Aceti Cells Research Articles

Towards a 2025 Global Beer Market: The Use of Aptamers in Biosensors to Detect Acetobacter Aceti in Beer - A Biotechnological Approach to Improve Food Hygiene and Safety in Beer Developing Markets and Beyond

Aptamers were studied to see if they could be designed as a rapid detector for viable spoilage organisms in beer. SELEX was used to obtain aptamers from random libraries of oligonucleotides that could bind to A. aceti. 13 SELEX rounds were performed to get specific aptamers, whole and counter cell-SELEX was employed. Aptamers’ binding ability to A. aceti was tested by flow cytometry and confirmed to bind by synthesizing them with fluorescent labels (5’FAM). ANOVA and a one-sample test revealed significant difference in the binding ability of selected aptamers (P < 0.05). A12 and A10 showed the highest fluorescent intensity (180 and 150) for binding ability. Flow cytometry test for the binding specificity of aptamers and their truncated sequence in comparison with other organisms revealed selected aptamers to be specific to A. aceti cells having the highest mean fluorescence intensity values of 120 ± 3.8 (A12), 119 ± 5.2 (TA12), 110 ± 4.5 (A10) and 111 ± 6.2 (TA10). The best predicted folding structure of selected aptamers from mFold was determined from the Gibbs free energy (A10: ΔG = -4.40, TA10: ΔG = 0.07, A12: ΔG = - 4.96, TA12: ΔG = 0.43). Sigmaplot showed no dose-dependent relationship for detection in candidate aptamers. A12 had a low KD value of 11.22 ± 1.32 nM which further validated it as the best aptamer for detecting A. aceti in beer.

The influence of cultivation conditions on the immobilized acetic acid bacteria metabolic activity

Brewing is an innovative sector of the food industry. Non-alcoholic beer production increases every year. The largest volumes of non-alcoholic beer are obtained by regular beer dialysis. The beer dialysate formed in production contains ethyl alcohol, which entails the need for its rational use. One of the effective ways to use beer dialysate is food biochemical vinegar production. The purpose of the work is to conduct research for biotechnology development of food vinegar from beer dialysates generation using acetic acid bacteria (AAB) immobilization on bio-carriers of various nature. The objects of research were samples of beer dialysates (BD), concentrated to a volume fraction of ethyl alcohol of 7.5%, Acetobacter aceti AAB, vinegar from BD. To study the changes in metabolic activity of immobilized AAB on a bio-carrier, acetic acid fermentation was performed using Acetobacter aceti cells, immobilized on beech shavings, zeolite, and high-pressure polyethylene. Air was supplied to a reservoir by a microcompressor from below through a finely porous ceramic nozzle, providing uniform air dispersion in cultural fluid. Graphs of the cells number and Acetobacter aceti biomass dependence on cultivation duration, nature of the bio-carrier and the aeration method when obtaining vinegar from beer dialysate are presented. The experimental data of the volatile components composition, organic acids and beer dialysate amino acids and vinegar from it are presented. Electron microscopic research of Acetobacter aceti AAB during beech shavings and zeolite cultivation is presented. The results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are presented.

Changes in the gene expression profile of Acetobacter aceti during growth on ethanol

Acetobacter aceti NBRC 14818 shows a diauxic growth profile and temporarily accumulates acetate when grown in medium containing ethanol. However, the mechanisms underlying the metabolic switching between the incomplete oxidation of ethanol and overoxidation of acetate, and the control of stress resistance systems in A. aceti cells grown on ethanol are not fully understood. In this study, time-dependent transcriptome changes in cells during growth on ethanol were analyzed by DNA microarray. In A. aceti, ethanol is oxidized to acetate via acetaldehyde by sequential reactions of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). We found that the genes encoding pyrroloquinoline quinone-dependent ADH, membrane-bound ALDH, and two NAD(+)-ADHs were expressed constitutively in cells throughout the culture period. In contrast, the expression levels of genes encoding tricarboxylic acid (TCA) cycle enzymes were low during acetate accumulation until ethanol was consumed, but were significantly upregulated after the accumulated acetate was started to be consumed. This result suggests that changes in the carbon metabolic flow through the TCA cycle are important for the metabolic switching from acetate accumulation to the overoxidation of acetate. In addition, the genes for glyoxylate pathway enzymes were significantly upregulated soon after the cells began oxidizing ethanol, indicating that this pathway is important for the utilization of ethanol as a carbon source.

Transcriptome response to different carbon sources in Acetobacter aceti

The draft genome sequence of Acetobacter aceti NBRC 14818 was determined by whole-genome shotgun sequencing and the transcriptome profile in cells exponentially grown on ethanol, acetate or glucose was analysed by using a DNA microarray. The genes for all enzymes that constitute the complete tricarboxylic acid (TCA) cycle and glyoxylate pathway were identified in the genome. The TCA cycle genes showed higher expression levels in A. aceti cells grown on acetate or glucose and the glyoxylate pathway genes were significantly induced by ethanol or acetate. Many SOS-response genes were upregulated in cells grown on ethanol, indicating that ethanol provoked damage of DNA and proteins. The superoxide dismutase and catalase genes showed high expression levels in culture on glucose, indicating that oxidation of glucose induced oxidative stress. A. aceti NBRC 14818 was found to have a highly branched respiratory chain. The genes for two type I and one type II NADH dehydrogenase were identified. The genes for one of the type I enzymes were highly expressed when cells were grown on acetate or glucose, but were significantly downregulated in culture on ethanol, probably because ubiquinones were directly reduced by pyrroloquinoline quinone-dependent alcohol dehydrogenase. Four sets of the genes for quinol oxidases, one bo(3)-type (BO3), one bd-type and two cyanide-insensitive-types (CIOs), were identified in the genome. The genes for BO3, which might have proton-pumping activity, were highly expressed under the conditions tested, but were downregulated in the glucose culture. In contrast, the genes for one of the CIOs were significantly upregulated in cells grown on glucose. The two CIOs, which are expected to have lower energy-coupling efficiency, seemed to have a higher contribution in glucose-grown cells. These results indicate that energy conservation efficiency is fine-tuned by changing the respiratory components according to the growth conditions in A. aceti cells.

Mediated catalytic current for the oxidation of ethanol produced by Acetobacter aceti cells suspended in solution

Mediated catalytic current for the oxidation of ethanol produced by Acetobacter aceti cells suspended in solution

Mediated catalytic current for the oxidation of ethanol produced by Acetobacter aceti cells suspended in solution

Mediated catalytic current for the oxidation of ethanol produced by Acetobacter aceti cells suspended in solution

Electrocatalytic properties of Acetobacter aceti cells immobilized on electrodes for the quinone-mediated oxidation of ethanol

Abstract Carbon paste electrodes on which intact cells of Acetobacter aceti (IFO3284) are immobilized behind a dialysis membrane produce a catalytic current for the oxidation of ethanol in the presence of 2-methyl-5,6-dimethoxy benzoquinone (Q 0 ). Analysis of the catalytic current reveals that the catalytic activity of the bacterial cells is due to alcohol dehydrogenase (ADH) existing in the cytoplasmic membranes. The membrane-bound ADH catalyzes the oxidation of ethanol by the use of Q 0 penetrating the membranes, in which Q 0 is reduced by accepting electrons from the enzyme. The reduced form of Q 0 , in turn, reaches the electrode and is oxidized there. Thus, Q 0 serves as an electron transfer mediator between the intact cells and the electrode. The mediated bioelectrocatalysis behavior can be described by an equation of the catalytic current derived for an enzyme-based electrocatalysis. Performance of the electrode as an ethanol sensor is compared with that of an electrode modified with purified ADH. An ADH deficient strain and the ADH mutant harboring ADH gene are also examined as biocatalysts in place of A. aceti .

Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti

Saccharomyces cerevisiae and Acetobacter aceti cells were immobilized by entrapment in Ca-alginate or by adsorption on to preformed cellulose beads and were treated with 0-20% (v/v) ethanol and 0-10% (v/v) acetic acid. At 20% (v/v) ethanol, lethal for free yeast cells, 62-72% of the immobilized cells survived. In 10% (v/v) acetic acid, free and adsorbed Acetobacter aceti cells ceased to grow but 69% of entrapped cells survived. Cells released from the carrier showed an intermediate survival (20-60%).

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

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.

Enhancement of acetic acid production in a high cell-density culture of Acetobacter aceti

The rate of production of acetic acid was greatly enhanced by a high cell-density culture of Acetobacter aceti. Acetobacter aceti cells were recycled through a hollow fiber filter module, and the high acetic acid production rate of 120 mg/ l/h (at an exit acetic acid concentration of 40 g/ l) at the dilution rate of 4.0 h −1 was obtained. High viable cell concentrations were maintained by using medium rich in yeast extract and controlling the DO concentration at an optimum level. Viable cell concentrations increased with increasing added amounts of yeast extract in the medium as long as the acetic acid concentration was not higher than 40 g/ l. When DO and yeast extract were not depleted from the culture, the correlation between the acetic acid production rate ( PD) and the dilution rate ( D) was given as, PD = 7.1 + 34.5 D (0.3 ≤ D ≤ 3)

Spheroplast of Acetic Acid Bacteria

A procedure, preparing spheroplast of acetic acid bacteria, was established to elucidate the membrane structure of the organisms. Of the acetic acid bacteria, only Acetobacter aceti cells were converted into spheroplasts by the sucrose-EDTA-lysozyme system. To Gluconobacter suboxydans, a method exchanging sucrose in the system for NaCl was indispensable. This NaCl-EDTA-lysozyme system was adequate for almost all acetic acid bacteria, which were converted efficiently into spheroplasts. The existence of EDTA was not essential to the genus Gluconobacter.

Oxaloacetate decarboxylase from Acetobacter aceti

An oxaloacetate (OAA) decarboxylase (EC 4.1.1.3) has been purified 72-fold from Acetobacter aceti cells grown on ethanol, and its molecular weight was estimated to be about 80,000 by gel filtration. Several properties distinguished this enzyme from the OAA decarboxylase from A. xylinum: a) It was not a constitutive enzyme; the activity was 6- to 20-fold higher in cells grown on a C2 substrate (acetate or ethanol) than in cells grown on a C3 compound (pyruvate or propionate). b) The optimum pH was 7.5; a value of 5.6 was reported for the enzyme from A. xylinum. c) The enzyme did not need a divalent cation and was not inhibited by EDTA. d) The Kmvalue for OAA was found to be 0.22 mM. It was not affected by the addition of nicotinamide adenine dinucleotide. e) The enzyme activity was neither inhibited by acetate nor by L-malate.

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.