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.     

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.     

Spheroplast Fusion of Acetobacter aceti and Its Application to the Breeding of Strains for Vinegar Production

Spheroplasts of auxotrophic mutants derived from Acetobacter aceti subsp. aceti No. 1023 were efficiently prepared by treatment with lysozyme, using sucrose as an osmotic stabilizer, and regenerated on an agar plate containing sorbitol and gelatin. In addition, spheroplast fusion between the several auxotrophic mutants was achieved in the presence of polyethylene glycol and CaCl₂. The frequency of fusion was found to be about 5 × 10 ⁵. Spheroplast fusion between A. aceti subsp. aceti No. 2 with the ability to grow at high temperature and A. aceti subsp. xylinum NBI1002 with high resistance to acetic acid was also achieved by the same method, with a frequency of 6.0 × 10 ⁶. The fusants showed various degrees of resistance to acetic acid and ability to grow at high temperature. One of the fusants, named No. 116, could produce acetic acid from ethanol continuously under conditions under which both parent strains were unable to grow. This suggests that spheroplast fusion is applicable to the breeding of strains for vinegar production.     

Localisation and distribution of alcohol-cytochrome 553 reductase in acetic acid bacteria

Alcohol-cytochrome 553 reductase was detected in several strains of the mesoxydans and oxydans group ofAcetobacter. A similar enzyme, but with a higher optimum pH, was detected inAcetobacter aceti (liquefaciens) and in two strains ofGluconobacter.Intermittent ultrasonic disruption ofAcetobacter aceti cells, strainsrancens T-5 andmobilis 6428, showed that the alcohol-cytochrome 553 reductase was mainly localized on the cell hull. The NADP-linked aldehyde dehydrogenase appeared to be present as a cytoplasmic component.The oxidation rate of ethanol and acetaldehyde by intact resting cells which have been grown with either glucose or ethanol as a carbon source under either neutral or acidic conditions was nearly identical. The ethanol oxidizing enzyme system thus behaved as constitutive enzymes, as would be expected if they were bound to the cell hull.The results support the hypothesis that the alcohol-cytochrome 553 reductase is one of the important components of the enzyme system responsible for the physiological production of acetic acid from ethanol by acetic acid bacteria.     

Nutritional requirements and biochemical activities of pineapple pink disease bacterial strains from Hawaii

Bacteria which cause pink disease of pineapple, identified on the basis of their nutritional and biochemical activities, were found to belong to three genera. These bacteria include the following species: Gluconobacter oxydans, Acetobacter aceti, and Erwinia herbicola. Several pink disease strains required one to three vitamins for growth. Both G. oxydans strains 303D and 180 required biotin, nicotinic acid, and pantothenic acid for growth; E. herbicola 189 required only nicotinic acid; however, A. aceti 295 was able to grow without any added supplements in glucose mineral salts medium. Optimal vitamin concentrations for maximal growth and optimal pH for the maximal number of generations per hour was established for a few pink disease strains.Journal Series Paper No. 2373 of the Hawaii Agricultural Experiment Station, Honolulu.     

Isolation and Identification of Acetic Acid Bacteria for Submerged Acetic Acid Fermentation at High Temperature

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.     

Glucose metabolism in Acetobacter aceti

Acetobacter aceti NCIB 8554 grows on a minimal medium with ethanol but not with glucose as carbon and energy source. Addition of glucose to a wild type culture on ethanol has no influence on growth of the organism. Growth of a glucose sensitive mutant A5 is inhibited by the addition of glucose until all glucose has disappeared from the medium. In order to determine the routes by which glucose is metabolised in wild type and mutant, radiorespirometric, enzymatic, and uptake experiments have been performed. For the radiorespirometric experiments of the continuous substrate feeding type an apparatus has been constructed.Of the glucose entering the cells about 30% is excreted as gluconate and 6% metabolised with liberation of C-1 as CO₂. The rest is accumulated intracellularly. No differences were found between wild type and mutant.Under different growth conditions and with different enzymatic assay methods no pyruvate kinase activity (EC 2.7.1.40) could be detected. This might explain the inability of A. aceti to grow on glucose.Abbreviations PPO 2,5-diphenyloxazole - DM-POPOP 2,2-p-phenylene bis(4-methyl-5-phenyloxazole) - TCA trichloroacetic acid     

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. Received: 31 March 2000 / Received revision: 22 May 2000 / Accepted: 3 June 2000     

Distribution and Solubilization of Particulate Gluconate Dehydrogenase and Particulate 2-Ketogluconate Dehydrogenase in Acetic Acid Bacteria

The distribution of two particulate enzymes, gluconate dehydrogenase (GDH) and 2-ketogluconate dehydrogenase (2KGDH), was investigated with cell free extract through 26 strains of genus Acetobacter and genus Gluconobacter. GDH activity was found in the cell free extracts from all strains of genus Gluconobacter and two species of genus Acetobacter, A. aceti and A. aurantium. High activity of 2KGDH was also found in the pigment-producing strains of genus Gluconobacter.

Best solubilization of particulate enzymes was attained with the highest recovery when 10 mg of Triton X–100 and 30 mg of protein of particulate fractions in 1 ml of 0.01 m phosphate buffer, pH 6.0, are incubated for 9 hr at 5°C with continuous stirring.

By comparison of the total enzyme activity of particulate enzymes with that of NAD(P)-linked enzymes in the cell free extract, it was obvious that the formation of ketogluconates by particulate enzymes was much more predominant, roughly over 100 times higher, as that of NAD(P)-linked enzymes.     

Stopped-Flow Measurement of Reaction Rate of Dihydroxyfumaric Acid with 2, 6-Dichlorophenolindophenol

The proline auxotrophic strain of Acetobacter aceti No. 1023 treated with CaCl₂ solution was transformed to the Pro⁺ phenotype at a frequency of up to 10²/μg DNA using chromosomal DNA prepared from the wild type prototrophic strain. The CaCl₂-treated cells of A. aceti No. 1023 could also be rendered competent for uptake of plasmid DNA. In an attempt to produce an appropriate cloning vector for A. aceti, the restriction patterns of the cryptic plasmids, pTA5001 A (23.5 Kb) and pTA5001B (23 Kb), found in A. aceti No. 1023 were determined. A selectable marker (ampicillin resistance) was introduced onto these cryptic plasmids by fusing them to vector pACYC177 from E. coli, using their single restriction site for XhoI. The hybrid plasmids generated could replicate in and confer ampicillin resistance to both A. aceti and E. coli. The maximum transformation frequency for A. aceti No. 1023 with these vectors was 10³/μg DNA.     

Pyruvate,orthophosphate dikinase from Acetobacter aceti

A pyruvate, orthophosphate dikinase (EC 2.7.9.1) has been isolated from Acetobacter aceti grown on pyruvate as the only source of carbon and energy. The enzyme was purified 65-fold, and its molecular weight was determined to be about 330,000 by gel filtration.The optimum pH was 8.0 in the forward direction [phosphoenolpyruvate (PEP) formation] and 7.1 for the backward reaction (pyruvate production). In both directions Mg²⁺ was required (forward K _m 1.70 mM; reverse K _m 0.87 mM) and no other divalent cation was able to replace it. The K _m values for pyruvate, ATP, and P_i were 27 M, 0.20 mM, and 0.83 mM, respectively, in the forward direction. The K _m values for PEP, AMP, and PP_i were 0.13 mM, 6 M, and 62 M, respectively, for the reverse reaction. The substrate-product pairs pyruvate-PEP, ATP-AMP, P_i-PP_i were competitive inhibitors to each other in both directions. These product inhibition studies suggest for the enzyme from A. aceti nonclassical three-site Tri (Uni Uni) Ping-Pong kinetics.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - SDS sodium dodecyl sulphate - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione     

Aflatoxin production by Aspergillus parasiticus in a competitive environment

Aspergillus parasiticus NRRL 2999 was grown in the presence of Rhizopus nigricans, Saccharomyces cerevisiae, Acetobacter aceti, or Brevibacterium linens and aflatoxin concentration was determined after 3,5,7, and 10 days of incubation at 28C. R. nigricans and S. cerevisiae inhibited growth and aflatoxin production by A. parasiticus. B. linens caused slight inhibition and A. aceti stimulated growth and aflatoxin production by A. parasiticus.     

Phosphoenolpyruvate carboxylase from Acetobacter aceti

In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K _m values for PEP and NaHCO₃ were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K _m for Mn²⁺, Co²⁺ and Mg²⁺ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg²⁺. Mn²⁺ and Co²⁺ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K _a 0.18 mM) or propionyl CoA (K _a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

The Phylogeny of Acetic Acid Bacteria Based on the Partial Sequences of 16S Ribosomal RNA: The Elevation of the Subgenus Gluconoacetobacter to the Generic Level

Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q₁₀-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q₉-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9–6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q₁₀-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q₁₀-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (≡ Acetobacter methanolicus), the type species of the genus Acidomonas, had 16–9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.     

Isolation of Clostridium propionicum strain 19acry3 and further characteristics of the species

Two mixed cultures able to ferment acrylate to equimolar acetate and propionate were enriched from anaerobic sediments. From one of these mixed cultures a pure culture of a Gram-positive, obligately anaerobic bacterium was isolated. This strain, designated 19acry3 (= DSM 6251) was identified as belonging to the species Clostridium propionicum. Only a narrow range of organic compounds supported growth, including acrylate and lactate. Acrylate and lactate were fermented to acetate and propionate in a 1:2 molar ratio. When co-cultured with the non-acrylate-fermenting Campylobacter sp. strain 19gly1 (DSM 6222), the fermentation balance shifted to almost equimolar acetate and propionate. Strain 19acry3 was compared with Clostridium propionicum type strain X2 (DSM 1682). The two strains displayed similar phenotypic properties. The mol% G+C of DNA isolated from both strains was 36–37 (by thermal denaturation). Both strains displayed a characteristic fluorescence when observed by fluorescence microscopy. Cell-free extracts of both strains were examined by spectrophotofluorimetry. In both strains, two excitation peaks were observed at 378 and 470 nm. Excitation at either of these wavelengths resulted in an emission maximum at 511 nm.     

Effects of immobilization on biomass production and acetic acid fermentation of Acetobacter aceti as a function of temperature and pH

Summary Acetobacter aceti cells were immobilized using entrapment in Ca-alginate gel and adsorption on preformed cellulose beads. The cell number within the supports showed no significant alterations on changing temperature or pH, whereas the acetic acid production was slightly increased by immobilization.     

Genetic organization ofAcetobacter for acetic acid fermentation

Plasmid vectors for the acetic acid-producing strains ofAcetobacter andGluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochromec, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene ofA. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of anA. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of theAcetobacter strains was developed and applied to improve their properties. ADH membrane-bound alcohol dehydrogenase - ALDH membrane-bound aldehyde dehydrogenase - IS insertion sequence - NTG N-methyl-N-nitro-N-nitrosoguanidine - PQQ pyrroloquinoline quinone     

Effect of mutation on DNA-composition of some bacteria

The aim of this study was to investigate whether DNA from bacteria would be drastically changed by spontaneous and induced mutation. Melting points of pure DNA were taken as a criterion to indicate possible changes. DNA from dark-blue Chromobacteria and from some pale-blue and non-pigmented spontaneous variants had the same melting point, indicating only a very small overall change in its base composition. A similar situation held forAcetobacter aceti (liquefaciens) and an Mn-induced mutant. A wild-typeAgrobacterium tumefaciens B6 and its streptomycin-resistant mutant S1 behaved similarly. However, pure DNA from another, UV-induced, mutant M39 had a melting point which was 1.5 C higher, showing that considerable alterations in the structure of DNA may occur upon mutation. The results are discussed.     

Evolution of Acetic Acid Bacteria During Fermentation and Storage of Wine

Acetic acid bacteria were present at all stages of wine making, from the mature grape through vinification to conservation. A succession of Gluconobacter oxydans, Acetobacter pasteurianus, and Acetobacter aceti during the course of these stages was noted. Low levels of A. aceti remained in the wine; they exhibited rapid proliferation on short exposure of the wine to air and caused significant increases in the concentration of acetic acid. Higher temperature of wine storage and higher wine pH favored the development and metabolism of these species.     

Production of acetic acid by a repeated batch culture with cell recycle ofAcetobacter aceti

Summary A comparison of volumetric production rates of acetic acid inAcetobacter aceti M23 was conducted for repeated batch (RB), cell-recycling repeated batch (CRB) and continuous (C) cultures. Best result was obtained with CRB culture. The magnification of productivity was 1.7 (to RB culture) and 3.3 (to C culture) for aiming final acetic acid concentration of 60 g/l and 42 g/l, respectively.