Enhancing small-scale acetification processes using adsorbed Acetobacter pasteurianus UMCC 2951 on κ-carrageenan-coated luffa sponge.

Background: This study explored the utilization of luffa sponge (LS) in enhancing acetification processes. LS is known for having high porosity and specific surface area, and can provide a novel means of supporting the growth of acetic acid bacteria (AAB) to improve biomass yield and acetification rate, and thereby promote more efficient and sustainable vinegar production. Moreover, the promising potential of LS and luffa sponge coated with κ-carrageenan (LSK) means they may represent effective alternatives for the co-production of industrially valuable bioproducts, for example bacterial cellulose (BC) and acetic acid. Methods: LS and LSK were employed as adsorbents for Acetobacter pasteurianus UMCC 2951 in a submerged semi-continuous acetification process. Experiments were conducted under reciprocal shaking at 1 Hz and a temperature of 32 °C. The performance of the two systems (LS-AAB and LSK-AAB respectively) was evaluated based on cell dry weight (CDW), acetification rate, and BC biofilm formation. Results: The use of LS significantly increased the biomass yield during acetification, achieving a CDW of 3.34 mg/L versus the 0.91 mg/L obtained with planktonic cells. Coating LS with κ-carrageenan further enhanced yield, with a CDW of 4.45 mg/L. Acetification rates were also higher in the LSK-AAB system, reaching 3.33 ± 0.05 g/L d as opposed to 2.45 ± 0.05 g/L d for LS-AAB and 1.13 ± 0.05 g/L d for planktonic cells. Additionally, BC biofilm formation during the second operational cycle was more pronounced in the LSK-AAB system (37.0 ± 3.0 mg/L, as opposed to 25.0 ± 2.0 mg/L in LS-AAB). Conclusions: This study demonstrates that LS significantly improves the efficiency of the acetification process, particularly when enhanced with κ-carrageenan. The increased biomass yield, accelerated acetification, and enhanced BC biofilm formation highlight the potential of the LS-AAB system, and especially the LSK-AAB variant, in sustainable and effective vinegar production. These systems offer a promising approach for small-scale, semi-continuous acetification processes that aligns with eco-friendly practices and caters to specialized market needs. Finally, this innovative method facilitates the dual production of acetic acid and bacterial cellulose, with potential applications in biotechnological fields.

Chemical Synthesis of Acetobacter pasteurianus Lipid A with a Unique Tetrasaccharide Backbone and Evaluation of Its Immunological Functions.

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, activates innate immunity. Its active principle is the terminal glycolipid lipid A. Acetobacter pasteurianus is a Gram-negative bacterium used in the fermentation of traditional Japanese black rice vinegar (kurozu). In this study, we focused on A. pasteurianus lipid A, which is a potential immunostimulatory component of kurozu. The active principle structure of A. pasteurianus lipid A has not yet been identified. Herein, we first systematically synthesized three types of A. pasteurianus lipid As containing a common and unique tetrasaccharide backbone. We developed an efficient method for constructing the 2-trehalosamine skeleton utilizing borinic acid-catalyzed glycosylation to afford 1,1'-α,α-glycoside in high yield and stereoselectivity. A common tetrasaccharide intermediate with an orthogonal protecting group pattern was constructed via [2+2] glycosylation. After introducing various fatty acids, all protecting groups were removed to achieve the first chemical synthesis of three distinct types of A. pasteurianus lipid As. After evaluating their immunological function using both human and murine cell lines, we identified the active principles of A. pasteurianus LPS. We also found the unique anomeric structure of A. pasteurianus lipid A contributes to its high chemical stability.

Investigation of Acid Tolerance Mechanism of Acetobacter pasteurianus under Different Concentrations of Substrate Acetic Acid Based on 4D Label-Free Proteomic Analysis.

Acetobacter pasteurianus is always used to brew vinegar because of its ability of producing and tolerating a high concentration of acetic acid. During vinegar fermentation, initial acetic acid contributes to acetic acid accumulation, which varies with initial concentrations. In this study, to investigate the mechanisms of tolerating and producing acetic acid of Acetobacter pasteurianus under different concentrations of substrate acetic acid, four-dimensional label-free proteomic technology has been used to analyze the protein profiles of Acetobacter pasteurianus at different growth stages (the lag and exponential phases) and different substrate acetic acid concentrations (0%, 3%, and 6%). A total of 2093 proteins were quantified in this study. The differentially expressed proteins were majorly involved in gene ontology terms of metabolic processes, cellular metabolic processes, and substance binding. Under acetic acid stress, strains might attenuate the toxicity of acetic acid by intensifying fatty acid metabolism, weakening the tricarboxylic acid cycle, glycerophospholipid and energy metabolism during the lag phase, while strains might promote the assimilation of acetic acid and inter-conversion of substances during the exponential phase by enhancing the tricarboxylic acid cycle, glycolysis, pyruvate, and energy metabolism to produce and tolerate acid. Besides, cell cycle regulation and protein translation might be potential acid tolerance pathways under high acid stress. The result contributes to the exploration of new potential acid tolerance mechanisms in Acetobacter pasteurianus from four-dimensional label-free relative quantitative proteomics analysis.

Alcohol degradation, learning, and memory-enhancing effect of Acetobacter pasteurianus BP2201 in Caenorhabditis elegans model.

AIMS: This study aimed to investigate the probiotic effects of Acetobacter pasteurianus BP2201, isolated from brewing mass, for the treatment of alcohol-induced learning and memory ability impairments in a Caenorhabditis elegans model. METHODS AND RESULTS: Acetobacter pasteurianus BP2201 was examined for probiotic properties, including acid and bile salt resistance, ethanol degradation, antioxidant efficacy, hemolytic activity, and susceptibility to antibiotics. The strain displayed robust acid and bile salt tolerance, efficient ethanol degradation, potent antioxidant activity, and susceptibility to specific antibiotics. Additionally, in the C. elegans model, administering A. pasteurianus BP2201 significantly improved alcohol-induced learning and memory impairments. CONCLUSIONS: Acetobacter pasteurianus BP2201 proves to be a promising candidate strain for the treatment of learning and memory impairments induced by alcohol intake.

Changes in the Bio-Compounds and Biological Activities of Eight Whole Grains Fermentation Starter with Different Oxidized Chin-Shin Oolong Teas.

Chin-shin oolong tea is the most widely planted variety in Taiwan. This study fermented eight whole grains fermentation starter (EGS) with light (LOT), medium (MOT), and fully (FOT) oxidized Chin-shin oolong teas for ten weeks. Comparing the three fermentation beverages, it was found that LOT fermentation can obtain the highest catechins (1644.56 ± 60.15 ppm) among the functional and antioxidant components. MOT can obtain the highest glucuronic acid (19,040.29 ± 2903.91 ppm), tannins, total phenols, flavonoids, and angiotensin-converting enzyme (ACE) inhibitory activity. FOT can obtain the highest GABA (1360.92 ± 123.24 ppm). In addition, both the LOT and MOT showed a significant increase in their ability to scavenge DPPH radicals after fermentation. EGS fermented with lightly or moderately oxidized Chin-shin oolong tea may be considered a novel Kombucha.

Genomic Plasticity of Acid-Tolerant Phenotypic Evolution in Acetobacter pasteurianus.

Acetic acid bacteria have a remarkable capacity to cope with elevated concentrations of cytotoxic acetic acid in their fermentation environment. In particular, the high-level acetate tolerance of Acetobacter pasteurianus that occurs in vinegar industrial settings must be constantly selected for. However, the improved acetic acid tolerance is rapidly lost without a selection pressure. To understand genetic and molecular biology of this acquired acetic acid tolerance in A. pasteurianus, we evolved three strains A. pasteurianus CICIM B7003, CICIM B7003-02, and ATCC 33,445 over 960 generations (4 months) in two initial acetic acids of 20 g·L-1 and 30 g·L-1, respectively. An acetic acid-adapted strain M20 with significantly improved specific growth rate of 0.159 h-1 and acid productivity of 1.61 g·L-1·h-1 was obtained. Comparative genome analysis of six evolved strains revealed that the genetic variations of adaptation were mainly focused on lactate metabolism, membrane proteins, transcriptional regulators, transposases, replication, and repair system. Among of these, lactate dehydrogenase, acetolactate synthase, glycosyltransferase, ABC transporter ATP-binding protein, two-component regulatory systems, the type II toxin-antitoxin system (RelE/RelB/StbE), exodeoxyribonuclease III, type I restriction endonuclease, tRNA-uridine 2-sulfurtransferase, and transposase might collaboratively contribute to the improved acetic acid tolerance in A. pasteurianus strains. The balance between repair factors and transposition variations might be the basis for genomic plasticity of A. pasteurianus strains, allowing the survival of populations and their offspring in acetic acid stress fluctuations. These observations provide important insights into the nature of acquired acetic acid tolerance phenotype and lay a foundation for future genetic manipulation of these strains.

Defining Paenibacillus azoreducens (P8) and Acetobacter pasteurianus (UMCC 2951) strains performances in producing acetic acid.

In this study, spore-forming bacteria isolated from saccharified rice were selected for producing acetic acid. From the screening of 15 strains, P8 strain was chosen as a candidate. The strain was identified as Paenibacillus azoreducens by 16S rRNA analysis (99.85% similarity with P. azoreducens CM1T). Acetic acid is the main component of vinegar but also an industrial commodity produced by chemical synthesis. Sustainable routes for obtaining acetic acid are of great interest for decreasing the environmental impact generated by chemical syntheses. Biological acetic acid production is effective for vinegar production by acetic acid bacteria, but it cannot economically compete with the chemical synthesis for producing it as a pure commodity. Considering the need to improve the yield of pure acetic acid produced by microbial conversions, in this study, P8 strain was chosen for designing processes in different fermentation conditions. Tests were conducted in single and semi-continuous systems, using rice wine as substrate. Acetic acid produced by P8 strain was compared with that of Acetobacter pasteurianus (UMCC 2951), a strain known for producing acetic acid from rice wine. Even though the fermentation performances of P. azoreducens P8 were slightly lower than those of acetic acid bacteria usually used for vinegar production, results highlight its suitability for producing acetic acid. The final acetic acid produced by P. azoreducens P8 was 73 g/L, in a single stage fermentation, without losses. In nine cycles of semi-continuous regime the average of acetification rate was 0.814 (g/L/days). Two main attributes of P. azoreducens P8 are of relevance for producing acetic acid, namely the ability to grow at temperature higher (+ 37°C), than mesophilic acetic acid bacteria, and the absence of cytoplasmic assimilation of acetic acid. These features allow to design multiple strains cultures, in which P. azoreducens can acts as a helper strain. Based on our results, the new isolate P. azoreducens P8 can be propagated in fermenting broths for boosting acetic acid production, under the selected conditions, and used in combination with acetic acid bacteria to produce biological acetic acid, as a non-food grade commodity.

Interaction of acetic acid bacteria and lactic acid bacteria in multispecies solid-state fermentation of traditional Chinese cereal vinegar.

The microbial community plays an important role on the solid-state fermentation (SSF) of Chinese cereal vinegar, where acetic acid bacteria (AAB) and lactic acid bacteria (LAB) are the dominant bacteria. In this study, the top-down (in situ) and bottom-up (in vitro) approaches were employed to reveal the interaction of AAB and LAB in SSF of Shanxi aged vinegar (SAV). The results of high-throughput sequencing indicates that Acetobacter pasteurianus and Lactobacillus helveticus are the predominant species of AAB and LAB, respectively, and they showed negative interrelationship during the fermentation. A. pasteurianus CGMCC 3089 and L. helveticus CGMCC 12062, both of which were isolated from fermentation of SAV, showed no nutritional competition when they were co-cultured in vitro. However, the growth and metabolism of L. helveticus CGMCC 12062 were inhibited during SSF due to the presence of A. pasteurianus CGMCC 3089, indicating an amensalism phenomenon between these two species. The transcriptomic results shows that there are 831 differentially expressed genes (|log2 (Fold Change)| > 1 and, p ≤ 0.05) in L. helveticus CGMCC 12062 under co-culture condition comparing to its mono-culture, which are mainly classified into Gene Ontology classification of molecular function, biological process, and cell composition. Of those 831 differentially expressed genes, 202 genes are up-regulated and 629 genes are down-regulated. The down-regulated genes were enriched in KEGG pathways of sugar, amino acid, purine, and pyrimidine metabolism. The transcriptomic results for A. pasteurianus CGMCC 3089 under co-culture condition reveals 529 differentially expressed genes with 393 up-regulated and 136 down-regulated, and the genes within KEGG pathways of sugar, amino acid, purine, and pyrimidine metabolism are up-regulated. Results indicate an amensalism relationship in co-culture of A. pasteurianus and L. helveticus. Therefore, this work gives a whole insight on the interaction between the predominant species in SSF of cereal vinegar from nutrient utilization, endogenous factors inhibition and the regulation of gene transcription.

Screening and Characterization of New Acetobacter fabarum and Acetobacter pasteurianus Strains with High Ethanol−Thermo Tolerance and the Optimization of Acetic Acid Production

The production of vinegar on an industrial scale from different raw materials is subject to constraints, notably the low tolerance of acetic acid bacteria (AAB) to high temperatures and high ethanol concentrations. In this study, we used 25 samples of different fruits from seven Moroccan biotopes with arid and semi-arid environmental conditions as a basic substrate to isolate thermo- and ethanol-tolerant AAB strains. The isolation and morphological, biochemical and metabolic characterization of these bacteria allowed us to isolate a total number of 400 strains with characters similar to AAB, of which six strains (FAGD1, FAGD10, FAGD18 and GCM2, GCM4, GCM15) were found to be mobile and immobile Gram-negative bacteria with ellipsoidal rod-shaped colonies that clustered in pairs and in isolated chains. These strains are capable of producing acetic acid from ethanol, growing on peptone and oxidizing acetate to CO2 and H2O. Strains FAGD1, FAGD10 and FAGD18 show negative growth on YPG medium containing D-glucose > 30%, while strains GCM2, GCM4 and GCM15 show positive growth. These six strains stand out on CARR indicator medium as isolates of the genus Acetobacter ssp. Analysis of 16S rDNA gene sequencing allowed us to differentiate these strains as Acetobacter fabarum and Acetobacter pasteurianus. The study of the tolerance of these six isolates towards pH showed that most of the six strains are unable to grow at pH 3 and pH 9, with an ideal pH of 5. The behavior of the six strains at different concentrations of ethanol shows an optimal production of acetic acid after incubation at concentrations between 6% and 8% (v/v) of ethanol. All six strains tolerated an ethanol concentration of 16% (v/v). The resistance of the strains to acetic acid differs between the species of AAB. The optimum acetic acid production is obtained at a concentration of 1% (v/v) for the strains of FAGD1, FAGD10 and FAGD18, and 3% (v/v) for GCM2, GCM4 and GCM15. These strains are able to tolerate an acetic acid concentration of up to 6% (v/v). The production kinetics of the six strains show the highest levels of growth and acetic acid production at 30 °C. This rate of growth and acetic acid production is high at 35 °C, 37 °C and 40 °C. Above 40 °C, the production of acid is reduced. All six strains continue to produce acetic acid, even at high temperatures up to 48 °C. These strains can be used in the vinegar production industry to minimize the load on cooling systems, especially in countries with high summer temperatures.

Evaluation of Bioactive Compounds and Antioxidative Activity of Fermented Green Tea Produced via One- and Two-Step Fermentation.

This study investigated the influence of one- and two-step fermentation on bioactive compound production in fermented green tea, i.e., one-step fermented green tea (OFG) and two-step fermented green tea (TFG). One-step fermentation entailed acetic acid fermentation, while two-step fermentation consisted of lactic acid fermentation followed by acetic acid fermentation. Acetobacter pasteurianus PCH 325, isolated from an over-ripened peach, was selected for acetic acid fermentation based on its growth and organic acid production characteristics. Acetic acid fermentation conditions were optimized for one- and two-step fermentation: 3% fermentation alcohol for both processes; 8% and 4% sucrose, respectively; and fermentation at 25 °C for both processes. For lactic acid fermentation of TFG, the inoculum and optimized conditions reported previously were used. Under the optimized conditions, the acetic acid content in OFG and TFG increased 21.20- and 29.51-fold, respectively. Furthermore, through two-step fermentation, γ-aminobutyric acid and lactic acid were produced up to 31.49 ± 1.17 mg/L and 243.44 ± 58.15 mg/L, respectively, which together with acetic acid could contribute to the higher DPPH scavenging activity of TFG. This study suggests that two-step fermentation may be a valuable strategy in industry for raising the amount of acetic acid and/or providing additional bioactive compounds.

Positive effects and mechanism of ultrasound on chitin preparation from shrimp shells by co-fermentation.

The objective of this study is to explore the effect and mechanism of ultrasound on chitin extraction from shrimp shells powder (SSP) by the co-fermentation of Bacillus subtilis and Acetobacter pasteurianus. After pre-treating the SSP with high-intensity ultrasound (HIU) at 800 W, the protease activity in the fermentation solution reached 96.9 U/mL on day 3, which was significantly higher than for SSP that had not been pre-treated with ultrasound (81.8 U/mL). The fermentation time of the chitin extraction process was 5.0 d without ultrasound pre-treatment, while it was shortened to 4.5 d when using ultrasound at 800 W to treat SSP. However, there were no obvious differences when we applied ultrasound at low power (200 W, 400 W). Furthermore, chitin purified from shrimp shells pre-treated with HIU at 800 W exhibited lower molecular weight (11.2 kDa), higher chitin purity (89.8%), and a higher degree of deacetylation (21.1%) compared to SSP with no ultrasound pre-treatment (13.5 kDa, 86.6%, 18.5%). Results indicate that HIU peels off the protein/CaCO3 matrix that covers the SSP surface. About 9.1% of protein and 4.7% of Ca2+ were released from SSP pre-treated with HIU at 800 W. These figures were both higher than with no ultrasound pre-treatment (4.5%, 3.2%). Additionally, the amount of soluble protein extracted from SSP through HIU at 800 W was 50% higher than for the control sample. SDS-PAGE analysis indicated that the soluble protein was degraded to the micromolecule. It also revealed that HIU (600, 800 W) induced the secondary and tertiary structure destruction of protein extracted from SSP. In conclusion, HIU-induced degradation and structural damage of protein enhances the protein/CaCO3 matrix to be peeled off from SSP. Also, in the co-fermentation process, an increase of protease activity further accelerates deproteinization.

Improving the Acetic Acid Fermentation of Acetobacter pasteurianus by Enhancing the Energy Metabolism.

Energy metabolism is important for cell growth and tolerance against environment stress. In acetic acid fermentation by Acetobacter pasteurianus, the correlation coefficients of acid production rate with energy charge and ATP content were 0.9981 and 0.9826, respectively. The main energy metabolism pathway, including glycolysis pathway, TCA cycle, ethanol oxidation, pentose phosphate pathway, and ATP production, was constructed by transcriptome analysis. The effects of fermentation conditions, including dissolved oxygen, initial acetic acid concentration, and total concentration, on acetic acid fermentation and energy metabolism of A. pasteurianus were analyzed by using the RT-PCR method. The results showed the high energy charge inhibited glucose catabolism, and associated with the high ethanol oxidation rate. Consequently, a virtuous circle of increased ethanol oxidation, increased energy generation, and acetic acid tolerance was important for improving acetic acid fermentation.

Acetic acid acting as a signaling molecule in the quorum sensing system increases 2,3-butanediol production in Saccharomyces cerevisiae.

2,3-Butanediol (2,3-BD) has been extensively used in chemical syntheses. This study aimed to explore acetic acid as a signaling molecule that activates a quorum sensing (QS) system to promote the production of 2,3-BD. The yield of 2,3-BD is proportional to the cell density. Saccharomyces cerevisiae W141 does not produce 2,3-BD when the cell density is lower than the threshold concentration (OD600 nm = 10 or cell density 4.4 × 108 CFU/mL). When 1.5 g/L acetic acid is added, the yield of 2,3-BD is 3.01 ± 0.04 g/L. Subsequently, S. cerevisiae W141 was cocultured with Acetobacter pasteurianus Huniang 1.01 under the optimal conditions, the acetic acid production was increased by 76.7% and 30.6% compared with the original strain and the strain cultivated with 1.5 g/L acetic acid, and the yield of 2,3-BD was increased by 81.9% and 3.3%, respectively. This difference is due to the activity of acetyl lactic acid synthase (ILV2) and 2,3-BD dehydrogenase (BDH1), as the relative expression of the ilv2 and bdh1 genes is increased. The results showed that the biosynthesis of 2,3-BD was regulated by acetic acid as a signaling molecule. S. cerevisiae is a promising host for producing 2,3-BD for industrial applications.

60Coγ induction improves the protective effect of Acetobacter pasteurianus against ionizing radiation in mice.

Exposure to ionizing radiation (IR) tends to cause serious health concerns. Thus, radioprotective agents are vital for the population exposed to radiation. As microorganisms have the advantages of fast reproduction and no geographical restrictions, direct microbe-based and environmental induction compounds are thriving radioprotectants resources. Oxidative system and oxidase in Acetobacter pasteurianus are unique and intriguing, the radioprotective effect of the cell-free extract from A. pasteurianus (APE) and 60Coγ-treated extract (IRE) were comparatively investigated in the present study. The survival rate of A. pasteurianus with IRE addition was 149.1% in H2O2 damage test, while that with APE was only 10.4%. The viability of 60Coγ-treated AML-12 cells was increased by 18.8% with IRE addition, yet APE showed no significant radioprotective effect. Moreover, in 60Coγ-treated mice, IRE could significantly protect the white blood cell, improve the liver index, and attenuate the injuries of immune organs in mice. Administration of IRE significantly raised the activities of superoxide dismutase (SOD) and reduced the products of lipid peroxidation. These results clarified that gavage with APE and IRE presented notable antioxidant and radioprotective efficacy. A. pasteurianus showed appealing potential to be novel radioprotective bioagents and 60Coγ treatment on microbe could be a new method for the development of better radioprotectant. KEY POINTS: • 60Coγ induction could improve the radioprotective effect of APE. • IRE protected white blood cell in mice under IR. • IRE products have broad application prospects in radioprotection based on microbes.

Two-Stage Polyhydroxyalkanoates (PHA) Production from Cheese Whey Using Acetobacter pasteurianus C1 and Bacillus sp. CYR1.

Cheese whey (CW) can be an excellent carbon source for polyhydroxyalkanoates (PHA)-producing bacteria. Most studies have used CW, which contains high amounts of lactose, however, there are no reports using raw CW, which has a relatively low amount of lactose. Therefore, in the present study, PHA production was evaluated in a two-stage process using the CW that contains low amounts of lactose. In first stage, the carbon source existing in CW was converted into acetic acid using the bacteria, Acetobacter pasteurianus C1, which was isolated from food waste. In the second stage, acetic acid produced in the first stage was converted into PHA using the bacteria, Bacillus sp. CYR-1. Under the condition of without the pretreatment of CW, acetic acid produced from CW was diluted at different folds and used for the production of PHA. Strain CYR-1 incubated with 10-fold diluted CW containing 5.7 g/L of acetic acid showed the higher PHA production (240.6 mg/L), whereas strain CYR-1 incubated with four-fold diluted CW containing 12.3 g/L of acetic acid showed 126 mg/L of PHA. After removing the excess protein present in CW, PHA production was further enhanced by 3.26 times (411 mg/L) at a four-fold dilution containing 11.3 g/L of acetic acid. Based on Fourier transform infrared spectroscopy (FT-IR), and 1H and 13C nuclear magnetic resonance (NMR) analyses, it was confirmed that the PHA produced from the two-stage process is poly-ß-hydroxybutyrate (PHB). All bands appearing in the FT-IR spectrum and the chemical shifts of NMR nearly matched with those of standard PHB. Based on these studies, we concluded that a two-stage process using Acetobacter pasteurianus C1 and Bacillus sp. CYR-1 would be applicable for the production of PHB using CW containing a low amount of lactose.

Characterization and Regulation of the Acetolactate Synthase Genes Involved in Acetoin Biosynthesis in Acetobacter pasteurianus.

Acetoin is an important aroma-active chemical in cereal vinegars. Acetobacter pasteurianus was reported to make a significant contribution to acetoin generation in cereal vinegars. However, the related acetoin biosynthesis mechanism was largely unknown. Two annotated acetolactate synthase (ALS) genes of A. pasteurianus were investigated in this study to analyze their functions and regulatory mechanisms. Heterologous expression in Escherichia coli revealed that only AlsS1 exhibited ALS activity and had the optimal activity at 55 °C and pH 6.5. Two alsS-defective mutants of A. pasteurianus CICC 22518 were constructed, and their acetoin yields were both reduced, suggesting that two alsS genes participated in acetoin biosynthesis. A total 79.1% decrease in acetoin yield in the alsS1-defective mutant revealed that alsS1 took a major role. The regulator gene alsR disruptant was constructed to analyze the regulation effect. The decline of the acetoin yield and down-regulation of the alsD and alsS1 gene transcriptions were detected, but the alsS2 gene transcription was not affected. Acetoin was an important metabolite of lactate catabolism in A. pasteurianus. The coexistence of two alsS genes can help strains rapidly and securely assimilate lactate to deal with the lactate pressure in a vinegar brewing environment, which represented a new genetic mode of acetoin production in bacteria.

Impacts of type II toxin-antitoxin systems on cell physiology and environmental behavior in acetic acid bacteria.

Acetic acid bacteria (AAB) are a group of Gram-negative and strictly aerobic microorganisms widely used in vinegar industry, especially the species belonging to the genera Acetobacter and Komagataeibacter. The environments inhabited by AAB during the vinegar fermentation, in particular those natural traditional bioprocesses, are complex and dynamically changed, usually accompanied by diverse microorganisms, bacteriophages, and the increasing acetic acid concentration. For this reason, how AAB survive to such harsh niches has always been an interesting research field. Previous omic analyses (e.g., genomics, proteomics, and transcriptomics) have provided abundant clues for the metabolic pathways and bioprocesses indispensable for the acid stress adaptation of AAB. Nevertheless, it is far from fully understanding what factors regulate these modular mechanisms overtly and covertly upon shifting environments. Bacterial toxin-antitoxin systems (TAS), usually consisting of a pair of genes encoding a stable toxin and an unstable antitoxin that is capable of counteracting the toxin, have been uncovered to have a variety of biological functions. Recent studies focusing on the role of TAS in Acetobacter pasteurianus suggest that TAS contribute substantially to the acid stress resistance. In this mini review, we discuss the biological functions of type II TAS in the context of AAB with regard to the acid stress resistance, persister formation and resuscitation, genome stability, and phage immunity. KEY POINTS: • Type II TAS act as regulators in the acid stress resistance of AAB. • Type II TAS are implicated in the formation of acid-tolerant persister cells in AAB. • Type II TAS are potential factors responsible for phage immunity and genome stability.

Thermal adaptation of acetic acid bacteria for practical high-temperature vinegar fermentation.

Thermotolerant microorganisms are useful for high-temperature fermentation. Several thermally adapted strains were previously obtained from Acetobacter pasteurianus in a nutrient-rich culture medium, while these adapted strains could not grow well at high temperature in the nutrient-poor practical culture medium, "rice moromi." In this study, A. pasteurianus K-1034 originally capable of performing acetic acid fermentation in rice moromi was thermally adapted by experimental evolution using a "pseudo" rice moromi culture. The adapted strains thus obtained were confirmed to grow well in such the nutrient-poor media in flask or jar-fermentor culture up to 40 or 39 °C; the mutation sites of the strains were also determined. The high-temperature fermentation ability was also shown to be comparable with a low-nutrient adapted strain previously obtained. Using the practical fermentation system, "Acetofermenter," acetic acid production was compared in the moromi culture; the results showed that the adapted strains efficiently perform practical vinegar production under high-temperature conditions.

Establishment of successive co-fermentation by Bacillus subtilis and Acetobacter pasteurianus for extracting chitin from shrimp shells.

To simplify the process of chitin bio-extraction from shrimp shells powder (SSP), successive co-fermentation using Bacillus subtilis and Acetobacter pasteurianus was explored in this work. Among three protease-producer (B. licheniformis, B. subtilis, and B. cereus), only B. subtilis exhibited high compatibility with A. pasteurianus in co-culture. Successive co-fermentation was constructed as follows: deproteinization was performed for 3 d by culturing B. subtilis in the medium containing 50 g·L-1 SSP, 50 g·L-1 glucose, and 1 g·L-1 yeast extracts; After feeding 5 g·L-1 KH2PO4 and 6 % (v/v) ethanol, A. pasteurianus was cultured for another 2 d without replacing and re-sterilizing medium. Through 5 d of fermentation, the final deproteinization, demineralization efficiency, and chitin yield reached 94.5 %, 92.0 %, and 18.0 %, respectively. This purified chitin had lower molecular weight (12.8 kDa) and higher deacetylation degree (19.6 %) compared with commercial chitin (18.5 kDa, 6.7 %), and showed excellent structural characterization of FESEM and FT-IR analysis.

Toxin-antitoxin HicAB regulates the formation of persister cells responsible for the acid stress resistance in Acetobacter pasteurianus.

Elucidation of the acetic acid resistance (AAR) mechanisms is of great significance to the development of industrial microbial species, specifically to the acetic acid bacteria (AAB) in vinegar industry. Currently, the role of population heterogeneity in the AAR of AAB is still unclear. In this study, we investigated the persister formation in AAB and the physiological role of HicAB in Acetobacter pasteurianus Ab3. We found that AAB were able to produce a high level of persister cells (10-2 to 100 in frequency) in the exponential-phase cultures. Initial addition of acetic acid and ethanol reduced the ratio of persister cells in A. pasteurianus by promoting the intracellular ATP level. Further, we demonstrated that HicAB was an important regulator of AAR in A. pasteurianus Ab3. Strains lacking hicAB showed a decreased survival under acetic acid exposure. Deletion of hicAB significantly diminished the acetic acid production, acetification rate, and persister formation in A. pasteurianus Ab3, underscoring the correlation between hicAB, persister formation, and acid stress resistance. By transcriptomic analysis (RNA-seq), we revealed that HicAB contributed to the survival of A. pasteurianus Ab3 under high acid stress by upregulating the expression of genes involved in the acetic acid over-oxidation and transport, 2-methylcitrate cycle, and oxidative phosphorylation. Collectively, the results of this study refresh our current understanding of the AAR mechanisms in A. pasteurianus, which may facilitate the development of novel ways for improving its industrial performance and direct the scaled-up vinegar production. KEY POINTS: • AAB strains form persister cells with different frequencies. • A. pasteurianus are able to form acid-tolerant persister cells. • HicAB contributes to the AAR and persister formation in A. pasteurianus Ab3.