Sugar and malic acid utilization and acetic acid formation byLeuconostoc oenos

Leuconostoc oenos ST8, isolated from an Argentinian red wine, utilized glucose, fructose and malic acid and produced acetic acid. Fructose was preferred to glucose as a source of carbohydrate. Malic acid was almost completely degraded in 3 days at 25°C. Acetic acid formation correlated with fructose utilization.     

Aerobic submerged fermentation by acetic acid bacteria for vinegar production: Process and biotechnological aspects

Strictly aerobic acetic acid bacteria (AAB) have a long history of use in fermentation processes, and the conversion of ethanol to acetic acid for the production of vinegar is the most well-known application.At the industrial scale, vinegar is mainly produced by submerged fermentation, which refers to an aerobic process in which the ethanol in beverages such as spirits, wine or cider is oxidized to acetic acid by AAB. Submerged fermentation requires robust AAB strains that are able to oxidize ethanol under selective conditions to produce high-titer acetic acid. Currently submerged fermentation is conducted by unselected AAB cultures, which are derived from previous acetification stocks and maintained by repeated cultivation cycles.In this work, submerged fermentation for vinegar production is discussed with regard to advances in process optimization and parameters (oxygen availability, acetic acid content and temperature) that influence AAB activity. Furthermore, the potential impact arising from the use of selected AAB is described.Overcoming the acetification constraints is a main goal in order to facilitate innovation in submerged fermentation and to create new industry-challenging perspectives.     

Cloning and characterization of the gene encoding phosphoketolase in <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

The gene encoding phosphoketolase, which is 2749 bp long and contains 814 amino acid polypeptides with a total molecular mass of 91.9 kDa, was cloned from Leuconostoc mesenteroides C7 (LMC7) and expressed in Escherichia coli. It exhibited a homology of >58% with phosphoketolases from other lactic acid bacteria. The phosphoketolase of LMC7 belongs to the xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) family, which is an enzyme with dual specificity for X5P and F6P. The members of this family contain typical thiamin pyrophosphate (TPP) binding sites as reported for other TPP-dependent enzymes, and several highly conserved regions as signature patterns for phosphoketolases. The plasmid pGPK containing the Xfp gene (xfp) exhibits phosphoketolase activity in E. coli. The specific activities of the enzyme from E. coli BL21 and E. coli EC101 harboring xfp were 0.28 and 0.14 units/mg, respectively. They both exhibited a 1.5-fold increase in the production of acetic acid from acetyl phosphate compared with their corresponding original strain.An erratum to this article can be found at     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Surface coat transformation and capsule formation by Leuconostoc mesenteroides NCDO 523 in the presence of sucrose

When Leuconostoc mesenteroides NCDO 523 was grown in MRS browth, electron microscopy of cells fixed in the presence of ruthenium red showed that the cell wall was covered with a thin layer of filamentous material. When MRS-grown cells were resuspended in the same medium supplemented with 3.6% sucrose, this surface coat doubled in thickness and a number of radial thickenings appeared within it. After 3 h the filamentous component of the surface coat had disappeared leaving only the radial projections. The progressive accumulation of polymer to produce a capsule visible by light microscopy was observed in only about 20% of the population. In this minority of cells, a dense globular dextran composed of fibrillar and particulate elements was always produced in the initial stages of synthesis. After 18 h, the dextran capsule was generally composed of an inner globular and outer fibrillar layer. It appeared that the outer layer was derived from the globular dextran of the capsule by a process of dispersion.     

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture, from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at 28 °C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.     

Fermentation of brined turnip roots using Lactobacillus plantarum and Leuconostoc mesenteroides starter cultures

The controlled fermentation of turnip slices using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures led to earlier acid production and earlier and more pronounced inhibition of Enterobacteriaceae than with uninoculated (natural) fermentation. Unlike the natural fermentation, the controlled fermentations did not show a yeast secondary fermentation and also had a better colour. Due to its ability to produce higher amounts of acid, the use of Lact. plantarum is more desirable than of Leuc. mesenteroides.     

Monitoring growth and acetic acid secretion by a thermotolerantBacillus using conduction microcalorimetry

Summary A method is described to determine power of heat-time curves by conduction microcalorimetry in order to monitor the viability and ability of a thermotolerantBacillus strain to secrete acetic acid both during exponential growth and during stationary-phase. In this system secreted acetic acid is neutralized by an insoluble source of lime (dolime) which results in a poor correlation between optical density and culture dry weight. As an alternative, cells and residual dolime were rapidly resuspended in isothermal fresh medium with glucose in a conduction microcalorimeter. Heat evolution was rapid over a period of 200–800 s. Steady state heat evolution rate decreased as a function of culture time and did not correlate with: 1) specific growth rate: 2) viable cell number: 3) glucose consumption rate; or 4) acetic acid secretion rate. Glucose consumption and acetic acid secretion during the stationary growth phase were correlated with specific heat evolution rate. These initial results indicate that this technique may be useful for further development as an on-line flow or stopped-flow method to monitor the physiology of bacilli in response to nutrient depletion or growth inhibition.     

Biofilm formation by exopolysaccharide mutants of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.     

Effect of phosphate concentration on the production of dextransucrase by<Emphasis Type="Italic"> Leuconostoc mesenteroides</Emphasis> NRRL B512F

Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran. This process has been studied since the Second World War, when it was used as blood plasma expander. A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work. Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals. Phosphate is currently used in order to buffer the culture medium. Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods. The standard medium for dextransucrase production is prepared using 0.1 M of K₂HPO₄. In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used. Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.     

Enhanced expression of aconitase raises acetic acid resistance in Acetobacter aceti

Acetobacter spp. are used for industrial vinegar production because of their high ability to oxidize ethanol to acetic acid and high resistance to acetic acid. Two-dimensional gel electrophoretic analysis of a soluble fraction of Acetobacter aceti revealed the presence of several proteins whose production was enhanced, to various extents, in response to acetic acid in the medium. A protein with an apparent molecular mass of 100 kDa was significantly enhanced in amount by acetic acid and identified to be aconitase by NH2-terminal amino acid sequencing and subsequent gene cloning. Amplification of the aconitase gene by use of a multicopy plasmid in A. aceti enhanced the enzymatic activity and acetic acid resistance. These results showed that aconitase is concerned with acetic acid resistance. Enhancement of the aconitase activity turned out to be practically useful for acetic acid fermentation, because the A. aceti transformant harboring multiple copies of the aconitase gene produced a higher concentration of acetic acid with a reduced growth lag-time.     

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO₂) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-¹³C]glucose, [U-¹³C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K _i=180 M) and uncompetitive (K_i=350 M) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K _i=1.3 M) with a low contribution of uncompetitive inhibition (K_i=13 M). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 M) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 M). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.     

Buffering capacity and membrane H+ conductance of acetic acid bacteria

Summary Buffering capacities and membrane conductance to H⁺ were measure inAcetobacter aceti ATCC 15973 andGluconobacter oxydans ATCC 621 by a pulse technique. In both strains the buffering capacity of intact cells was a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied between one species and another. Over the pH range studied, 4.02 to 8.15,Gluconobacter oxydans, which oxidizes sugars and alcohols to acids and accumulates them, showed lower values of buffering capacities and membrane conductance to protons thanAcetobacter aceti, which oxidizes these substrates completely to CO₂ and H₂O.     

The internal pH of Acetobacterium wieringae and Acetobacter aceti during growth and production of acetic acid

The intracellular pH was measured in growing Acetobacterium wieringae and Acetobacter aceti with an acid equilibrium distribution method. [¹⁴C]-acetylsalicylic acid, [¹⁴C-benzoic acid and [¹⁴C]-acetic acid were used as pH-indicators. The extracellular pH of Acetobacterium wieringae decreased from 7.0 to 5.0 during growth; accordingly, the intracellular pH changed from 7.1 to 5.5, and a pH between 0.1 and 0.65 (interior more alkaline) was maintained. Corresponding results were obtained for Acetobacter aceti. The external pH and the internal pH decreased in parallel from 6.2 to 3.5 and from 5.8 to 3.9, respectively.This demonstrates that neither the anaerobic nor the aerobic acetogen was able to maintain a large pH in the presence of high concentrations of acetic acid.     

Production of acetic acid by Clostridium thermoaceticum in electrodialysis culture using a fermenter equipped with an electrodialyser

Electrodialysis culture of Clostridium thermoaceticum increased the yield of acetate by its continuous removal. In normal batch cultures without pH control the yield was 4.2 g acetic acid/800 ml, while in pH-controlled culture it was 16.8 g/800 ml. Although electrodialysis cultures gave almost the same yield (15.4 g/800 ml) as that in pH-controlled cultures, sparging CO₂ into the broth in electrodialysis culture increased the amount of acetic acid to 22.3 g/800 ml. CO₂ sparging into normal cultures with or without pH control did not significantly increase the amount of acetate produced but yields, in terms of amounts of glucose consumed, were higher than without sparging. The theoretical yield was almost obtained in pH-controlled, electrodialysis cultures with CO₂ sparging.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan     

Growth characteristics of Geotrichum ingens on propionic acid and acetic acid in batch and continuous culture

Growth of Geotrichum ingens in batch cultures was completely inhibited by 47 g acetic acid/l or 33 g propionic acid/I. With mixtures of acetic and propionic acids, however, growth only ceased at 55 g/l. Acetic acid inhibited growth linearly, whereas propionic acid inhibited growth non-linearly. In continuous culture, two steady states at each dilution rate were observed at high dilution rates for acetic acid and propionic acid. The highest yield coefficient (0.69 g cells/g substrate) was achieved with propionic acid as substrate. On both substrates and their mixtures, the protein content of the biomass increased when the dilution rate was increased.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa     

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.