1) h(-1)). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100-200 microm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO2 enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing.
Abstract: This work deals with changes in microbial phenol degradation and cell proliferation caused by immobilization into silica gel. Mixed microbial culture and the yeast Candida tropicalis were immobilized in silica layers and pieces prepared by mixing of prepolymerized tetraethoxysilane with cell suspension. The phenol degradation rate of cells entrapped in silica gel was compared with those immobilized into an organic polymer-polyurethane. The phenol degradation efficiency decreased in the following order: free cell suspension > cells entrapped into polyurethane foam similar to cells entrapped into prepolymerized TEOS. Inside the silica there was no growth observed by optical microscope. The immobilization of bacterium Pseudomonas species 2 into silica gel, cells which co-metabolize PCBs with biphenyl, did not result in substantial change of intermediate concentration.
