Formation Of Diacetyl Research Articles

Effect of α-acetolactate decarboxylase on diacetyl content of beer

The effect of a-acetolactate decarboxylase (ALDC) on the diacetyl content of beer was studied. Diacetyl is known as an off-flavor compound in beer due to its buttery flavor. ALDC is known to reduce diacetyl by bypassing the diacetyl formation pathway. Herein, beer was brewed with two-row barley (Jinyang) and six-row barley (Dahyang) cultured in Korea with the addition of ALDC. The formation of diacetyl is reversely affected by free amino nitrogen, such as valine. ALDC (0.02, 0.04, and 0.06 unit/mL) was added to the samples, and the physicochemical properties of the resulting beer were analyzed to determine the quality. Diacetyl content was decreased in proportion to the enzyme concentration. The diacetyl content in samples made from Jinyang and Dahyang decreased by 25 and 15%, respectively. The diacetyl content in both was acceptable for microbrewery.

An Improved Model for Prediction of Wort Fermentation Progress and Total Diacetyl Profile

Diacetyl is normally considered to be an off-flavor in lager beer, and its removal prolongs the overall brewing process. Here, the effects of fermentation temperature (9, 12, and 15°C), initial wort pH (4.8, 5.1, and 5.3), and wort free amino nitrogen (FAN) content (222, 252, 287, and 366 ppm) on diacetyl formation and removal in lager beer fermentations were studied in order to develop an enhanced model for predicting fermentation progress (alcohol content, biomass, and pH) and diacetyl concentration. The relationships between model coefficients, temperature, pH, and FAN were calculated, and models predicted fermentation and diacetyl profiles with a good fit (overall relative mean square error less than 10.1%). The model was validated, and also applied to a larger-scale fermentation involving a different wort and yeast strain by adjusting model coefficients. The model can be used for predicting diacetyl concentrations and for brewing process parameter optimization in industrial fermentations.

Suppression of microbial metabolic pathways inhibits the generation of the human body odor component diacetyl by Staphylococcus spp.

Diacetyl (2,3-butanedione) is a key contributor to unpleasant odors emanating from the axillae, feet, and head regions. To investigate the mechanism of diacetyl generation on human skin, resident skin bacteria were tested for the ability to produce diacetyl via metabolism of the main organic acids contained in human sweat. l-Lactate metabolism by Staphylococcus aureus and Staphylococcus epidermidis produced the highest amounts of diacetyl, as measured by high-performance liquid chromatography. Glycyrrhiza glabra root extract (GGR) and α-tocopheryl-l-ascorbate-2-O-phosphate diester potassium salt (EPC-K1), a phosphate diester of α-tocopherol and ascorbic acid, effectively inhibited diacetyl formation without bactericidal effects. Moreover, a metabolic flux analysis revealed that GGR and EPC-K1 suppressed diacetyl formation by inhibiting extracellular bacterial conversion of l-lactate to pyruvate or by altering intracellular metabolic flow into the citrate cycle, respectively, highlighting fundamentally distinct mechanisms by GGR and EPC-K1 to suppress diacetyl formation. These results provide new insight into diacetyl metabolism by human skin bacteria and identify a regulatory mechanism of diacetyl formation that can facilitate the development of effective deodorant agents.

Diacetyl Formation by Oenococcus oeni during Winemaking Induced by Exogenous Pyruvate

Pyruvate is the central metabolite in diacetyl synthesis by <i>Oenococcus oeni.</i> Therefore, any substrate that increases intracellular pyruvate concentration can induce diacetyl accumulation. This study evaluates the effect of exogenous pyruvate on diacetyl formation and the expression of diacetyl-related genes in <i>Oenococcus oeni</i> during winemaking. Diacetyl formation by <i>Oenococcus oeni</i> was induced by yeast-derived pyruvate in the early stage of winemaking. Furthermore, when additional pyruvate was added, α-acetolactate synthase (<i>alsS</i>) gene expression increased 1.6-fold and the diacetyl concentration increased from 0.4 mg/L to 2.3 mg/L. Although the highest <i>alsS</i> expression (a 10-fold increase) was found 24 hr after pyruvate addition, no further increase in diacetyl concentration was found. In addition, <i>alsD</i> was overexpressed (9-fold) at that point, indicating that α-acetolactate was converted into acetoin. Together, the results show that exogenous pyruvate induces diacetyl formation by <i>Oenococcus oeni</i> in the early stage of winemaking<i>.</i> Furthermore, pyruvate-derived diacetyl accumulates because of a delayed <i>alsD</i> response that prevents acetoin formation.

Fed-batch fermentation with glucose syrup as an adjunct for high-ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed-batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC-9, with a pitching rate at 7.0 × 106 cells/mL. An all-malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed-batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 106 cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling

Time course of diacetyl formation during vinification withSaccharomyces cerevisiaeandOenococcus oenico-cultivation

Background and Aims Diacetyl accumulation in wine, which has undergone malolactic fermentation by Oenococcus oeni, is often associated with aromatic off-flavours. Characterisation of the diacetyl-related metabolic pathway helps to explain bacterial diacetyl formation during winemaking. The present study describes the time-dependent formation of diacetyl during the vinification process after simultaneous, induced malolactic fermentation by freeze-dried O. oeni on the basis of gene-expression analysis of the citrate- and pyruvate-derived pathways. Methods and Results After simultaneously induced malolactic fermentation in Pinot Blanc by O. oeni, the dynamics of diacetyl formation were compared with citrate consumption and gene-expression of the diacetyl-related metabolic pathways. Diacetyl concentration showed two maxima: the first increase was primarily influenced by the activity of Saccharomyces cerevisiae; however, the second increase was induced only by O. oeni and correlates with bacterial citrate degradation. Expression of the alsS gene showed two significant responses; however, only the second response was affected by the citrate-associated genes maeP and citE. Additionally, alsD and butA2 were found to be continuously underexpressed during the winemaking process. Conclusions Taken together, we suggest that diacetyl accumulation during the vinification process by O. oeni is affected by citrate fermentation. The diacetyl-related alsS gene, however, is also overexpressed independently by other substrates, which may also increase the intracellular pyruvate level resulting in diacetyl formation. Significance of the Study Characterisation of the time-dependent diacetyl accumulation and its degradation during the vinification process is essential for the development of strategies that focus on the suppression of the diacetyl concentration below the sensory threshold.

125thAnniversary Review: Diacetyl and its control during brewery fermentation

Diacetyl is a butter-tasting vicinal diketone produced as a by-product of yeast valine metabolism during fermentation. Concentration is dependent on a number of factors including rate of formation of the precursor α-acetolactate by yeast, spontaneous decarboxylation of this acetohydroxy acid to diacetyl and removal of diacetyl by yeast via the action of various reductase enzymes. Lowering concentrations of diacetyl in green beer represents an expensive and time-consuming part of the brewing process and strategies to minimize diacetyl formation or hasten its reduction have potential for improving overall efficiency of the lager brewing system. Here we review the processes that determine diacetyl levels in green beer as well as the various ways in which diacetyl levels can be controlled. The amount of diacetyl produced during fermentation can be affected by modifying process conditions, wort composition or fermentation technique, or by yeast strain development through genetic engineering or adaptive evolution. The process of diacetyl reduction by yeast is not as well understood as the process of formation, but is dependent on factors such as physiological condition, cell membrane composition, temperature and pH. The process of diacetyl removal is typically rate-limited by the reaction rate for the spontaneous decarboxylation of α-acetolactate to diacetyl. Copyright © 2013 The Institute of Brewing & Distilling

Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort

Undesirable butter-tasting vicinal diketones are produced as by-products of valine and isoleucine biosynthesis during wort fermentation. One promising method of decreasing diacetyl production is through control of wort valine content since valine is involved in feedback inhibition of enzymes controlling the formation of diacetyl precursors. Here, the influence of valine supplementation, wort amino acid profile and free amino nitrogen content on diacetyl formation during wort fermentation with the lager yeast Saccharomyces pastorianus was investigated. Valine supplementation (100 to 300 mg L−1) resulted in decreased maximum diacetyl concentrations (up to 37 % lower) and diacetyl concentrations at the end of fermentation (up to 33 % lower) in all trials. Composition of the amino acid spectrum of the wort also had an impact on diacetyl and 2,3-pentanedione production during fermentation. No direct correlation between the wort amino acid concentrations and diacetyl production was found, but rather a negative correlation between the uptake rate of valine (and also other branched-chain amino acids) and diacetyl production. Fermentation performance and yeast growth were unaffected by supplementations. Amino acid addition had a minor effect on higher alcohol and ester composition, suggesting that high levels of supplementation could affect the flavour profile of the beer. Modifying amino acid profile of wort, especially with respect to valine and the other branched-chain amino acids, may be an effective way of decreasing the amount of diacetyl formed during fermentation.

Diacetylplatinum(II) and ‐platinum(IV) Complexes Bearing κ2‐ and κ3‐Coordinated Tris(pyrazolyl)methane Ligands: Investigations on the Synthesis, Fluxionality, and Reactivity in Relation to the Substitution Pattern of the Ligands

AbstractReactions of the dinuclear platina‐β‐diketone [Pt2{(COMe)2H}2(μ‐Cl)2] (1) with HC(pz)3 and HC(3,5‐Me2pz)3 (pz = pyrazol‐1‐yl; 3,5‐Me2pz = 3,5‐dimethylpyrazol‐1‐yl) afforded cationic, thermally labile diacetyl(hydrido)platinum(IV) complexes [Pt(COMe)2H{(pz)3CH}]Cl (3a) and [Pt(COMe)2H{(3,5‐Me2pz)3CH}]Cl (3b) with κ3‐coordinated tris(pyrazolyl)methane ligands, which were found to react with NaOH or NEt3 to yield neutral diacetylplatinum(II) complexes with κ2‐coordinated tris(pyrazolyl)methane ligands {[Pt(COMe)2{(pz)3CH}] (4a); [Pt(COMe)2{(3,5‐Me2pz)3CH}] (4b)}. In 4a/b, a molecular rearrangement (decoordination of a pyrazolyl ring and coordination of the originally pendant one) has been found that has been investigated by variable‐temperature 1H NMR spectroscopic measurements (coalescence method) as well as by DFT calculations. Diacetylplatinum(II) complexes 4 were found to react in oxidative addition reactions with ROTf (R = H, Me; OTf = trifluoromethanesulfonate) and methyl iodide to yield cationic diacetylplatinum(IV) complexes of the type [Pt(COMe)2R{(pz)3CH}]X (R/X = H/OTf, 5a; Me/OTf, 6a; Me/I, 7a) and [Pt(COMe)2R{(3,5‐Me2pz)3CH}]X [R/X = H/OTf (5b), Me/OTf (6b), Me/I (7b)] with κ3‐bonded tris(pyrazolyl)methane ligands. Treatment of 4b with alkynyliodine(III) reagents of the type [IPh(C≡CR)]X (R/X = SiMe3/OTf, Ph/OTf, tBu/OTos, iPr/OTos; OTos = p‐toluenesulfonate) led to the formation of cationic diacetyl(alkynyl)platinum(IV) complexes [Pt(COMe)2(C≡CR){(3,5‐Me2pz)3CH}]X [R/X = SiMe3/OTf (8a), Ph/OTf (8b), tBu/OTos (8c), iPr/OTos (8d)]. The identities of all platinum complexes were unambiguously proven by high‐resolution mass spectrometric investigations, by NMR (1H, 13C, 195Pt) and IR spectroscopy, as well as by single‐crystal X‐ray diffraction analyses (4a, 4b, 7a, 8a/d). The constitution of the thermally labile complexes 3a/b has been confirmed by low‐temperature (–80 °C) NMR (1H, 13C) spectroscopic measurements. The electronic and steric influence of the additional methyl groups in HC(3,5‐Me2pz)3 on reactivity, stability, and properties of the investigated compounds will be discussed.

Monitoring the effect of high pressure and transglutaminase treatment of milk on the evolution of flavour compounds during lactic acid fermentation using PTR-ToF-MS

In this study, the effects of thermal or high hydrostatic pressure (HHP) treatment of a milk base in the absence or presence of a transglutaminase (TGase) protein cross-linking step on the flavour development of yoghurt were investigated. The presence of several tentatively identified volatile flavour compounds (VOCs), both during the enzymatic treatment and the lactic acid fermentation of the milk base, were monitored using a proton transfer reaction time-of-flight mass spectrometer (PTR-ToF-MS). The formation of the major flavour compounds (acetaldehyde, diacetyl, acetoin, and 2-butanone) followed a sigmoidal trend described by the modified Gompertz model. The HHP treatment of milk increased significantly the volatile compound formation rate whereas it did not affect the duration of the lag phase of formation, with the exception of acetaldehyde and diacetyl formation. On the contrary, the TGase cross-linking of milk did not significantly modify the formation rate of the volatile compounds but shortened the duration of the lag phase of their formation.

Structural insights into the mechanism of acetolactate decarboxylase

Acetolactate decarboxylase (ALDC) is a bacterial enzyme of the butanediol fermentation pathway that decarboxylates S‐acetolactate into R‐acetoin. The remarkable feature of this enzyme is that it also catalyses the decarboxylation of the opposite enantiomer R‐acetolactate to give the same product R‐acetoin. The enzyme is widely used commercially by the brewing industry. It shortens the maturation step by preventing the formation of diacetyl, which gives an off taste to beer. The structure of Bacillus brevis ALDC has been solved using X‐ray crystallography, in the presence of a series of chiral substrate analogues. In the resting state the active site zinc ion is coordinated to three conserved histidines and a glutamate. Upon binding of the substrate analogues the C‐terminal loop moves away losing the glutamate coordination bond. The structural information combined with experimental results from active site mutants allows us to present a mechanism for the stereospecific decarboxylation of acetolactate. The structural and biochemical data we have obtained will be used to develop further modifications and applications of the enzyme in future work.The authors thank EPSRC for funding through the Molecular Organisation and Assembly in Cells (MOAC) Doctoral Training Centre.

Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers' yeast

Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc -ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc -ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc -ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization.

Cytosolic Localization of Acetohydroxyacid Synthase Ilv2 and Its Impact on Diacetyl Formation during Beer Fermentation

Diacetyl (2,3-butanedione) imparts an unpleasant "butterscotch-like" flavor to alcoholic beverages such as beer, and therefore its concentration needs to be reduced below the sensory threshold before packaging. We examined the mechanisms that lead to highly elevated diacetyl formation in petite mutants of Saccharomyces cerevisiae during beer fermentations. We present evidence that elevated diacetyl formation is tightly connected to the mitochondrial import of acetohydroxyacid synthase (Ilv2), the key enzyme in the production of diacetyl. Our data suggest that accumulation of the matrix-targeted Ilv2 preprotein in the cytosol is responsible for the observed high diacetyl levels. We could show that the Ilv2 preprotein accumulates in the cytosol of petite yeasts. Furthermore, expression of an Ilv2 variant that lacks the N-terminal mitochondrial targeting sequence and thus cannot be imported into mitochondria led to highly elevated diacetyl levels comparable to a petite strain. We further show that expression of a mutant allele of the γ-subunit of the F(1)-ATPase (ATP3-5) could be an attractive way to reduce diacetyl formation by petite strains.

Breeding of a Low Pyruvate-Producing Sake Yeast by Isolation of a Mutant Resistant to Ethyl α-Transcyanocinnamate, an Inhibitor of Mitochondrial Pyruvate Transport

Pyruvate is the key substance controlling the formation of diacetyl, acetaldehyde, and acetate during alcoholic fermentation. Here we report the breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport. Mitochondrial function was involved in resistance to this substance and in the production of pyruvate by the mutants.

Expected and unexpected photochemical reactions of acyl iodides

Photolysis of acyl iodides RCOI (R = Me, Me2CH, Ph) under UV irradiation in toluene environment for 20–55 h proved to be a simple and efficient method of preparation of symmetrical α-diketones RCOCOR. In contrast, the photolysis under the same conditions of acyl iodides RCOI [R = Me(CH2)3, Me3C] did not lead to the formation of the corresponding diacyls, and the reaction products were unexpected 1,1-bis(4-methylphenyl)pentane and a mixture of isomeric 3- and 4-methyl(tert-butyl)benzenes respectively. The most probable mechanism of their formation is the primary photochemical acylation of toluene in the aromatic ring followed by the photochemical reduction of the arising butyl 4-methylphenyl ketone in the case of the valeroyl iodide or the photochemical Norrish type I cleavage of isomeric 3- and 4-methylphenyl (tert-butyl) ketones in event of the pivaloyl iodide. In the photolysis of acetyl iodide (R = Me) in benzene or toluene alongside the diacetyl formation polyarylation process was observed of acylated and iodinated into the aromatic ring solvents with the formation of polymeric products with semiconductor and paramagnetic properties.

From platina-β-diketones to diacetylplatinum(II) complexes – Synthesis, characterization and structural features

The reaction of the electronically unsaturated platina-β-diketone [Pt 2{(COMe) 2H} 2(μ-Cl) 2] ( 1a) with N ⌢N donors led to the formation of diacetyl(hydrido)platinum(IV) complexes [Pt(COMe) 2Cl(H)(N ⌢N)] ( 2). By the reaction of these complexes with NaOH in a two-phase system (H 2O/CH 2Cl 2) diacetylplatinum(II) complexes [Pt(COMe) 2(N ⌢N)] (N ⌢N = bpy, 4a; 4,4′-Me 2-bpy, 4b; 4,4′- t-Bu 2-bpy, 4c; 4,4′-Ph 2-bpy, 4d; 4,4′- t-Bu 2-6- n-Bu-bpy, 4e; bpym, 4f; bpyr, 4g; phen, 4h; 4-Me-phen, 4i; 5-Me-phen, 4j) were obtained. All complexes were characterized by microanalysis, IR and 1H and 13C NMR spectroscopy. Additionally, complexes 4a, 4c, 4d and 4e were characterized by single-crystal X-ray diffraction analysis. The observed variety of packing patterns resulting from π–π stacking and hydrogen bonding is discussed.

Formation of diacetyl and acetoin by Lactococcus lactis via aspartate catabolism

To verify whether diacetyl can be produced by Lactococcus lactis via amino acid catabolism, and to investigate the impact of the pH on the conversion. Resting cells of L. lactis were incubated in reaction media at different pH values, containing L-aspartic acid or L-alanine as a substrate. After incubation, the amino acid and metabolites were analysed by HPLC and GC/MS. At pH 5 about 75% of aspartic acid and only 40% of alanine was degraded to pyruvate via a transamination step that requires the presence of alpha-ketoglutarate in the medium, but diacetyl was only produced from aspartic acid. Three per cent of pyruvate was transformed to acetolactate of which 50% was converted into diacetyl. At pH 5 x 5 and above the pyruvate conversion into acetolactate was less efficient than at pH 5, and acetolactate was mainly decarboxylated to acetoin. Acetoin and diacetyl can be formed as a result of aspartate or alanine catabolism by L. lactis in the presence of alpha-ketoglutarate in the medium. Lactic acid bacteria exhibiting both glutamate dehydrogenase activity and high aspartate aminotransferase activity are expected to be good diacetyl producers during cheese ripening at pH close to 5.

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man-Rogosa-Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.

Method for the simultaneous assay of diacetyl and acetoin in the presence of α-acetolactate: Application in determining the kinetic parameters for the decomposition of α-acetolactate

A simultaneous assay method for diacetyl and acetoin was developed to investigate the formation of diacetyl during the brewing of alcoholic beverages. A GC-MS analysis after the extraction from neutralized sample by ethyl acetate gave accurate assay results. The detection limit was below 0.1 mg/l and the assay was quantitative from 0.1 to 100 mg/l for both compounds. Unlike other methods, the assay results were unaffected by the presence of alpha-acetolactate (up to 26 mg/l), which easily decomposes to diacetyl or acetoin, because the extraction condition prevents the decomposition and extraction of this acidic compound. Since our assay is compatible with samples that contain alpha-acetolactate, the kinetic parameters for decomposition of alpha-acetolactate to diacetyl and acetoin were determined. The decomposition rate constants were affected by the ethanol concentration. Overall kinetics for the decomposition of alpha-acetolactate was formulated as a function of ethanol concentration, pH and temperature. The kinetics can be applied to alcoholic beverages such as sake.

Continuous Primary Fermentation of Beer with Yeast Immobilized on Spent Grains—The Effect of Operational Conditions

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 13–16°C. 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.