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The activity of bacterial strains significantly influences the quality and the taste of vinegar. Previous studies of acetic acid bacteria have primarily focused on the ability of bacterial strains to produce high amounts of acetic acid. However, few studies have examined the production of gluconic acid during acetous fermentation at high temperatures. The production of vinegar at high temperatures by two strains of acetic acid bacteria isolated from apple and cactus fruits, namely AF01 and CV01, respectively, was evaluated in this study. The simultaneous production of gluconic and acetic acids was also examined in this study. Biochemical and molecular identification based on a 16s rDNA sequence analysis confirmed that these strains can be classified as Acetobacter pasteurianus. To assess the ability of the isolated strains to grow and produce acetic acid and gluconic acid at high temperatures, a semi-continuous fermentation was performed in a 20-L bioreactor. The two strains abundantly grew at a high temperature (41°C). At the end of the fermentation, the AF01 and CV01 strains yielded acetic acid concentrations of 7.64% (w/v) and 10.08% (w/v), respectively. Interestingly, CV01 was able to simultaneously produce acetic and gluconic acids during acetic fermentation, whereas AF01 mainly produced acetic acid. In addition, CV01 was less sensitive to ethanol depletion during semi-continuous fermentation. Finally, the enzymatic study showed that the two strains exhibited high ADH and ALDH enzyme activity at 38°C compared with the mesophilic reference strain LMG 1632, which was significantly susceptible to thermal inactivation., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Enantiopure (S)-1-(4-methoxyphenyl) ethanol {(S)-MOPE} can be employed as an important synthon for the synthesis of cycloalkyl [b] indoles with the treatment function for general allergic response. To date, the biocatalytic resolution of racemic MOPE through asymmetric oxidation in the biphasic system has remained largely unexplored. Additionally, deep eutectic solvents (DESs), as a new class of promising green solvents, have recently gained increasing attention in biocatalysis for their excellent properties and many successful examples in biocatalytic processes. In this study, the biocatalytic asymmetric oxidation of MOPE to get (S)-MOPE using Acetobacter sp. CCTCC M209061 cells was investigated in different two-phase systems, and adding DES in a biphasic system was also explored to further improve the reaction efficiency of the biocatalytic oxidation., Results: Of all the examined water-immiscible organic solvents and ionic liquids (ILs), 1-butyl-3-methylimidazolium hexafluorophoshpate ([C4MIM][PF6]) afforded the best results, and consequently was selected as the second phase of a two-phase system for the asymmetric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061 cells. For the reaction performed in the [C4MIM][PF6]/buffer biphasic system, under the optimized conditions, the initial reaction rate, the maximum conversion and the residual substrate e.e. recorded 97.8 μmol/min, 50.5 and >99.9 % after 10 h reaction. Furthermore, adding the DES [ChCl][Gly] (10 %, v/v) to the aqueous phase, the efficiency of the biocatalytic oxidation was rose markedly. The optimal substrate concentration and the initial reaction rate were significantly increased to 80 mmol/L and 124.0 μmol/min, respectively, and the reaction time was shortened to 7 h with 51.3 % conversion. The immobilized cell still retained over 72 % of its initial activity after 9 batches of successive reuse in the [C4MIM][PF6]/[ChCl][Gly]-containing buffer system. Additionally, the efficient biocatalytic process was feasible up to a 500-mL preparative scale., Conclusion: The biocatalytic asymmetric oxidation of MOPE with Acetobacter sp. CCTCC M209061 cells was successfully conducted in the [C4MIM][PF6]-containing biphasic system with high conversion and enantioselectivity, and the reaction efficiency was further enhanced by adding [ChCl][Gly] to the reaction system. The efficient biocatalytic process was promising for the preparation of enantiopure (S)-MOPE.

Environmental adaptation is considered as one of the most challenging subjects in biology to understand evolutionary or ecological diversification processes and in biotechnology to obtain useful microbial strains. Temperature is one of the important environmental stresses; however, microbial adaptation to higher temperatures has not been studied extensively. For industrial purposes, the use of thermally adapted strains is important, not only to reduce the cooling expenses of the fermentation system, but also to protect fermentation production from accidental failure of thermal management. Recent progress in next-generation sequencing provides a powerful tool to track the genomic changes of the adapted strains and allows us to compare genomic DNA sequences of conventional strains with those of their closely related thermotolerant strains. In this article, we have attempted to summarize our recent approaches to produce thermotolerant strains by thermal adaptation and comparative genomic analyses of Acetobacter pasteurianus for high-temperature acetic acid fermentations, and Zymomonas mobilis and Kluyveromyces marxianus for high-temperature ethanol fermentations. Genomic analysis of the adapted strains has found a large number of mutations and/or disruptions in highly diversified genes, which could be categorized into groups related to cell surface functions, ion or amino acid transporters, and some transcriptional factors. Furthermore, several phenotypic and genetic analyses revealed that the thermal adaptation could lead to decreased ROS generation in cells that produce higher ROS levels at higher temperatures. Thus, it is suggested that the thermally adapted cells could become robust and resistant to many stressors, and thus could be useful for high-temperature fermentations.

Acetobacter pasteurianus (Ap) CICC 20001 and CGMCC 1.41 are two acetic acid bacteria strains that, because of their strong abilities to produce and tolerate high concentrations of acetic acid, have been widely used to brew vinegar in China. To globally understand the fermentation characteristics, acid-tolerant mechanisms and genetic stabilities, their genomes were sequenced. Genomic comparisons with 9 other sequenced Ap strains revealed that their chromosomes were evolutionarily conserved, whereas the plasmids were unique compared with other Ap strains. Analysis of the acid-tolerant metabolic pathway at the genomic level indicated that the metabolism of some amino acids and the known mechanisms of acetic acid tolerance, might collaboratively contribute to acetic acid resistance in Ap strains. The balance of instability factors and stability factors in the genomes of Ap CICC 20001 and CGMCC 1.41 strains might be the basis for their genetic stability, consistent with their stable industrial performances. These observations provide important insights into the acid resistance mechanism and the genetic stability of Ap strains and lay a foundation for future genetic manipulation and engineering of these two strains.

As a traditional Asian food for several centuries, vinegar is known to be produced by acetic acid bacteria. The Acetobacter species is the primary starter for vinegar fermentation and has evolutionarily acquired acetic acid resistance, in which Acetobacter pasteurianus Ab3 is routinely used for industrial production of rice vinegar with a high acidity (9 %, w/v). In contrast to the documented short-term and low acetic acid effects on A. pasteurianus, here we investigated the molecular and cellular signatures of long-term and high acetic acid responses by proteomic profiling with bidimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS) analyses. Protein spots of interest were selected based on the threshold ANOVA p value of 0.05 and minimal twofold of differential expression, leading to the identification of 26 proteins that are functionally enriched in oxidoreductase activity, cell membrane, and metabolism. The alterations in protein functioning in respiratory chain and protein denaturation may underlay cellular modifications at the outer membrane. Significantly, we found that at higher acidity fermentation phase, the A. pasteurianus Ab3 cells would adapt to distinct physiological processes from that of an ordinary vinegar fermentation with intermediate acidity, indicating increasing energy requirement and dependency of membrane integrity during the transition of acetic acid production. Together, our study provided new insights into the adaptation mechanisms in A. pasteurianus to high acetic acid environments and yield novel regulators and key pathways during the development of acetic acid resistance.

Aims: To improve the thermotolerant properties (TTP) of acetic acid bacteria (AAB) cells for high temperature acetification., Methods and Results: At high temperature (36 ± 1°C), the acetification rate (ETA) is usually lower than at 30 ± 1°C. The addition of 0·15% calcium chloride (CaCl2 ) may decrease the negative effect of the increase of temperature from 30 ± 1°C to 36 ± 1°C on the ETA. The effect of CaCl2 on the thermotolerant properties of acetic acid bacteria cells was investigated. The CaCl2 increased the content of phospholipids (phosphotidylcholine and phosphatidylglycerol), fatty acids (cis-vaccenic acid, palmitic acid and myristic acid) and the activities of membrane-bound enzymes involved in acetification, alcohol dehydrogenase and aldehyde dehydrogenase. Transmission electron microscope images revealed a more compact cell wall with CaCl2. Process consistency at 36 ± 1°C was tested in nine sequential acetification cycles using 0·15% (w/v) CaCl2. High ETAs (9·33 ± 0·6; 8·67 ± 0·8 and 9·67 ± 0·7 g l(-1) day(-1)) were obtained during the last three cycles., Conclusions: The results confirm that changes of the content of lipid, activities of membrane-bound enzymes and cell-wall thickness occurred with added CaCl2., Significance and Impact of the Study: High temperature acetification (HTA) with additions of CaCl2 was investigated. Significant reductions in the overall production costs result from lower cooling costs associated with HTA., (© 2015 The Society for Applied Microbiology.)

A gene encoding a putative Na(+)/H(+) antiporter was previously proposed to be involved in the thermotolerance mechanism of Acetobacter tropicalis SKU 1100. The results of this study show that disruption of this antiporter gene impaired growth at high temperatures with an external pH>6.5. The growth impairment at high temperatures was much more severe in the absence of Na(+) (with only the presence of K(+)); under these conditions, cells failed to grow even at 30°C and neutral to alkaline pH values, suggesting that this protein is also important for K(+) tolerance. Functional analysis with inside-out membrane vesicles from wild type and mutant strains indicated that the antiporter, At-NhaK2 operates as an alkali cation/proton antiporter for ions such as Na(+), K(+), Li(+), and Rb(+) at acidic to neutral pH values (6.5-7.5). The membrane vesicles were also shown to contain a distinct pH-dependent Na(+)(specific)/H(+) antiporter(s) that might function at alkaline pH values. In addition, phylogenetic analysis showed that At-NhaK2 is a novel type of Na(+)/H(+) antiporter belonging to a phylogenetically distinct new clade. These data demonstrate that At-NhaK2 functions as a Na(+)(K(+))/H(+) antiporter and is essential for K(+) and pH homeostasis during the growth of A. tropicalis SKU1100, especially at higher temperatures., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Gut microorganisms are essential for the nutritional health of many animals, but the underlying mechanisms are poorly understood. This study investigated how lipid accumulation by adult Drosophila melanogaster is reduced in flies associated with the bacterium Acetobacter tropicalis which displays oral-faecal cycling between the gut and food. We demonstrate that the lower lipid content of A. tropicalis-colonized flies relative to bacteria-free flies is linked with a parallel bacterial-mediated reduction in food glucose content; and can be accounted for quantitatively by the amount of glucose acquired by the flies, as determined from the feeding rate and assimilation efficiency of bacteria-free and A. tropicalis-colonized flies. We recommend that nutritional studies on Drosophila include empirical quantification of food nutrient content, to account for likely microbial-mediated effects on diet composition. More broadly, this study demonstrates that selective consumption of dietary constituents by microorganisms can alter the nutritional balance of food and, thereby, influence the nutritional status of the animal host., (© 2015 The Author(s).)

Solid-state fermentation of traditional Chinese vinegar is a mixed-culture refreshment process that proceeds for many centuries without spoilage. Here, we investigated bacterial community succession and flavor formation in three batches of Zhenjiang aromatic vinegar using pyrosequencing and metabolomics approaches. Temporal patterns of bacterial succession in the Pei (solid-state vinegar culture) showed no significant difference (P > 0.05) among three batches of fermentation. In all the batches investigated, the average number of community operational taxonomic units (OTUs) decreased dramatically from 119 ± 11 on day 1 to 48 ± 16 on day 3, and then maintained in the range of 61 ± 9 from day 5 to the end of fermentation. We confirmed that, within a batch of fermentation process, the patterns of bacterial diversity between the starter (took from the last batch of vinegar culture on day 7) and the Pei on day 7 were similar (90%). The relative abundance dynamics of two dominant members, Lactobacillus and Acetobacter, showed high correlation (coefficient as 0.90 and 0.98 respectively) among different batches. Furthermore, statistical analysis revealed dynamics of 16 main flavor metabolites were stable among different batches. The findings validate the batch-to-batch uniformity of bacterial community succession and flavor formation accounts for the quality of Zhenjiang aromatic vinegar. Based on our understanding, this is the first study helps to explain the rationality of age-old artistry from a scientific perspective., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The current investigation analyzes metabolites of Acetobacter aceti to explore chemical compounds responsible for the induction of vitamins in barley seeds. A bioactivity guided assay of bacterial extracts and chromatographic analyses of barley produce revealed 13 chemical compounds, which were subjected to principal component analysis (PCA). PCA determined four chemical compounds (i.e., quinolinic acid, pyridoxic acid, p-aminobenzoate, and α-oxobutanoic acid) highly associated with increased quantities of vitamins. Further experimentations confirmed that quinolinic acid and p-aminobenzoate were the most efficient vitamin inducers. The results indicated chloroform/ethanol (4:1) as the best solvent system for the extraction of active compounds from crude metabolites of A. aceti. Significant quantities of mevalonic acid were detected in the extracted fraction, indicating the possible induction of the isoprenoid pathway. Altogether, the current investigation broadens the frontiers in plant-microbe interaction.

Cocoa beans (Theobroma cacao L.) are the raw material for chocolate production. Fermentation of cocoa pulp by microorganisms is crucial for developing chocolate flavor precursors. Yeasts conduct an alcoholic fermentation within the bean pulp that is essential for the production of good quality beans, giving typical chocolate characters. However, the roles of bacteria such as lactic acid bacteria and acetic acid bacteria in contributing to the quality of cocoa bean and chocolate are not fully understood. Using controlled laboratory fermentations, this study investigated the contribution of lactic acid bacteria to cocoa bean fermentation. Cocoa beans were fermented under conditions where the growth of lactic acid bacteria was restricted by the use of nisin and lysozyme. The resultant microbial ecology, chemistry and chocolate quality of beans from these fermentations were compared with those of indigenous (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii, Kluyveromyces marxianus and Saccharomyces cerevisiae, the lactic acid bacteria Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in control fermentations. In fermentations with the presence of nisin and lysozyme, the same species of yeasts and acetic acid bacteria grew but the growth of lactic acid bacteria was prevented or restricted. These beans underwent characteristic alcoholic fermentation where the utilization of sugars and the production of ethanol, organic acids and volatile compounds in the bean pulp and nibs were similar for beans fermented in the presence of lactic acid bacteria. Lactic acid was produced during both fermentations but more so when lactic acid bacteria grew. Beans fermented in the presence or absence of lactic acid bacteria were fully fermented, had similar shell weights and gave acceptable chocolates with no differences in sensory rankings. It was concluded that lactic acid bacteria may not be necessary for successful cocoa fermentation., (Copyright © 2015. Published by Elsevier B.V.)

This paper studies the amino acid profile of beverages obtained through the fermentation of strawberry purée by a surface culture using three strains belonging to different acetic acid bacteria species (one of Gluconobacter japonicus, one of Gluconobacter oxydans and one of Acetobacter malorum). An HPLC-UV method involving diethyl ethoxymethylenemalonate (DEEMM) was adapted and validated. From the entire set of 21 amino acids, multiple linear regressions showed that glutamine, alanine, arginine, tryptophan, GABA and proline were significantly related to the fermentation process. Furthermore, linear discriminant analysis classified 100% of the samples correctly in accordance with the microorganism involved. G. japonicus consumed glucose most quickly and achieved the greatest decrease in amino acid concentration. None of the 8 biogenic amines were detected in the final products, which could serve as a safety guarantee for these strawberry gluconic fermentation beverages, in this regard., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The utilization of fermentation media derived from waste and by-product streams from biodiesel and confectionery industries could lead to highly efficient production of bacterial cellulose. Batch fermentations with the bacterial strain Komagataeibacter sucrofermentans DSM (Deutsche Sammlung von Mikroorganismen) 15973 were initially carried out in synthetic media using commercial sugars and crude glycerol. The highest bacterial cellulose concentration was achieved when crude glycerol (3.2 g/L) and commercial sucrose (4.9 g/L) were used. The combination of crude glycerol and sunflower meal hydrolysates as the sole fermentation media resulted in bacterial cellulose production of 13.3 g/L. Similar results (13 g/L) were obtained when flour-rich hydrolysates produced from confectionery industry waste streams were used. The properties of bacterial celluloses developed when different fermentation media were used showed water holding capacities of 102-138 g · water/g · dry bacterial cellulose, viscosities of 4.7-9.3 dL/g, degree of polymerization of 1889.1-2672.8, stress at break of 72.3-139.5 MPa and Young's modulus of 0.97-1.64 GPa. This study demonstrated that by-product streams from the biodiesel industry and waste streams from confectionery industries could be used as the sole sources of nutrients for the production of bacterial cellulose with similar properties as those produced with commercial sources of nutrients.

Cellulose producing bacteria were isolated from fruit samples and kombucha tea (a fermented beverage) using CuSO4 solution in modified Watanabe and Yamanaka medium to inhibit yeasts and molds. Six bacterial strains showing cellulose production were isolated and identified by 16S rRNA gene sequencing as Gluconacetobacter xylinus strain DFBT, Ga. xylinus strain dfr-1, Gluconobacter oxydans strain dfr-2, G. oxydans strain dfr-3, Acetobacter orientalis strain dfr-4, and Gluconacetobacter intermedius strain dfr-5. All the cellulose-producing bacteria were checked for the cellulose yield. A potent cellulose-producing bacterium, i.e., Ga. xylinus strain DFBT based on yield (cellulose yield 5.6 g/L) was selected for further studies. Cellulose was also produced in non- conventional media such as pineapple juice medium and hydrolysed corn starch medium. A very high yield of 9.1 g/L cellulose was obtained in pineapple juice medium. Fourier transform infrared spectrometer (FT-IR) analysis of the bacterial cellulose showed the characteristic peaks. Soft cellulose with a very high water holding capacity was produced using limited aeration. Scanning electron microscopy (SEM) was used to analyze the surface characteristics of normal bacterial cellulose and soft cellulose. The structural analysis of the polymer was performed using (13)C solid-state nuclear magnetic resonance (NMR). More interfibrillar space was observed in the case of soft cellulose as compared to normal cellulose. This soft cellulose can find potential applications in the food industry as it can be swallowed easily without chewing.

Bacteria cellulose membranes (BCM) are used for wound dressings, bone grafts, tissue engineering, artificial vessels, and dental implants because of their high tensile strength, crystallinity and water holding ability. In this study, the effects of BCM application for 15 days on healing of burn wounds were investigated based on evaluation of skin regeneration and angiogenesis in burn injury skin of Sprague-Dawley (SD) rats. BCM showed a randomly organized fibrils network, 12.13 MPa tensile strength, 12.53% strain, 17.63% crystallinity, 90.2% gel fraction and 112.14 g × m(2)/h highest water vapor transmission rate (WVTR) although their swelling ratio was enhanced to 350% within 24h. In SD rats with burned skin, the skin severity score was lower in the BCM treated group than the gauze (GZ) group at all time points, while the epidermis and dermis thickness and number of blood vessels was greater in the BCM treated group. Furthermore, a significant decrease in the number of infiltrated mast cells and in vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) expression was observed in the BCM treated group at day 10 and 15. Moreover, a significant high level in collagen expression was observed in the BCM treated group at day 5 compared with GZ treated group, while low level was detected in the same group at day 10 and 15. However, the level of metabolic enzymes representing liver and kidney toxicity in the serum of BCM treated rats was maintained at levels consistent with GZ treated rats. Overall, BCM may accelerate the process of wound healing in burn injury skin of SD rats through regulation of angiogenesis and connective tissue formation as well as not induce any specific toxicity against the liver and kidney., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Zhenjiang vinegar, the grains of which contain a unique microbial flora, is one of the four famous traditional Chinese vinegars. We investigated the components of Zhenjiang vinegar grains. Unique acetic acid bacteria were randomly isolated from Zhenjiang vinegar grains, and the obtained strains were qualitatively analyzed to compare their capacities for acetate decomposition and acid production. Acetic acid bacteria with a high acid-producing rate were identified by 16S rDNA sequencing, and further confirmation was performed using the Basic Local Alignment Search Tool comparison method. Six significant strains of acetic acid bacteria were isolated. Qualitative analysis showed that these strains produced no brown precipitate and had a capacity for acetate decomposition. Based on physiological and biochemical evaluation, the two strains with the highest acid yield were sequenced, and the results identified strain W1 as Acetobacter aceti and strain W6 as A. pasteurianus.

Acetobacter pasteurianus plays an important role in the process of traditional vinegar production and is also essential for the fermentation of Zhenjiang aromatic vinegar. In this study, we utilized the proteomic approach to analyze the proteomic profile of A. pasteurianus HSZ3-21, and 258 proteins were successfully identified by MALDI-TOF-MS and database search. The hydropathy and GO analyse combined with COG results of the identified proteins revealed the molecular biological characteristics of A. pasteurianus proteins, that is, most proteins of A. pasteurianus were related to metabolic process, binding, catalytic or cellular response. Meanwhile, our results also showed that some proteins of A. pasteurianus may be responsible for acetic acid tolerance, thermotolerance, and stress response. Therefore, the identification of 258 proteins not only deciphers protein composition and functional classification of A. pasteurianus, but also provides useful information for improving quality of Zhenjiang aromatic vinegar.

Aims: To evaluate the comparative impact of high initial concentrations of acetic acid (AAi ) and of ethanol (ETi ) on acetification rate (ETA)., Methods and Results: Acetic acid bacteria (AAB) were cultivated in a 100-l internal Venturi injector bioreactor. To quantify the oxygen availability, the 1.0 l min(-1) air inflow rate for the start-up phase (25 l) while 3·0 l min(-1) for the operational phase (75 l) achieved a high oxygen transfer coefficient (kL a). Changes in cell wall by TEM images and the remained ADH and ALDH activities confirmed the high acid tolerance ability of AAB. While ETAs using high AAi at 65 g l(-1) could be processed of 9.57 ± 0.19 g l(-1) day(-1) , which is just higher than 9.12 ± 0.12 g l(-1) day(-1) using high ETi at 55 g l(-1) . The average biotransformation yields were at 96.3 ± 0.1% and 94.4 ± 0.1% for high AAi and ETi , respectively., Conclusions: Results confirm that high oxygenation was generated in the bioreactor. Both high AAi and ETi were important in increasing ETA under stress 100 g l(-1) total concentration., Significance and Impact of the Study: High acid-tolerant AAB contains the high ADH and ALDH activities causing higher ETAs in HIA process. It is a competitive commercialized acetification process., (© 2014 The Society for Applied Microbiology.)

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.

Nanocomposites of polymethylmethacrylate (PMMA) and cellulose were made by a solution casting method using acetone as the solvent. The nanofiber networks were prepared using three different types of cellulose nanofibers: (i) nanofibrillated cellulose (NFC), (ii) cellulose nanocrystals (CNC) and (iii) bacterial cellulose from nata de coca (NDC). The loading of cellulose nanofibrils in the PMMA varied between 0.25 and 0.5 wt%. The mechanical properties of the composites were evaluated using a dynamic mechanical thermal analyzer (DMTA). The flexural modulus of the nanocomposites reinforced with NDC at the 0.5 wt% loading level increased 23% compared to that of pure PMMA. The NFC composite also exhibited a slightly increased flexural strength around 60 MPa while PMMA had a flexural strength of 57 MPa. The addition of NDC increased the storage modulus (11%) compared to neat PMMA at room temperature while the storage modulus of PPMA/CNC nanocomposite containing 0.25 and 0.5 wt% cellulose increased about 46% and 260% to that of the pure PMMA at the glass transition temperature, respectively. Thermogravimetric analysis (TGA) indicated that there was no significant change in thermal stability of the composites. The UV-vis transmittance of the CNF nanocomposites decreased by 9% and 27% with the addition of 0.25 wt% CNC and NDC, respectively. This work is intended to spur research and development activity for application of CNF reinforced PMMA nanocomposites in applications such as: packaging, flexible screens, optically transparent films and light-weight transparent materials for ballistic protection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: To maximize acetification rate (ETA) by adsorption of acetic acid bacteria (AAB) on loofa sponge matrices (LSM)., Methods and Results: AAB were adsorbed on LSM, and the optimal shaking rate was determined for maximized AAB growth and oxygen availability. Results confirm that the 1 Hz reciprocating shaking rate with 40% working volume (liquid volume 24 l, tank volume 60 l) achieved a high oxygen transfer coefficient (k(L)a). The highest ETA was obtained at 50% (w:v) LSM-AAB:culture medium at 30 ± 2°C (P ≤ 0·05). To test process consistency, nine sequential acetification cycles were run using LSM-AAB and comparing it with no LSM. The highest ETA (1·701-2·401 g l(-1) d(-1)) was with LSM-AAB and was associated with the highest biomass of AAB, confirmed by SEM images., Conclusions: Results confirm that LSM-AAB works well as an inert substrate for AAB. High oxygenation was maintained by a reciprocating shaker. Both shaking and LSM were important in increasing ETA., Significance and Impact of the Study: High cell biomass in LSM-AAB provides good conditions for higher ETAs of quick acetification under adequate oxygen transfer by reciprocating shaker. It is a sustainable process for small-scale vinegar production system requiring minimal set-up cost., (© 2014 The Society for Applied Microbiology.)

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Acetobacter species are well known to have the ability to grow floating on the surface of the medium by producing pellicle, which consists of cells and a self-produced matrix of cell-attached polysaccharide. We previously isolated three thermotolerant strains (SL13E-2, SL13E-3, and SL13E-4) from Sri Lankan coconut vinegar and identified all these strains as Acetobacter pasteurianus. The pellicle polysaccharides of these three strains and of A. pasteurianus SKU1108, which was originally isolated from Thailand, were characterized. The monosaccharide composition of the pellicle polysaccharides of these A. pasteurianus strains was found to be varied. For example, the pellicle polysaccharide of SL13E-2 is composed of rhamnose and glucose in the ratio 1:8, and that of SL13E-4 and mesophilic A. pasteurianus NBRC3191 consists of rhamnose, glucose and xylose in the ratio 1:5:2 and 1:4:2, respectively. On the other hand, the pellicle polysaccharides of SL13E-3 and SKU1108 strains are composed of rhamnose, glucose and galactose in the ratio 2:2:1 and 1:5:2.5, respectively. The pellicle formation of thermotolerant SL13E-2, SL13E-3, and SL13E-4 was found to be significantly induced by the addition of ethanol, while poor induction was observed with SKU1108. The size and sugar composition of the polysaccharides obtained from cells induced by ethanol and by uninduced cells were the same, indicating that the number of molecules of the polysaccharides had increased but the polysaccharide molecule remained unchanged. The addition of a sugar source such as glucose, sucrose or fructose slightly induced pellicle formation in SKU1108, especially at 40°C., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Enantiomerically pure alcohols are important building blocks for production of chiral pharmaceuticals, flavors, agrochemicals and functional materials and appropriate whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to these valuable compounds. At present, most of these biocatalysts follow Prelog's rule, and thus the (S)-alcohols are usually obtained when the smaller substituent of the ketone has the lower CIP priority. Only a few anti-Prelog (R)-specific whole cell biocatalysts have been reported. In this paper, the biocatalytic anti-Prelog reduction of 2-octanone to (R)-2-octanol was successfully conducted with high enantioselectivity using whole cells of Acetobacter pasteurianus GIM1.158., Results: Compared with other microorganisms investigated, Acetobacter pasteurianus GIM1.158 was shown to be more effective for the reduction reaction, affording much higher yield, product enantiomeric excess (e.e.) and initial reaction rate. The optimal temperature, buffer pH, co-substrate and its concentration, substrate concentration, cell concentration and shaking rate were 35°C, 5.0, 500 mmol/L isopropanol, 40 mmol/L, 25 mg/mL and 120 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 89.5% and >99.9%, respectively, in 70 minutes. Compared with the best available data in aqueous system (yield of 55%), the yield of (R)-2-octanol was greatly increased. Additionally, the efficient whole-cell biocatalytic process was feasible on a 200-mL preparative scale and the chemical yield increased to 95.0% with the product e.e. being >99.9%. Moreover, Acetobacter pasteurianus GIM1.158 cells were proved to be capable of catalyzing the anti-Prelog bioreduction of other prochiral carbonyl compounds with high efficiency., Conclusions: Via an effective increase in the maximum yield and the product e.e. with Acetobacter pasteurianus GIM1.158 cells, these results open the way to use of whole cells of this microorganism for challenging enantioselective reduction reactions on laboratory and commercial scales.

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together. Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together. The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

This study investigated the potential of Acetobacterium woodii, a homoacetogen, in co-culture with common acetogens for acetate production during glucose fermentation. Three types of inocula, A. woodii (AW), heat-treated sludge (HTS) and co-culture of A. woodii and heat-treated sludge (AW-HTS) were investigated. Results showed that ∼ 150 mM of glucose was almost completely converted to biomass, gases and other products in co-culture. The addition of A. woodii induced homoacetogenic fermentation in AW-HTS during the first 3 days, as evidenced by the decreased hydrogen production and acetate dominance (>90%, corresponding to 1.19 mol acetate/mol glucose) in total soluble products. However, due to the unfavorable environmental conditions, metabolic pathway in AW-HTS treatment shifted towards butyrate type at the end of the experiment. Bacterial diversity analysis indicated that species supporting growth of A. woodii were dominant during the first several days and their abundance gradually decreased until the end of experiment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Objective: The aim of the study is to propose a dynamic acetic acid resistance mechanism through analysis on response of cellular morphology, physiology and metabolism of A. pasteurianus CICIM B7003 during vinegar fermentation., Methods: Vinegar fermentation was carried out in a Frings 9 L acetator by strain B7003 and cultures were sampled at different cellular growth phases. Simultaneously, percentage of capsular polysaccharide versus dry cells weight, ratio of unsaturated fatty acids to saturated fatty acids, transcription of acetic acid resistance genes, activity of alcohol respiratory chain enzymes and ATPase were detected for these samples to assay the responses of bacterial morphology, physiology and metabolism., Results: When acetic acid was existed, no obvious capsular polysaccharide was secreted by cells. As vinegar fermentation proceeding, percentage of capsular polysaccharide versus dry cells weight was reduced from 2.5% to 0.89%. Ratio of unsaturated fatty acids to saturated fatty acids was increased obviously which can improve membrane fluidity. Also transcription level of acetic acid resistance genes was promoted. Interestingly, activity of alcohol respiratory chain and ATPase was not inhibited but promoted obviously with acetic acid accumulation which could provide enough energy for acetic acid resistance mechanism., Conclusion: On the basis of the results obtained from the experiment, A. pasteurianus CICIM B7003 relies mainly on the cooperation of changes of extracellular capsular polysaccharide and membrane fatty acids, activation of acid resistance genes transcription, enhancement of activity of alcohol respiratory chain and rapid energy production to tolerate acidic environment.

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848(T), Acetobacter fabarum LMG 24244(T), and Acetobacter senegalensis 108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848(T) oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848(T) and Lactobacillus fermentum 222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight that A. pasteurianus 386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates for A. pasteurianus in its indispensable cross-feeding interactions with yeast and LAB during cocoa bean fermentation.

Background: Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (-21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis., Results: Heterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at -21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred., Conclusions: High storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced that even moderate carbonylation of some critical cellular proteins (like ribosomal proteins) may lead to VBNC formation or death of freeze-dried bacteria. Moreover, it seems that other mechanisms of biomolecule deterioration preceding protein carbonylation lead to VBNC formation under very low storage temperature.

In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5'-TCCAAATTTGGAT'-3') and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.

The animal gut is perpetually exposed to microorganisms, and this microbiota affects development, nutrient allocation, and immune homeostasis. A major challenge is to understand the contribution of individual microbial species and interactions among species in shaping these microbe-dependent traits. Using the Drosophila melanogaster gut microbiota, we tested whether microbe-dependent performance and nutritional traits of Drosophila are functionally modular, i.e., whether the impact of each microbial taxon on host traits is independent of the presence of other microbial taxa. Gnotobiotic flies were constructed with one or a set of five of the Acetobacter and Lactobacillus species which dominate the gut microbiota of conventional flies (Drosophila with untreated microbiota). Axenic (microbiota-free) flies exhibited prolonged development time and elevated glucose and triglyceride contents. The low glucose content of conventional flies was recapitulated in gnotobiotic Drosophila flies colonized with any of the 5 bacterial taxa tested. In contrast, the development rates and triglyceride levels in monocolonized flies varied depending on the taxon present: Acetobacter species supported the largest reductions, while most Lactobacillus species had no effect. Only flies with both Acetobacter and Lactobacillus had triglyceride contents restored to the level in conventional flies. This could be attributed to two processes: Lactobacillus-mediated promotion of Acetobacter abundance in the fly and a significant negative correlation between fly triglyceride content and Acetobacter abundance. We conclude that the microbial basis of host traits varies in both specificity and modularity; microbe-mediated reduction in glucose is relatively nonspecific and modular, while triglyceride content is influenced by interactions among microbes.

Unpolished rice black vinegar (kurozu), a traditional Japanese vinegar, is considered to have beneficial health effects. Kurozu is produced via a static fermentation process involving the saccharification of rice by Aspergillus oryzae, alcohol fermentation by Saccharomyces cerevisiae, and the oxidation of ethanol to acetic acid by acetic acid bacteria such as Acetobacter pasteurianus. Since this process requires about 6 months' fermentation and then over a year of aging, most of these organisms die during the production process and so microbial components, which might stimulate the innate immune system, are expected to be present in the vinegar. In this study, we investigated whether microbial components are present in kurozu, and after confirming this we characterized their immunostimulatory activities. Lyophilized kurozu stimulated murine spleen cells to produce tumor necrosis factor (TNF)-α, at least in part, via Toll-like receptor (TLR) 2 and the Nod-like receptors NOD1 and 2. The active components associated with TLR2 activation were concentrated by Triton X-114-water phase partitioning and hydrophobic interaction chromatography on Octyl Sepharose. TLR4-activating components were also enriched by these methods. The concentrated preparation stimulated murine spleen cells to produce TNF-α and interferon (IFN)-γ. These results indicate that long-term fermented kurozu contains immunostimulatory components and that the TLR2 and TLR4-activating immunostimulatory components of kurozu are hydrophobic. These components might be responsible for the beneficial health effects of kurozu., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This project aimed to evaluate the effects 8 additives on the fermentation, dry matter (DM) losses, nutritive value, and aerobic stability of corn silage. Corn forage harvested at 31% DM was chopped (10mm) and treated with (1) deionized water (control); (2) Buchneri 500 (BUC; 1×10(5) cfu/g of Pediococcus pentosaceus 12455 and 4×10(5) cfu/g of Lactobacillus buchneri 40788; Lallemand Animal Nutrition, Milwaukee, WI); (3) sodium benzoate (BEN; 0.1% of fresh forage); (4) Silage Savor acid mixture (SAV: 0.1% of fresh forage; Kemin Industries Inc., Des Moines, IA); (5) 1×10(6) cfu/g of Acetobacter pasteurianus-ATCC 9323; (6) 1×10(6) cfu/g of Gluconobacter oxydans-ATCC 621; (7) Ecosyl 200T (1×10(5) cfu/g of Lactobacillus plantarum MTD/1; Ecosyl Products Inc., Byron, IL); (8) Silo-King WS (1.5×10(5) cfu/g of L. plantarum, P. pentosaceus and Enterococcus faecium; Agri-King, Fulton, IL); and (9) Biomax 5 (BIO; 1×10(5) cfu/g of L. plantarum PA-28 and K-270; Chr. Hansen Animal Health and Nutrition, Milwaukee, WI). Treated forage was ensiled in quadruplicate in mini silos at a density of 172 kg of DM/m(3) for 3 and 120 d. After 3 d of ensiling, the pH of all silages was below 4 but ethanol concentrations were least in BEN silage (2.03 vs. 3.24% DM) and lactic acid was greatest in SAV silage (2.97 vs. 2.51% DM). Among 120-d silages, additives did not affect DM recovery (mean=89.8% ± 2.27) or in vitro DM digestibility (mean=71.5% ± 0.63). The SAV silage had greater ammonia-N (0.85 g/kg of DM) and butyric acid (0.22 vs. 0.0% DM) than other treatments. In contrast, BEN and Silo-King silages had the least ammonia-N concentration and had no butyric acid. The BEN and A. pasteurianus silages had the lowest pH (3.69) and BEN silage had the least ethanol (1.04% DM) and ammonia nitrogen (0.64 g/kg DM) concentrations, suggesting that fermentation was more extensive and protein degradation was less in BEN silages. The BUC and BIO silages had greater acetic acid concentrations than control silages (3.19 and 3.19 vs. 2.78% DM), but yeast counts did not differ. Aerobic stability was increased by 64% by BUC (44.30 h) and by 35% by BEN (36.49 h), but other silages had similar values (27.0±1.13 h)., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The coproduction of a useful material and electricity via a novel application of microbial fuel cell (MFC) technology to oxidative fermentation was investigated. We focused on vinegar production, i.e., acetic acid fermentation, as an initial and model useful material that can be produced by oxidative fermentation in combination with MFC technology. The coproduction of acetic acid and electricity by applying MFC technology was successfully demonstrated by the simultaneous progress of acetic acid fermentation and electricity generation through a series of repeated batch fermentations. Although the production rate of acetic acid was very small, it increased with the number of repeated batch fermentations that were conducted. We obtained nearly identical (73.1%) or larger (89.9%) acetic acid yields than that typically achieved by aerated fermentation (75.8%). The open-cycle voltages measured before and after fermentation increased with the total fermentation time and reached a maximum value of 0.521 V prior to the third batch fermentation. The maximum current and power densities measured in this study (19.1 μA/cm² and 2.47 μW/cm², respectively) were obtained after the second batch fermentation., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process., Results: The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition., Conclusions: Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.

Red-koji vinegar is a Monascus -involved and acetic acid fermentation-derived traditional product, in which the presence of monacolin K and citrinin has attracted public attention. In this study, red-koji wine was prepared as the substrate and artificially supplemented with monacolin K and citrinin and subjected to vinegar fermentation with Acetobacter starter. After 30 days of fermentation, 43.0 and 98.1% of the initial supplements of monacolin K and citrinin were decreased, respectively. During fermentation, acetic acid contents increased, accompanied by decreases of ethanol and lactic acid contents and pH values. The contents of free amino acids increased while the contents of other organic acids, including fumaric acid, citric acid, succinic acid, and tartaric acid, changed limitedly. Besides, increased levels of total phenolics in accordance with increased antioxidative potency, α,α-diphenyl-β-picrylhydrazyl scavenging, and xanthine oxidase inhibitory (XOI) activities were detected. It is of merit that most citrinin was eliminated and >50% of the monacolin K was retained; contents of free amino acids and total phenolics along with antioxidant and XOI activities of the red-koji vinegar were increased after fermentation.

In vitro adaptation is one of the most challenging subjects in biology to understand adaptive evolution. Microbial adaptation to temperature is not only interesting in terms of understanding the adaptation mechanism, but also useful for industrial applications. In this study, we attempted the in vitro adaptation of Acetobacter pasteurianus SKU1108 by repeating its cultivation under high-temperature acetic acid fermentation conditions. As a result, thermo-adapted strains having the higher fermentation ability than the wild-type strain were obtained. Mutations and/or disruptions in several proteins of the adapted strains were detected with NGS sequencing technology. In particular, two different adapted strains had mutations or disruptions in three specific genes in common, suggesting that these genes are essential for thermotolerance or fermentation at higher temperature. In order to clarify their involvement in thermotolerance, two of the three genes were disrupted and their phenotype was examined. The results showed that mutations of the two proteins, MarR and an amino acid transporter, are partly responsible for higher fermentation ability and/or thermotolerance. Thus, it was suggested that these elevated abilities of the adapted strains are acquired by assembling several single gene mutations including the above two mutations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of this work was to analyze the microbiota involved in the traditional vinegar elaboration of strawberry fruit during a spontaneous and inoculated process. In the spontaneous processes, low biodiversity was detected in both alcoholic fermentation (AF) and acetification. Nevertheless, a strain of Saccharomyces cerevisiae and of Acetobacter malorum were selected and tested as starter cultures in the inoculation study. The inoculated processes with these strains were compared with another spontaneous process, yielding a significant reduction in time for AF with a total imposition of the S. cerevisiae strain. The resulting strawberry wine was acetified in different containers (glass and wood) yielding an initial imposition of the A. malorum inoculated strain, although displacement by Gluconacetobacter species was observed in the wood barrels., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acetobacter aceti MTCC 2623 was studied as an alternative microbial source for bacterial cellulose (BC) production. Effect of media components on cell growth rate, BC production and cellulose characteristics were studied. FTIR results showed significant variations in cellulose characteristics produced by A. aceti in different media. Results have shown the role of fermentation time on crystallinity ratio of BC in different media. Further, effect of six different media components on cell growth and BC production was studied using fractional factorial design. Citric acid was found to be the most significant media component for cell growth rate (95% confidence level, R(2)=0.95). However, direct role of these parameters on cellulose production was not established (p-value>0.05)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dental root canal treatment is required when dental caries progress to infection of the dental pulp. A major goal of this treatment is to provide complete decontamination of the dental root canal system. However, the morphology of dental root canal systems is complex, and many human dental roots have inaccessible areas. In addition, dental reinfection is fairly common. In conventional treatment, a cotton pellet and paper point made from plant cellulose is used to dry and sterilize the dental root canal. Such sterilization requires a treatment material with high absorbency to remove any residue, the ability to improve the efficacy of intracanal medication and high biocompatibility. Bacterial cellulose (BC) is produced by certain strains of bacteria. In this study, we developed BC in a pointed form and evaluated its applicability as a novel material for dental canal treatment with regard to solution absorption, expansion, tensile strength, drug release and biocompatibility. We found that BC has excellent material and biological characteristics compared with conventional materials, such as paper points (plant cellulose). BC showed noticeably higher absorption and expansion than paper points, and maintained a high tensile strength even when wet. The cumulative release of a model drug was significantly greater from BC than from paper points, and BC showed greater compatibility than paper points. Taken together, BC has great potential for use in dental root canal treatment., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The biocatalytic anti-Prelog stereoselective reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate {(R)-EHB} was successfully conducted in the aqueous system using immobilized Acetobacter sp. CCTCC M209061 cells. Among various microorganisms tested, Acetobacter sp. CCTCC M209061 gave the best performance and showed great potential for the bioreduction of EAA to (R)-EHB. Acetobacter sp. CCTCC M209061 cells were immobilized on calcium alginate and then coated with chitosan, and the immobilized cells clearly surpassed the free cells in terms of better thermal stability, storability and recyclability. The optimal co-substrate for the reaction was found to be glucose. The optimal substrate concentration, buffer pH, glucose concentration, reaction temperature, biocatalyst concentration and shaking speed were 35.0 mmol/L, 5.5, 80.0 mmol/L, 35 °C, 0.45 g/mL and 200 rpm, respectively. Under the optimized conditions, the initial reaction rate, the yield and the enantiomeric excess (e.e.) of the product were 1.28 μmol/min, 82.6% and above 99.0%, respectively, which were much higher than those reported previously. The efficient whole-cell biocatalytic process was feasible on a 500-mL preparative scale, and the immobilized cells showed excellent operational stability and could be re-used for at least 10 batches. Additionally, the product e.e. constantly remained above 99.0% regardless of re-use cycles of the biocatalyst., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose utilization. However, in medium containing both ethanol and glucose, the mutant exhibited significantly poorer growth and lower glucose consumption compared to the wild-type strain. Notably, the mutant oxidized ethanol nearly stoichiometrically to acetate, which was retained in the medium for a longer period of time than the acetate produced by wild-type strain. The features of the aceA-glcB knockout mutant revealed here indicate that the lack of the glyoxylate pathway is advantageous for industrial vinegar production by A. aceti., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: The bacterium Acetobacter sp. CCTCC M209061 is a promising whole-cell biocatalyst with exclusive anti-Prelog stereoselectivity for the reduction of prochiral ketones that can be used to make valuable chiral alcohols such as (R)-4-(trimethylsilyl)-3-butyn-2-ol. Although it has promising catalytic properties, its stability and reusability are relatively poor compared to other biocatalysts. Hence, we explored various materials for immobilizing the active cells, in order to improve the operational stability of biocatalyst., Results: It was found that Ca-alginate give the best immobilized biocatalyst, which was then coated with chitosan to further improve its mechanical strength and swelling-resistance properties. Conditions were optimized for formation of reusable immobilized beads which can be used for repeated batch asymmetric reduction of 4'-chloroacetophenone. The optimized immobilized biocatalyst was very promising, with a specific activity of 85% that of the free-cell biocatalyst (34.66 μmol/min/g dw of cells for immobilized catalyst vs 40.54 μmol/min/g for free cells in the asymmetric reduction of 4'-chloroacetophenone). The immobilized cells showed better thermal stability, pH stability, solvent tolerance and storability compared with free cells. After 25 cycles reaction, the immobilized beads still retained >50% catalytic activity, which was 3.5 times higher than degree of retention of activity by free cells reused in a similar way. The cells could be recultured in the beads to regain full activity and perform a further 25 cycles of the reduction reaction. The external mass transfer resistances were negligible as deduced from Damkohler modulus Da < <1, and internal mass transfer restriction affected the reduction action but was not the principal rate-controlling step according to effectiveness factors η < 1 and Thiele modulus 0.3<∅ <1., Conclusions: Ca-alginate coated with chitosan is a highly effective material for immobilization of Acetobacter sp. CCTCC M209061 cells for repeated use in the asymmetric reduction of ketones. Only a small cost in terms of the slightly lower catalytic activity compared to free cells could give highly practicable immobilized biocatalyst.

This study demonstrated electricity generation in a thermophilic microbial fuel cell (MFC) operated on synthesis gas (syngas) as the sole electron donor. At 50°C, a volumetric power output of 30-35 mWL(R)(-1) and a syngas conversion efficiency of 87-98% was achieved. The observed pathway of syngas conversion to electricity primarily consisted of a two-step process, where the carbon monoxide and hydrogen were first converted to acetate, which was then consumed by the anodophilic bacteria to produce electricity. A denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rDNA revealed the presence of Geobacter species, Acetobacter, methanogens and several uncultured bacteria and archaea in the anodic chamber., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.

Acetobacter species are members of the α-subclass of Proteobacteria, which harbors a large number of bacteria recalcitrant to cultivation. Strain AB0220 was isolated from a superficial acetification system and preserved for 9 years by short and long time methods. Under short time preservation it was estimated that 540.54 number of generations occurred, whereas in long time preservation conditions the number of generations was 17.40. Ethanol oxidation to acetic acid was stable and confirmed, as well as acetate assimilation during long time preservation. Cultivability checks showed persistence of phenotypic traits (growth on ethanol and methanol, growth on different carbon sources and cellulose production) over the extended preservation time. 16S rRNA gene sequences analysis showed 100 % of similarity with A. pasteurianus (Accession number GQ240636). Stability of subcultures related to the culture age and subcultures frequency, tested by ERIC/PCR, confirmed the suitability of long term preservation at least over a period of 9 years.

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic microorganisms that show a unique resistance to ethanol (EtOH) and acetic acid (AcH). Members of the Acetobacter and Gluconacetobacter genera are capable of transforming EtOH into AcH via the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes and are used for the industrial production of vinegar. Several mechanisms have been proposed to explain how AAB resist high concentrations of AcH, such as the assimilation of acetate through the tricarboxylic acid (TCA) cycle, the export of acetate by various transporters and modifications of the outer membrane. However, except for a few acetate-specific proteins, little is known about the global proteome responses to AcH. In this study, we used 2D-DIGE to compare the proteome of Acetobacter pasteurianus LMG 1262(T) when growing in glucose or ethanol and in the presence of acetic acid. Interesting protein spots were selected using the ANOVA p-value of 0.05 as threshold and 1.5-fold as the minimal level of differential expression, and a total of 53 proteins were successfully identified. Additionally, the size of AAB was reduced by approximately 30% in length as a consequence of the acidity. A modification in the membrane polysaccharides was also revealed by PATAg specific staining., (Copyright © 2011 Elsevier B.V. All rights reserved.)