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Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria.

In this study, we compared the growth properties and molecular characteristics of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) among highly acetic acid-resistant strains of acetic acid bacteria. Gluconacetobacter europaeus exhibited the highest resistance to acetic acid (10%), whereas Gluconacetobacter intermedius and Acetobacter pasteurianus resisted up to 6% of acetic acid. In media with different concentrations of acetic acid, the maximal acetic acid production rate of Ga. europaeus slowly increased, but specific growth rates decreased concomitant with increased concentration of acetic acid in medium. The lag phase of A. pasteurianus was twice and four times longer in comparison to the lag phases of Ga. europaeus and Ga. intermedius, respectively. PQQ-dependent ADH activity was twice as high in Ga. europaeus and Ga. intermedius as in A. pasteurinus. The purified enzymes showed almost the same specific activity to each other, but in the presence of acetic acid, the enzyme activity decreased faster in A. pasteurianus and Ga. intermedius than in Ga. europaeus. These results suggest that high ADH activity in the Ga. europaeus cells and high acetic acid stability of the purified enzyme represent two of the unique features that enable this species to grow and stay metabolically active at extremely high concentrations of acetic acid.

Acetic Acid↗

Bacterial response to acetate challenge: a comparison of tolerance among species.

Although acetate formation and tolerance are important criteria for various aspects of biotechnological process development, available studies on acetate tolerance in different species are disparate. We evaluate the response of eight bacterial strains, including two variants of Escherichia coli, two variants of Staphylococcus capitis, and one each of Acetobacter aceti, Gluconobacter suboxydans, Lactobacillus acetotolerans, and L. bulgaricus, to acetate challenges under identical conditions. Our findings were: (1) wild-type organisms of species that are considered tolerant of acetate perform only slightly better than E. coli in unadapted shaker cultures; (2) the ability to tolerate acetate is strongly dependent on the carbon source, and is, especially for E. coli, much greater on glycerol than on glucose; (3) respiration is not as important to acetate tolerance in E. coli and S. capitis as has been reported for the acetic acid bacteria; (4) S. capitis was the least affected by acetate under all conditions and grew at up to 44 g/l acetate without any preconditioning; and (5) qualitative high-throughput screening of growth characteristics can be achieved with relatively inexpensive multiwell plate readers.

Acetates↗

Effect of glycine betaine on osmoadaptation of Propionibacterium acidipropionici cultivated in elevated osmolarities.

The sensitivity of industrial strains Acetobacter aceti, Gluconobacter frateurii, and Propionibacterium acidipropionici to osmotic stress was studied. Growth of A. aceti and G. frateurii was totally inhibited at 0.4 M NaCl concentration, but P. acidipropionici was able to grow on a medium containing 1.2 M NaCl. Addition of glycine betaine to the medium had no detectable osmoprotective effect on A. aceti and G. frateurii cultivations in elevated NaCl concentrations, but it enabled cells of P. acidipropionici to achieve faster the maximum specific growth rate after the prolonged lag phase and therefore to gain faster the final biomass and product concentrations. The final concentrations of biomass and product of P. acidipropionici were the same as for the cultivations of the bacterium without NaCl and glycine betaine present in the medium. Intracellular accumulation of glycine betaine was detected in P. acidipropionici cells cultivated in the medium containing glycine betaine. The amount accumulated increased with NaCl concentration, suggesting that glycine betaine plays an important role in the osmoadaptation.

Acetobacter↗

Bacterial cellulose production under oxygen-enriched air at different fructose concentrations in a 50-liter, internal-loop airlift reactor.

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 was carried out in a 50-1 internal-loop airlift reactor in air at an initial fructose concentration of 40 g/l. The BC production rate was 0.059 g/l per h. When oxygen-enriched air was supplied instead of air, the BC production rate increased to 0.093 g/l per h, and the BC yield was enhanced from 11% in air to 18%. When the initial fructose concentrations were varied from 30 to 70 g/l, the highest BC yield (35%) the highest production rate (0.22 g/l x per h), and the highest concentration of BC produced (10.4 g/l) were observed at 60-70 g/l fructose. From the carbon mass balance calculated at the final stage of cultivation, it was observed that enhanced BC production was reflected as a decrease in both CO2 evolution and the concentration of other unknown substances, suggesting the efficient utilization of energy for BC synthesis despite O2 limitation.

Acetobacter↗

Mechanical properties of primary plant cell wall analogues.

Mechanical effects of turgor pressure on cell walls were simulated by deforming cell wall analogues based on Acetobacter xylinus cellulose under equi-biaxial tension. This experimental set-up, with associated modelling, allowed quantitative information to be obtained on cellulose alone and in composites with pectin and/or xyloglucan. Cellulose was the main load-bearing component, pectin and xyloglucan leading to a decrease in modulus when incorporated. The cellulose-only system could be regarded as an essentially linear elastic material with a modulus ranging from 200 to 500 MPa. Pectin incorporation modified extensibility properties of the system by topology/architecture changes of cellulose fibril assemblies, but the cellulose/pectin composites could still be described as a linear elastic material with a modulus ranging from 120 to 250 MPa. The xyloglucan/cellulose composite could not be modelled as a linear elastic material. Introducing xyloglucan into a cellulose network or a cellulose/pectin composite led to very compliant materials characterised by time-dependent creep behaviour. Modulus values obtained for the composite materials were compared with mechanical data found for plant-derived systems. After comparing bi-axial and uni-axial behaviour of the different composites, structural models were proposed to explain the role of each polysaccharide in determining the mechanical properties of these plant primary cell wall analogues.

Acetobacter↗

Induced circular dichroism study of the aqueous solution complexation of cello-oligosaccharides and related polysaccharides with aromatic dyes.

Acetobacter xylinum, grown in the presence of low levels of the water-soluble dye Calcofluor White ST produces a pellicle of cellulose that has no detectable crystallinity. Biological factors of this sort are probably more important than physical factors in controlling the higher order structures of celluloses. Circular dichroism (CD) is induced by complexes that are formed by specific interactions between chiral oligosaccharides and dye molecules. Using CD, equilibrium constants were measured for the association reactions between various dyes with a series of cello-oligosaccharides (n = 2-6), methylcellulose, hydroxypropylcellulose (HPC), amylose, cyclomalto-oligosaccharides (cyclodextrins), and the linear malto-oligosaccharides (n = 3-7). Possible structural features of the complexes are discussed. Dyes that are capable of binding to the higher cello-oligomers in aqueous solutions are the same dyes that modify the solid structure of bacterial cellulose. An analogy between the binding of water-soluble dyes to cello-oligosaccharides and the binding of the cellulose-degrading enzyme, cellobiohydrolase I, to cellulose is discussed.

Acetobacter↗

[Biosynthesis of bacterial cellulose from glucose selectively deuterated in position 6: NMR study].

D-glucose specifically deuterated at C-6 was prepared and used for the biosynthesis of bacterial cellulose with Acetobacter xylinum. The material obtained was converted into glucitol hexaacetate and analyzed by 250 MHz n.m.r. and mass spectrometry. These spectra indicated that about 70% of the starting D-glucose was incorporated without modification of deuteriation at the C-6 position. However an explanation is required of the finding that deuterium was also incorporated at the C-2 and C-1 positions of the cellulose, arising respectively from the 6 pro-R and 6 pro-S positions of the initial D-glucose.

Acetobacter↗

Direct demonstration of enol-oxaloacetate as an immediate product of malate oxidation by the mammalian succinate dehydrogenase.

Rapid malonate-sensitive transitory formation of enol-oxaloacetate followed by slow ketonization of the product was observed after addition of malate to the mammalian succinate-ubiquinone reductase in the presence of electron acceptor. The initial rate of enol-oxaloacetate production was equal to that of malate oxidation. Oxaloacetate keto-enol tautomerase had no effect on the initial rate of enol-oxaloacetate production nor on the kinetics of malate oxidation; the enzyme drastically accelerated the ketonization of the product. The solubilized and partially purified membrane-bound flavine adenine dinucleotide-dependent malate dehydrogenase from Acetobacter xylinum catalyzed oxidation of L- and D-malate without formation of enol-oxaloacetate as an intermediate of the reaction.

Acetobacter↗

Molecular origins of acetan solution properties.

Acetan is a branched acidic heteropolysaccharide secreted by Acetobacter xylinum. X-ray diffraction studies of oriented fibres suggest non-crystalline helices with fivefold symmetry and a pitch of 4.8 nm. Optical rotation and circular dichroism studies are consistent with the retention of the helical structure in solution and a helix-coil transition upon heating and cooling. Aqueous solutions yield high 'low shear rate' viscosity and shear-thin upon shearing.

Acetobacter↗

Hemicelluloses as structure regulators in the aggregation of native cellulose.

The influence of hemicelluloses on the aggregation of cellulose in higher plant cell walls was modelled by adding hemicelluloses to cultures of the cellulose producer Acetobacter xylinum. Characterization of the celluloses by X-ray diffractometry showed them to be more like those that occur in higher plants; the coaggregation of the hemicelluloses suggests their occlusion within and between the crystalline domains of the celluloses. The authors propose that hemicelluloses may be primary moderators of the tertiary structure of cell wall celluloses, allowing the development of a wide range of properties.

Acetobacter↗

Formation, derivatization and applications of bacterial cellulose.

Acetobacter xylinum produces highly crystalline cellulose extracellularly using glucose as a carbon source. The polymer formed is free of other biogenic compounds, separable in a simple way and characterized by its high water-absorption capacity. Stepwise solvent exchange from water to unpolar solvents leads to a drastic decrease of the water content of the bacterial cellulose without decrease of the highly swollen and activated state. Heterogeneous as well as homogeneous derivatizations, e.g. carboxymethylation, silylation and acetylation, were performed on the wet or dried biopolymer. Furthermore, different methods for formation of hollow fibres during biosynthesis were investigated. Such tubes may have applications as biocompatible material in medicine.

Absorption↗

Ionic adsorption of catalase on bioskin: kinetic and ultrastructural studies.

Bioskin is a natural polymer produced by Acetobacter xylinum and several yeasts in culture. It contains glucosamine and N-acetyl galactosamine which promote ionic adsorption of catalase at the adequate pH value. High values of ionic strength are required to enzyme desorption. Adsorption of catalase on bioskin fibers has been visualized by scanning electron microscopy associated to a dispersion X-ray analyzer. At low enzyme density, the affinity of the immobilized catalase for hydrogen peroxide was 30% lower than that of the free enzyme. This affinity decreased dramatically at higher density of immobilized enzyme and could not be increased by agitation of the enzyme reaction mixture. Immobilized catalase retains about 70% of its initial activity after 16 d storage, whereas soluble enzyme is completely inactivated after 3 d at room temperature. The haeme group of catalase is not protected after immobilization since it is accessible to both EDTA and phloroglucinol, chelating agents which inactivate catalase by removing the iron atom from the haeme group.

Acetobacter↗

Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production.

The growth of acetic acid bacteria on grapes or throughout the winemaking process influences the quality of wine, mainly because it increases the volatile acidity. The objective of this study was to analyse how the acetic acid bacteria population evolves in the changing environment of the grape surface and during wine fermentation. We have analysed the influence of yeast inoculation and SO2 addition on acetic acid bacteria populations. These bacteria were analysed at both the species and the strain level by molecular methods such as Restriction Fragment Length Polimorfism (RFLP) of amplified 16S rDNA, and amplification by polymerase chain reaction of Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) and Repetitive Extragenic Palindromic (REP-PCR). Our results show that the increases in population size are normally accompanied by a proliferation of Acetobacter aceti, which is the main species during fermentation. The diversity of strains is considerable in natural environments such as the grape surface. Changes in the environment during alcoholic fermentation substantially reduce the survival and the diversity of acetic acid bacteria. Few strains are able to survive these conditions and they seem to originate from both the grapes and the winery. To the best of our knowledge this is the first time that acetic acid bacteria are analysed at the strain level in grape surfaces and during winemaking.

Acetic Acid↗

Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.

Acetic Acid↗

The quinohemoprotein alcohol dehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site.

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. In addition to the reduction of ubiquinone, ADHs of Gluconobacter suboxydans and Acetobacter aceti were shown to have a novel function in the oxidation of ubiquinol. The oxidation activity of ubiquinol was detected as an ubiquinol:ferricyanide oxidoreductase activity, which can be monitored by selected wavelength pairs at 273 and 298 nm with a dual-wavelength spectrophotometer. The ubiquinol oxidation activity of G. suboxydans ADH was shown to be two times higher in 'inactive ADH', whose ubiquinone reductase activity is 10 times lower, than with normal 'active' ADH. No activity could be detected in the isolated subunit II or subunit I/III complex, but activity was detectable in the reconstituted ADH complex. Inactive and active ADHs exhibited a 2-3-fold difference in their affinity to ubiquinol despite having the same affinity to ubiquinone. Furthermore, the ubiquinol oxidation site in ADH could be distinguished from the ubiquinone reduction site by differences in their sensitivity to ubiquinone-related inhibitors and by their substrate specificity with several ubiquinone analogues. Thus, the results strongly suggest that the reactions occur at different sites. Furthermore, in situ reconstitution experiments showed that ADH is able to accept electrons from ubiquinol present in Escherichia coli membranes, suggesting the ubiquinol oxidation activity of ADH has a physiological function. Thus, ADH of acetic acid bacteria, which has ubiquinone reduction activity, was shown to have a novel ubiquinol oxidation activity, of which the physiological function in the respiratory chain of the organism is also discussed.

Acetobacter↗

ORF2 gene involves in the construction of high-order structure of bacterial cellulose.

An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.

Acetobacter↗

Molecular interactions in bacterial cellulose composites studied by 1D FT-IR and dynamic 2D FT-IR spectroscopy.

Specific strain-induced orientation and interactions in three Acetobacter cellulose composites: cellulose (C), cellulose/pectin (CP) and cellulose/xyloglucan (CXG) were characterized by FT-IR and dynamic 2D FT-IR spectroscopies. On the molecular level, the reorientation of the cellulose fibrils occurred in the direction of the applied mechanical strain. The cellulose-network reorientation depends on the composition of the matrix, including the water content, which lubricates the motion of macromolecules in the network. At the submolecular level, dynamic 2D FT-IR data suggested that there was no interaction between cellulose and pectin in CP and that they responded independently to a small amplitude strain, while in CXG, cellulose and xyloglucan were uniformly strained along the sample length.

Acetobacter↗

The enumeration and identification of acetic acid bacteria from South African red wine fermentations.

Acetic acid bacteria are microorganisms that can profoundly influence the quality of wine. Surprisingly, little research has been done on these microorganisms in the winemaking field. The object of this study was to investigate the occurrence of acetic acid bacteria in South African red wine fermentations and to identify the dominant species occurring. Acetic acid bacteria were isolated and enumerated from small-scale and commercial red must fermentations in 1998 and 1999, respectively. The initial occurrence of acetic acid bacteria in the must was shown to vary with cell numbers ranging from 10(6)-10(7) to 10(4)-10(5) cfu/ml for the 1998 and 1999 musts, respectively. The acetic acid bacteria decreased to 10(2)-10(3) cfu/ml in musts having a low pH (< or = 3.6), whereas in some musts having a high pH (> or = 3.7), the cell numbers increased during fermentation. During the process of cold soaking, the cell numbers of acetic acid bacteria also increased until inoculation with commercial wine yeast. Gluconobacter oxydans dominated in the fresh must and Acetobacter pasteurianus and A. liquefaciens during fermentation. This study showed that A. liquefaciens and A. hansenii were present in significant numbers, which has not been reported before.

Acetic Acid↗