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1.  Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes. 
Journal of Bacteriology  1975;123(3):1124-1130.
Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.
PMCID: PMC235836  PMID: 239921
2.  Effects of pH and Sugar on Acetoin Production from Citrate by Leuconostoc lactis 
The relationship between acetoin production and citrate utilization in Leuconostoc lactis NCW1 was studied. In a complex medium the organism utilized citrate at neutral pH (initial pH, 6.3) and at acid pH (initial pH, 4.5) but produced nine times more acetoin at the latter pH. In resting cells the utilization of citrate was optimum at pH 5.3. Production of acetoin as a function of citrate utilization increased as the pH decreased, and at pH 4.3 all of the citrate utilized was recovered as acetoin. Glucose (10 mM) and lactose (10 mM) markedly stimulated citrate utilization but totally inhibited acetoin production in glucose- and lactose-grown cells. Addition of glucose to cells actively metabolizing citrate caused an immediate increase in citrate uptake and a reduction in the level of acetoin. The apparent Km values of lactic dehydrogenase for pyruvate were 1.05, 0.25, and 0.15 mM at pH 7.5, 6.5, and 5.0, respectively. Several heterofermentation intermediates inhibited α-acetolactate synthetase and decarboxylase activities. The implications of these results in regulating acetoin formatin are discussed.
PMCID: PMC243632  PMID: 16345676
3.  Conversion of Pyruvate to Acetoin Helps To Maintain pH Homeostasis in Lactobacillus plantarum† 
Pyruvate is the substrate for diacetyl and acetoin synthesis by lactobacilli. Exogenous pyruvate stimulates acetoin production when glucose is present as an energy source. In Lactobacillus plantarum ATCC 8014, the energy derived from glucose via glycolysis generated a constant proton motive force of about -120 mV. At a low external pH, energized cells rapidly transported and accumulated pyruvate but did not do so when they were deenergized by nigericin. When large amounts of pyruvate were transported and subsequently accumulated internally, the cotransported protons rapidly lowered the internal pH. The conversion of pyruvate to acetoin instead of acidic end products contributed to the maintenance of pH homeostasis. This is the first report showing that the conversion of pyruvate to acetoin serves as a mechanism of pH homeostasis.
PMCID: PMC195350  PMID: 16348677
4.  Acetoin production by wild-type strains and a lactate dehydrogenase-deficient mutant of Streptococcus mutans. 
Infection and Immunity  1987;55(6):1399-1402.
Eleven different laboratory strains of Streptococcus mutans representing the various serogroups were found to produce an average of 6.0 +/- 4.8 mM acetoin when grown in glucose-containing medium under aerobic conditions. None of the strains produced detectable acetoin when grown anaerobically. A lactate dehydrogenase-deficient mutant produced acetoin both aerobically and anaerobically and in substantially greater amounts than the wild-type strains did. Substitution of mannitol for glucose resulted in decreased acetoin production by wild-type strains and the lactate dehydrogenase-deficient mutant, indicating a role for NADH2 in the regulation of the acetoin pathway. Pyruvate incorporated into the growth medium of a wild-type strain caused acetoin to be produced anaerobically and stimulated acetoin production aerobically. Cell extracts of a wild-type S. mutans strain were capable of producing acetoin from pyruvate and were (partly) dependent on thiamine PPi. Extracts prepared from aerobically grown cells had approximately twice the acetoin-producing activity as did extracts prepared from anaerobically grown cells. The results indicate that acetoin production by S. mutans may represent an auxiliary reaction of pyruvate dehydrogenase in this organism.
PMCID: PMC260527  PMID: 3570471
5.  Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilis ZM4(pZB5) 
The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by 13C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo 31P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.
PMCID: PMC91804  PMID: 10618222
6.  Microbial Production of 2,3-Butylene Glycol from Cheese Whey 
Six microorganisms that produced acetoin or diacetyl or both from glucose were tested for the production of 2,3-butylene glycol from lactose. Bacillus polymyxa and Streptococcus faecalis gave positive results and were tested in unmodified wheys. Cottage cheese whey was unsatisfactory, but B. polymyxa produced large amounts of the glycol in sweet whey, about 60 mmol of glycol per 100 mmol of lactose utilized. Aeration and an increased ratio of surface area to volume of whey enhanced the production of glycol. 2,3-Butylene was separated from the spent whey and from acetoin and diacetyl with a Sephadex G-10 column.
PMCID: PMC244211  PMID: 16346020
7.  Intracellular Carbon Fluxes in Riboflavin-Producing Bacillus subtilis during Growth on Two-Carbon Substrate Mixtures 
Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional 13C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.
doi:10.1128/AEM.68.4.1760-1771.2002
PMCID: PMC123836  PMID: 11916694
8.  Biometric Study of Acetoin Production in Hanseniaspora guilliermondii and Kloeckera apiculata 
Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of Hanseniaspora guilliermondii and Kloeckera apiculata were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (P = 0.001) in acetoin production and K. apiculata exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with Saccharomyces cerevisiae seems promising for optimization of wine bouquet.
PMCID: PMC182169  PMID: 16348961
9.  Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation 
Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.
PMCID: PMC184489  PMID: 16348209
10.  Diacetyl and Acetoin Production by Lactobacillus casei 
Applied Microbiology  1971;22(4):517-521.
Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension.
PMCID: PMC376354  PMID: 4943268
11.  d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum ▿ †  
Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.
doi:10.1128/AEM.01616-10
PMCID: PMC3126369  PMID: 21335380
12.  Two-Stage pH Control Strategy Based on the pH Preference of Acetoin Reductase Regulates Acetoin and 2,3-Butanediol Distribution in Bacillus subtilis 
PLoS ONE  2014;9(3):e91187.
Acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), which catalyzes the interconversion between acetoin and 2,3-butanediol, plays an important role in distribution of the products pools. This work characterized the Bacillus subtilis AR/BDH for the first time. The enzyme showed very different pH preferences of pH 6.5 for reduction and pH 8.5 for oxidation. Based on these above results, a two-stage pH control strategy was optimized for acetoin production, in which the pH was controlled at 6.5 for quickly converting glucose to acetoin and 2,3-butanediol, and then 8.0 for reversely transforming 2,3-butanediol to acetoin. By over-expression of AR/BDH in the wild-type B. subtilis JNA 3-10 and applying fed-batch fermentation based on the two-stage pH control strategy, acetoin yield of B. subtilis was improved to a new record of 73.6 g/l, with the productivity of 0.77 g/(l·h). The molar yield of acetoin was improved from 57.5% to 83.5% and the ratio of acetoin/2,3-butanediol was switched from 2.7∶1 to 18.0∶1.
doi:10.1371/journal.pone.0091187
PMCID: PMC3946754  PMID: 24608678
13.  Deoxyribonucleic Acid Base Composition of Some Members of the Micrococcaceae 
Journal of Bacteriology  1966;92(1):28-34.
Auletta, Angela E. (Catholic University, Washington, D.C.), and E. R. Kennedy. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92:28–34. 1966.—Thirty-seven strains from the genera Micrococcus, Staphylococcus, Gaffkya, and Sarcina were examined for deoxyribonucleic acid base composition and biochemical activity. Organisms were tested for production of catalase, coagulase, deoxyribonuclease, oxidase, phosphatase, hydrogen sulfide, indole, and acetoin; nitrate reduction; gelatin, starch, and urea hydrolysis; citrate and ammonium phosphate utilization; NaCl tolerance; growth at 10 and 45 C, and growth in litmus milk. They were tested for production of acid from dextrose and mannitol under anaerobic conditions, and for aerobic production of acid from dextrose, mannitol, lactose, sucrose, raffinose, maltose, xylose, and glycerol. Organisms could be divided into two groups on the basis of guanine-cytosine (GC) content. Group I had an average GC content of 32%, and included all organisms which produced acid from dextrose. Group II had an average GC content of 62%, and included those organisms incapable of producing acid from dextrose under anaerobic conditions. Sarcina ureae had a GC content of 43%.
PMCID: PMC276191  PMID: 5941282
14.  Production of d-Mannitol and Glycerol by Yeasts 
Applied Microbiology  1968;16(12):1847-1852.
D-Mannitol has not so far been known as a major product of sugar metabolism by yeasts. Three yeast strains, a newly isolated yeast from soy-sauce mash, Torulopsis versatilis, and T. anomala, were found to be good mannitol producers. Under optimal conditions, the isolate produced mannitol at good yield of 30% of the sugar consumed. Glucose, fructose, mannose, galactose, maltose, glycerol, and xylitol were suitable substrates for mannitol formation. High concentrations of yeast extract, Casamino Acids, NaCl, and KCl in media affected significantly the mannitol yield, whereas high levels of inorganic phosphate did not show any detrimental effect.
PMCID: PMC547782  PMID: 5749751
15.  Thermophilic fermentation of acetoin and 2,3-butanediol by a novel Geobacillus strain 
Background
Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination.
Results
This work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7 g/L of acetoin and 14.5 g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography–mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. α-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C.
Conclusions
Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work.
doi:10.1186/1754-6834-5-88
PMCID: PMC3538569  PMID: 23217110
Acetoin; 2,3-butanediol; Thermophilic fermentation; Geobacillus; Novel metabolite; Key enzymes
16.  Ca2+-Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334 
Applied and Environmental Microbiology  2013;79(15):4603-4612.
The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca2+ and not as free citrate or the Mg2+-citrate complex, thereby identifying Ca2+-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca2+ and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca2+-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca2+-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca2+-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca2+-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages.
doi:10.1128/AEM.00925-13
PMCID: PMC3719530  PMID: 23709502
17.  Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+ 
Citr+Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu2+. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu2+, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu2+. On the other hand, the YGlu value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu2+ enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu2+ stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PPi. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.
PMCID: PMC184819  PMID: 16348274
18.  ROLES OF CITRATE AND ACETOIN IN THE METABOLISM OF STREPTOCOCCUS DIACETILACTIS 
Journal of Bacteriology  1963;86(6):1301-1307.
Harvey, R. J. (University of California, Davis), and E. B. Collins. Roles of citrate and acetoin in the metabolism of Streptococcus diacetilactis. J. Bacteriol. 86:1301–1307. 1963.—Streptococcus diacetilactis was unable to use citrate as a source of energy for growth, but the addition of citrate to a lactose-containing medium increased the specific growth rate 35%. Besides serving as the precursor of acetoin, some of the pyruvate formed from citrate was incorporated into cell material, primarily into lipids. A constant fraction of the weight of new cells was synthesized from the pyruvate formed from citrate. The rate of entry of citrate into cells was independent of the growth rate, and the usual result was that more pyruvate was formed from citrate than was required for cell synthesis. All excess pyruvate was converted to acetoin. Thus, acetoin formation acts as a detoxification mechanism, a means of removing intracellular pyruvate not required for synthesis of cell material.
PMCID: PMC283645  PMID: 14086105
19.  Regulation of the Acetoin Catabolic Pathway Is Controlled by Sigma L in Bacillus subtilis 
Journal of Bacteriology  2001;183(8):2497-2504.
Bacillus subtilis grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. The acoABCL operon encodes the E1α, E1β, E2, and E3 subunits of the acetoin dehydrogenase complex in B. subtilis. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The acoR gene is located downstream from the acoABCL operon and encodes a positive regulator which stimulates the transcription of the operon. The product of acoR has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a −12, −24 promoter, and transcription is abolished in acoR and sigL mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an acoR′-′lacZ strain of B. subtilis showed that the expression of acoR is not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of acoR is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region of acoR was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of acoR is mediated by the binding of CcpA to the promoter region of acoR.
doi:10.1128/JB.183.8.2497-2504.2001
PMCID: PMC95166  PMID: 11274109
20.  Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar diacetylactis 
The production of aroma compounds (acetoin and diacetyl) in fresh unripened cheese by Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 was studied at 30°C at different initial oxygen concentrations (0, 21, 50, and 100% of the medium saturation by oxygen). Regardless of the initial O2 concentration, maximal production of these compounds was reached only after all the citrate was consumed. Diacetyl and acetoin production was 0.01 and 2.4 mM, respectively, at 0% oxygen. Maximum acetoin concentration reached 5.4 mM at 100% oxygen. Diacetyl production was increased by factors of 2, 6, and 18 at initial oxygen concentrations of 21, 50, and 100%, respectively. The diacetyl/acetoin concentration ratio increased linearly with initial oxygen concentration: it was eight times higher at 100% (3.3%) than at 0% oxygen (0.4%). The effect of oxygen on diacetyl and acetoin production was also shown with other lactococci. At 0% oxygen, specific activity of α-acetolactate synthetase (0.15 U/mg) and NADH oxidase (0.04 U/mg) was 3.6 and 5.4 times lower, respectively, than at 100% oxygen. The increasing α-acetolactate synthetase activity in the presence of oxygen would explain the higher production of diacetyl and acetoin. The NADH oxidase activity would replace the role of the lactate dehydrogenase, diacetyl reductase, and acetoin reductase in the reoxidation of NADH, allowing accumulation of these two aroma compounds.
PMCID: PMC182177  PMID: 16348966
21.  Carbohydrate fermentation by gut microflora in preterm neonates. 
Archives of Disease in Childhood  1989;64(10 Spec No):1367-1373.
To study organic acid excretion, urine was collected from 52 preterm infants at weekly intervals and analysed by capillary gas chromatography-mass spectrometry. Twelve of 22 babies born before 33 weeks' gestation excreted 2,3-butanediol, as did six born between 33 and 36 weeks. Six very immature babies also excreted acetoin, the metabolic precursor of the diol. Other products derived from carbohydrate included methylmalonic and ethylmalonic acids in one baby, and D-lactic acid in five. Acetoin has never been found in urine before, and the other four acids have been found only rarely. Excretion of these metabolites by preterm babies can be explained by increased intestinal permeability, unabsorbed lactose in the colon, and colonisation with certain opportunistic micro-organisms prevalent in neonatal units, including klebsiella, serratia, and enterobacter. The findings support evidence from breath hydrogen analysis that carbohydrate fermentation takes place in the gut of preterm infants.
PMCID: PMC1590111  PMID: 2589871
22.  FUMARATE REDUCTION AND ITS ROLE IN THE DIVERSION OF GLUCOSE FERMENTATION BY STREPTOCOCCUS FAECALIS1 
Journal of Bacteriology  1964;88(4):858-864.
Deibel, R. H. (American Meat Institute Foundation, Chicago, Ill.), and M. J. Kvetkas. Fumarate reduction and its role in the diversion of glucose fermentation by Streptococcus faecalis. J. Bacteriol. 88:858–864. 1964.—Fumarate diverts the normal fermentation of glucose by Streptococcus faecalis FB82, as shown by the production of increased amounts of CO2, formate, acetate, and acetoin, and decreased formation of lactate and ethanol. Experiments with d-glucose-1-C14, in which low levels of labeled CO2 were recovered, indicated that C-1 cleavage of the glucose molecule was not involved. The presence of fumarate afforded consistently larger cell crops in growth studies with glucose and other energy sources. On a molar growth-yield basis, anaerobically grown, glucose-fumarate cultures were equivalent to aerobically grown, glucose cultures. The reduction of fumarate by cell suspensions indicated that glucose, gluconate, and, to a lesser extent, glycerol and mannitol could serve as hydrogen donors. Several common metabolic inhibitors had no effect upon the fumarate reductase system in cell suspensions, although some sensitivity to acidic pH was noted. Significant levels of succinate oxidation activity were not detected. Fumarate reductase activity was demonstrated in all five S. faecalis strains tested. Distribution of this ability in S. faecium strains was variable, ranging from activity comparable with that of S. faecalis to total inactivity. The observations support the conclusion that fumarate functions as an alternate hydrogen acceptor, thus allowing pyruvate to participate in the energy-yielding phosphoroclastic and dismutation pathways.
PMCID: PMC314825  PMID: 14219047
23.  Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate 
Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h−1. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, α-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.
doi:10.1128/AEM.67.6.2677-2682.2001
PMCID: PMC92924  PMID: 11375180
24.  Glucosyltransferase production by Klebsiella sp. K18 and conversion of sucrose to palatinose using immobilized cells 
The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL-1) was achieved using the optimized medium composed by sugar cane molasses (80 g L-1), bacteriological peptone (7 g L-1) and yeast extract (20 g L-1), after 8 hours of fermentation at 28°C. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%.
doi:10.1590/S1517-838220090001000010
PMCID: PMC3768490  PMID: 24031319
cell immobilization; glucosyltransferase; Klebsiella sp; palatinose; response surface methodology
25.  Evaluation of methods for differentiation of coagulase-positive staphylococci. 
Journal of Clinical Microbiology  1992;30(12):3217-3219.
The purpose of this study was to determine the minimum number of tests that could be used to differentiate between the coagulase-positive strains of staphylococcus: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius. Eighty coagulase-positive strains of each of the three species were examined. The five tests conducted were growth on modified Baird-Parker agar, growth on P agar supplemented with acriflavin, production of acetoin, anaerobic fermentation of mannitol, and presence of beta-galactosidase. Positive test percentages for S. aureus were 100% for growth on modified Baird-Parker agar, 100% for growth on P agar supplemented with acriflavin, 94% for production of acetoin, 99% for anaerobic fermentation of mannitol, and 0% for presence of beta-galactosidase. Positive test percentages for S. intermedius were 0% for growth on modified Baird-Parker agar, 0% for growth on P agar supplemented with acriflavin, 1% for production of acetoin, 0% for anaerobic fermentation of mannitol, and 100% for presence of beta-galactosidase. S. hyicus isolates were negative in all five tests. Results from the 240 coagulase-positive staphylococcus strains tested would suggest correct identification of coagulase-positive staphylococci with P agar supplemented with acriflavin and the beta-galactosidase test. These two tests are simple to conduct and result in quick and easy differentiation of the three coagulase-positive staphylococcal species.
PMCID: PMC270634  PMID: 1452705

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