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A 16S rRNA gene-based fingerprinting method was developed for the preliminary characterization of native diazotrophs bacterial isolates Azotobacter, Acetobacter and Azospirillum tested with reference strains belonging to the free-living nitrogen-fixing bacterial group. According to this method, the 16S rRNA gene was amplified using universal primers and PCR products were subsequently digested with TaqI, BamHI, HinfI and HaeIII. The analysis of the restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the native diazotrophic bacterial isolates. Phylogenetic analysis of 16S rRNA indicated that these diazotrophic bacterial isolates belonged to respective genus Azotobacter, Acetobacter and Azospirillum and closely related to standard strains. [ABSTRACT FROM AUTHOR]

Objective: The objective of the study was to evaluate the effectiveness of chitosan and chitosan-ethylenediamine tetraacetic acid (EDTA) (3:1,1:1,1:3) in comparison with 5.2% sodium hypochlorite (NaOCl) in disinfecting Enterococcus faecalis biofilm on root canal dentin and in the removal of smear layer with minimal erosion. Materials and Methods: Seventy single-rooted extracted human mandibular premolars (n = 70) were selected for the study. Forty tooth samples were biomechanically prepared, vertically sectioned, and sterilized by autoclaving. The tooth sections were artificially infected with E. faecalis (ATCC 29212 [n = 35] and clinical isolate [SBEF2, n = 35]) to form mature dentinal biofilm in vitro. The tooth samples were treated with the test solutions: chitosan and chitosan-EDTA (3:1, 1:1, 1:3), and the killing time was determined. The smear layer removal ability of the test solutions (Group A: chitosan-EDTA [1:1], Group B: EDTA, Group C: control) (n = 10 tooth/group) was assessed. Results: Chitosan and chitosan-EDTA (3:1, 1:1, 1:3) exhibited antibacterial activity against both the strains of E. faecalis. Chitosan and chitosan-EDTA caused 3 log reduction in the viable count of the sessile cells of E. faecalis at 15 min while 5.2% NaOCl exhibited 99.98% inhibition at 15 min. Chitosan-EDTA (1:1) was found to be effective in removing the smear layer and showed lesser erosion than EDTA at the coronal and middle portions. Conclusion: Chitosan-EDTA (1:1) is a potential root canal irrigant that performs a dual role – root canal disinfection and smear layer removal. [ABSTRACT FROM AUTHOR]

3-hydroxypropionic acid (3-HP) is an important chemical platform organic in material industry, daily chemical industry and biomedicine field due to its numerous valuable derivatives. However, no mature methods have been established in the synthesis industry for direct large scale production. The bacterium Acetobacter sp. CGMCC 8142 with high efficiency of alcohols oxidation property was immobilized for biosynthesis of 3-HP from 1, 3-propanediol (1, 3-PDO). Parameter values in mass transfer modeling indicated that mass transfer of immobilized biocatalysts affected the oxidation reaction (the internal effectiveness factor η < 1) but was not the rate-limiting step if Thiele modulus 1 > φ > 0.3. The optimal immobilization conditions for 3-HP biocatalysis was sodium alginate 40 g/L, gel beads diameter 1 mm, cross-linkage time 2 h and 0.1mM FeCl. Immobilized cells showed promising substrate tolerance, pH stability, thermal stability and storability. After 5 cycles of reaction, 3-HP molar yield of immobilized beads was retained to 80.26%, and 66.95 g/L 3-HP were produced from 70 g/L 1, 3-PDO. The biocatalysis process of immobilized cells introduced in this study may provide an economical and efficiency alternative route for practical production of 3-HP. [ABSTRACT FROM AUTHOR]

Cell immobilization comprises the retention of metabolically active cells inside a polymeric matrix. In this study, the production of jabuticaba (Myrciaria jaboticaba) vinegar using immobilized Acetobacter aceti and Gluconobacter oxydans cells is proposed as a new method to prevent losses of jabuticaba fruit surplus. The pulp of jabuticaba was processed and Saccharomyces cerevisiae CCMA 0200 was used to ferment the must for jabuticaba wine production. Sugars, alcohols (ethanol and glycerol) and organic acids were assayed by high-performance liquid chromatography. Volatile compounds were determined by gas chromatography-fl ame ionization detector. The ethanol content of the produced jabuticaba wine was approx. 74.8 g/L (9.5% by volume) aft er 168 h of fermentation. Acetic acid fermentation for vinegar production was performed using a mixed culture of immobilized A. aceti CCT 0190 and G. oxydans CCMA 0350 cells. The acetic acid yield was 74.4% and productivity was 0.29 g/(L·h). The vinegar had particularly high concentrations of citric (6.67 g/L), malic (7.02 g/L) and succinic (5.60 g/L) acids. These organic acids give a suitable taste and fl avour to the vinegar. Seventeen compounds (aldehydes, higher alcohols, terpene, acetate, diether, furans, acids, ketones and ethyl esters) were identified in the jabuticaba vinegar. In conclusion, vinegar was successfully produced from jabuticaba fruits using yeast and immobilized mixed cultures of A. aceti and G. oxydans. To the best of our knowledge, this is the fi rst study to use mixed culture of immobilized cells for the production of jabuticaba vinegar. [ABSTRACT FROM AUTHOR]

Some species of acetic acid bacteria (AAB) play relevant roles in the metabolism and physiology of Drosophila spp. and in some cases convey benefits to their hosts. The pest Drosophila suzukii harbors a set of AAB similar to those of other Drosophila species. Here, we investigate the potential to exploit the ability of AAB to produce volatile substances that attract female D. suzukii. Using a two-way olfactometer bioassay, we investigate the preference of D. suzukii for strains of AAB, and using solid-phase microextraction gas chromatography-mass spectrometry we specifically characterize their volatile profiles to identify attractive and non-attractive components produced by strains from the genera Acetobacter, Gluconobacter, and Komagataeibacter. Flies had a preference for one strain of Komagataeibacter and two strains of Gluconobacter. Analyses of the volatile profiles from the preferred Gluconobacter isolates found that acetic acid is distinctively emitted even after 2 days of bacterial growth, confirming the relevance of this volatile in the profile of this isolate for attracting flies. Analyses of the volatile profile from the preferred Komagataeibacter isolate showed that a different volatile in its profile could be responsible for attracting D. suzukii. Moreover, variation in the concentration of butyric acid derivatives found in some strains may influence the preference of D. suzukii. Our results indicate that Gluconobacter and Komagataeibacter strains isolated from D. suzukii have the potential to provide substances that could be exploited to develop sustainable mass-trapping-based control approaches. [ABSTRACT FROM AUTHOR]

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro-organisms, namely yeasts, lactic acid bacteria ( LAB) and acetic acid bacteria ( AAB), which results in well-fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp-bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa-derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully. [ABSTRACT FROM AUTHOR]

The aim of this study was to understand the correlation between ethanol resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Two ethanol-tolerant strains, FY-24 and DY-5, identified as Acetobacter pasteurianus were isolated from Chinese vinegar Pei. FY-24 and DY-5 maintained the growth and fermentation ability with 12 % (v/v) ethanol at 37 °C, but A. pasteurianus AS1.41 (widely used in China) could only be resistant to 10 % ethanol. ADH activities of FY-24 (6.13 U/mg) and DY-5 (5.41 U/mg) were much higher than that of AS1.41 (1.57 U/mg) at 8 % ethanol. Using purified ADH, it was shown that the ADHs from FY-24 and DY-5 exhibited a higher ethanol tolerance as ethanol increasing from 10 to 40 %. The optimum ethanol concentration for ADHs of FY-24 and DY-5 was 10 %, but was 5 % for that of AS1.41. Additionally, ADHs from FY-24 and DY-5 exhibited a higher resistance to temperature and acetic acid than that of AS1.41. From the amino acid sequence of ADH subunits, a certain amount of residues were found in the ADHs of FY-24 and DY-5 (411th D and 731th N in subunit I, 170th D in subunit II and 40th G in subunit III), which were different from that of AS1.41. The difference in amino acid residues might partly relate to the ethanol tolerance. Our results suggested that FY-24 and DY-5 could be developed for high ethanol acetic acid fermentation. [ABSTRACT FROM AUTHOR]

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine ( Pinus flexilis) and potential N2-fixing acetic acid bacteria ( AAB) indicates that native foliar endophytes may supply subalpine forests with N., To assess whether the P. flexilis- AAB association is consistent across years, we re-sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N2, we incubated twigs with 13N2-enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using 13N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September., We found evidence for N2 fixation in P. flexilis foliage. N2 diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6 μg N m−2 d−1 to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community., We propose that foliar endophytes represent a low-cost, evolutionarily stable N2-fixing strategy for long-lived conifers. This novel source of biological N2 fixation has fundamental implications for understanding forest N budgets. [ABSTRACT FROM AUTHOR]

Classical studies concerning ' Acetobacter xylinum' focus on bacterial cellulose 'BC' yield and rate in broth, after a long period of incubation (7-14 days). Such observations do not highlight bacterial physiology in the first incubation hours and its impact on BC production. In this study, the growth of a wild strain of Acetobacter was monitored in the first incubation hours. We showed the presence of two different physiologies; the first extends from the incubation moment till the formation of a sparse BC. Sparse BC modifies surface viscosity, and stabilizes hydrodynamic conditions to initiate compact BC production that marks the second physiology. Two containers, of different shapes, were used to confirm our findings, one of which is a culture tube with high drift currents on the broth-air interface, and the other is a conical flask with more stable hydrodynamic conditions at the culture's surface. We showed that Acetobacter always follows two physiologies independent of the container shape. Logistic model, FTIR, XRD and SEM analysis are used to confirm the results. [ABSTRACT FROM AUTHOR]

The ion-translocating c ring of the Na+ F1Fo ATP synthase of the anaerobic bacterium Acetobacterium woodii is the first heteromeric c ring found in nature that contains one V- ( c1) and two F-type-like c subunits ( c2/ c3), the latter of identical amino acid sequence. To address whether they are of equal or different importance for function, they were deleted in combination or individually. Deletion of c1 was compensated by incorporation of two c2/ c3 subunits but the enzyme was unstable and largely impaired in Na+ transport. Deletion of c2 was compensated by incorporation of c3 but also led to a reduction of Na+ transport. Deletion of c3 had no effect. In contrast, deletion of both c2 and c3 led to a complete loss of ATPase activity at the cytoplasmic membrane. Mass spectrometric analysis of c2+1 Ala and c2+2 Ala variants revealed a copy number of 8 : 1 for c2/ c3 which is consistent with the biochemical characteristics of the variants. These data indicate a role of c1 in assembly and a function of c2 as the predominant c ring constituent. [ABSTRACT FROM AUTHOR]

Cross-linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross-linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7-aminocephalosporanic acid. The CLEAs had a Kcat/KM of 0.9 M−1/S−1 for 7-ACA (7-aminocephalosporanic acid) and 0.1 M−1/S−1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis-Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36-42, 2016 [ABSTRACT FROM AUTHOR]

Organic apple cider vinegar is produced from apples that go through very restricted treatment in orchard. During the first stage of the process, the sugars from apples are fermented by yeasts to cider. The produced ethanol is used as a substrate by acetic acid bacteria in a second separated bioprocess. In both, the organic and conventional apple cider vinegars the ethanol oxidation to acetic acid is initiated by native microbiota that survived alcohol fermentation. We compared the cultivable acetic acid bacterial microbiota in the production of organic and conventional apple cider vinegars from a smoothly running oxidation cycle of a submerged industrial process. In this way we isolated and characterized 96 bacteria from organic and 72 bacteria from conventional apple cider vinegar. Using the restriction analysis of the PCR-amplified 16S-23S rRNA gene ITS regions, we identified four different HaeIII and five different HpaII restriction profiles for bacterial isolates from organic apple cider vinegar. Each type of restriction profile was further analyzed by sequence analysis of the 16S-23S rRNA gene ITS regions, resulting in identification of the following species: Acetobacter pasteurianus (71.90 %), Acetobacter ghanensis (12.50 %), Komagataeibacter oboediens (9.35 %) and Komagataeibacter saccharivorans (6.25 %). Using the same analytical approach in conventional apple cider vinegar, we identified only two different HaeIII and two different HpaII restriction profiles of the 16S-23S rRNA gene ITS regions, which belong to the species Acetobacter pasteurianus (66.70 %) and Komagataeibacter oboediens (33.30 %). Yeasts that are able to resist 30 g/L of acetic acid were isolated from the acetic acid production phase and further identified by sequence analysis of the ITS1-5.8S rDNA-ITS2 region as Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. This study has shown for the first time that the bacterial microbiota for the industrial production of organic apple cider vinegar is clearly more heterogeneous than the bacterial microbiota for the industrial production of conventional apple cider vinegar. Further chemical analysis should reveal if a difference in microbiota composition influences the quality of different types of apple cider vinegar. [ABSTRACT FROM AUTHOR]

The involvement of microorganisms in corrosion refers to microbiologically influenced corrosion (MIC). We have previously succeeded in a lab-scale corrosion test using bottom water from oil-storage tanks and shown the change in the microbial community after corrosion using denaturing gradient gel electrophoresis analysis. However, those community structures have not yet been correlated with accelerated corrosion. Therefore, we conducted further analysis of the microbial community by pyrosequencing using a next-generation sequencer. In the corrosion sample, an acetic acid bacterium Acetobacterium sp. and a sulfate-reducing bacterium Desulfovibrio sp. were enriched. In contrast, only Desulfovibrio sp. bacterium was enriched in the noncorroded culture, and Acetobacterium sp. bacterium was not observed. These results indicate that the Acetobacterium sp. bacterium may be involved in the accelerated corrosion. This is first report of corrosion by Acetobacterium sp. The combination of a lab-scale corrosion test and microbial community analysis by pyrosequencing would be useful to correlate between corrosion behavior and a change in microbial community. In the future, such investigation would contribute to understanding of MIC. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria (AAB) have been used in various fermentation processes. Of several ABB, the bacterial nanocellulose (BNC) producers, notably Komagataeibacter xylinus, appears as an interesting species, in large part because of their ability in the secretion of cellulose as nano/microfibrils. In fact, BNC is characterized by a native nanofibrillar structure, which may outperform the currently used celluloses in the food industry as a promising novel hydrocolloid additive. During the last couple of years, a number of companies worldwide have introduced some BNC-based products to the market. The main aim of this editorial is to underline the BNC potentials. [ABSTRACT FROM AUTHOR]

The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well. [ABSTRACT FROM AUTHOR]

Microbially influenced corrosion (MIC) is a costly problem that impacts hydrocarbon production and processing equipment, water distribution systems, ships, railcars, and other types of metallic infrastructure. In particular, MIC is known to cause considerable damage to hydrocarbon fuel infrastructure including production, transportation, and storage systems, often times with catastrophic environmental contamination results. As the production and use of alternative fuels such as fuel-grade ethanol (FGE) increase, it is important to consider MIC of engineered materials exposed to these 'newer fuels' as they enter existing infrastructure. Reports of suspected MIC in systems handling FGE and water prompted an investigation of the microbial diversity associated with these environments. Small subunit ribosomal RNA gene pyrosequencing surveys indicate that acetic-acid-producing bacteria ( Acetobacter spp. and Gluconacetobacter spp.) are prevalent in environments exposed to FGE and water. Other microbes previously implicated in corrosion, such as sulfate-reducing bacteria and methanogens, were also identified. In addition, acetic-acid-producing microbes and sulfate-reducing microbes were cultivated from sampled environments containing FGE and water. Results indicate that complex microbial communities form in these FGE environments and could cause significant MIC-related damage that may be difficult to control. How to better manage these microbial communities will be a defining aspect of improving mitigation of global infrastructure corrosion. [ABSTRACT FROM AUTHOR]

Acetic acid bacterial strains were investigated for their ability to grow in aerobic and anaerobic sourdoughs (dough yield = 500) produced from wheat, whole wheat, spelt and rye flour and to produce effectual levels of exopolysaccharides (EPS) within 48 h of fermentation. The strains Kozakia ( K.) baliensis DSM 14400 and Neoasaia ( N.) chiangmaiensis NBRC 101099 were chosen. Both strains remained in aerobic doughs produced with any of the tested flours. For EPS optimization, 50, 75 and 100 g/l sucrose (sucrose) were added to the doughs and the parameters EPS, pH value, sucrose, acetic acid and gluconic acid were analysed. Results were subjected to statistical data analysis. In sucrose-enriched spelt dough, K. baliensis reached up to 49 g/kg EPS, followed by whole wheat (33 g/kg), wheat and rye dough (32-36 g/kg). N. chiangmaiensis produced 24-29 g/kg EPS in spelt and whole wheat doughs. EPS formation was positively correlated with start sucrose dosage. In contrast to this, a rather negative relation between acetic acid formation and start sucrose was found for both strains, and a positive relation with fermentation time. In spelt doughs, K. baliensis and N. chiangmaiensis produced up to 100 and 21-38 mM/kg acetic acid, respectively. Formation of gluconic acid was positively correlated with start sucrose dosage. K. baliensis produced at maximum 165 mM/kg flour and N. chiangmaiensis at maximum 119 mM/kg flour. Remaining sucrose contents were always positively correlated with start sucrose dosage, whereby both tested strains reached remaining sucrose amounts below 50 g/kg. A subsequent experiment with K. baliensis in backslopped spelt dough showed that this strain can remain in the dough over at least 8 days. [ABSTRACT FROM AUTHOR]

The anaerobic, acetogenic bacterium Acetobacterium woodii grows on hydrogen and carbon dioxide and uses the Wood-Ljungdahl pathway to fix carbon but also to synthesize ATP. The free energy change of acetogenesis from H + CO allows for synthesis of only a fraction of an ATP under environmental conditions, and A. woodii is clearly a paradigm for microbial life under extreme energy limitation. However, it was unknown how much energy is required to make ATP under these conditions. In the present study, we determined the phosphorylation potential in cells metabolizing three different acetogenic substrates. It accounts to 37.9 ± 1.3 kJ/mol ATP during acetogenesis from fructose, 32.1 ± 0.3 kJ/mol ATP during acetogenesis from H + CO and 30.2 ± 0.9 kJ/mol ATP during acetogenesis from CO, the lowest phosphorylation potential ever described. The physiological consequences in terms of energy conservation under extreme energy limitation are discussed. [ABSTRACT FROM AUTHOR]

The asymmetric reduction of ethyl acetoacetate (EAA) to ethyl ( R)-3-hydroxybutyrate [( R)-EHB] using immobilized Acetobacter sp. CCTCC M209061 cells was successfully conducted in a hydrophilic ionic liquid (IL)-containing system. The best one of all the tested watermiscible ILs was 1-(hydroxyethyl)-3-methylimidazolium hydrochloride (COHMIM·Cl). In COHMIM·Cl-aqueous buffer hybrid system, it was found that the optimal IL concentration, substrate and co-substrate concentration, reaction temperature, buffer pH and shaking rate were 0.5mol/L, 45 mmol/L, 80 mmol/L, 35°C, pH 5.5 and 200 rpm, respectively. Under the optimized reaction conditions, the initial reaction rate, the yield and the product e.e. reached 4.90 µmol/min, 95.3 and > 99.0%, respectively, which were much higher than the corresponding values reported previously. The efficient biocatalytic process mediated by the immobilized cells was feasible on 500 mL preparative scale, and the biocatalysts showed good operational stability and could be recycled for at least 10 batches. [ABSTRACT FROM AUTHOR]

Among probiotics, acetic acid bacteria (AAB) can be used as preservative agents because of their high fermentation and acidification activities. This study aimed to isolate, identify and biologically characterise Acetobacter strains from traditional Iranian dairy products. Acetobacter strains were identified by catalase assay, Gram staining, and combined repetitive sequence-based PCR [(GTG)5-PCR fingerprints] and 16S rDNA gene sequencing. We identified eight strains belonging to four species, including Acetobacter aceti, Acetobacter indonesiensis, Acetobacter cibinongensis and Acetobacter syzygii. The molecular techniques could be used as an effective and rapid alternative tool to identify and characterise dairy-associated AAB. Primary probiotic assessments, including low pH and high bile salt tolerance tests, antagonistic activity test against pathogens, and antibiotic susceptibility confirmed the probiotic properties of these AAB, particularly A. cibinongensis 34L strain, which was isolated from curd. Therefore, this strain can be introduced as novel candidate probiotics that could be used in the food industry. [ABSTRACT FROM AUTHOR]

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose. [ABSTRACT FROM AUTHOR]

The diversity of yeasts and bacterial species on grapes has been investigated in vineyards worldwide. For winemaking are very important three groups of microorganisms. First two includes acetic acid and lactic acid bacteria; they live and grow normally on grape surface. The third group includes more than 20 detectable genera of yeasts. There are three principal genera found on grapes Hanseniaspora uvarum (Kloeckera apiculata), Metschnikowia pulcherrima (Candida pulcherrima), and Candida stellata or new descripted Candida zemplinina. Aim of this study was investigate of number of three major groups of microorganisms which are important for grapes and winemaking. The number of bacteria on Acetobacter agar (AA) ranged from 1.76 log CFU/mL to 2.80 log CFU/mL. Lactic acid bacteria were counted on MRS agar and the number of detectable colonies ranged from 0.48 log CFU/mL to 2.06 log CFU/mL. Sabouraud dextrose agar (SDA) was used for cultivation of yeast and the number of yeasts ranged from 2.47 log CFU/mL to 2.76 log CFU/mL. For identification of yeast species were used different types of agar media with acid base indicator bromocresol green. Identified 10 yeasts species includes to genus: Candida, Metschnikowia, Pichia, Kluyveromyces, Hanseniaspora, Hansenula, Candida, Debaromyces, Rhodotorula and Saccharomyces. We identified only few bacterial species includes to genus Lactobacillus, Pediococcus, Gluconobacter and Acetobacter. [ABSTRACT FROM AUTHOR]

The acsD gene is involved in cellulose biosynthesis among the Acetobacter species. In the current study, we created an acsD disruption mutant in the acsABCD cellulose synthase operon of Gluconacetobacter xylinus and characterized the resulting cellulose to aid in providing insight into the function of the acsD gene. Both the wild type G. xylinus AY201 (derivative of Gluconacetobacter hansenii ATCC 23769) and the acsD disruption mutant produced crystalline cellulose I microfibrils. The cellulose produced by both appeared to be synthesized from an aggregate of pores known as a linear terminal complex; however the total cellulose synthesized was 10 % that of the wild type G. xylinus AY201. TEM observations of the acsD disruption mutant confirmed that microfibrils and bundles of microfibrils were similar in size to the G. xylinus AY201 wild type; however, the final ribbon dimensions were narrower (53.4 ± 13.1 nm wt, vs. 28.2 ± 8.2 nm). Additional TEM observations of the mutant cells incubated at 4 °C revealed an abnormal linear terminal complex orientation whereby the resulting band material could be observed in a transverse orientation as well as longitudinally to the long axis of the cell. Taken together, these data strongly suggest that acsD aids in the proper orientation of the linear terminal complexes along the longitudinal axis of the cell indicating the AcsD protein is involved in the final level of the hierarchical assembly of cellulose resulting in highly efficient cellulose synthesis. [ABSTRACT FROM AUTHOR]

Bacteria of the species Gluconobacter oxydans are applied in the industrial production of dihydroxyacetone (DHA) via glycerol oxidation. The major problem of this biotransformation involves process inhibition by substrate and/or product. Improper initial concentration of glycerol and increasing DHA concentration may inhibit the metabolic activity of bacterial cells and impede further course of the reaction. An attempt was, therefore, undertaken in this study to determine which concentrations of glycerol (30, 50, 70, 100 g L) and DHA (10-100 g L) may inhibit the growth of acetic acid bacteria of G. oxydans ATCC 621 species. Cultures of this strain were run in the Bioscreen C MBR apparatus on experimental culture media with various initial concentrations of glycerol and DHA. Analyses were also carried out to examine the impact of pH (5.0, 7.0, 8.0) of glycerol-containing culture media on cell growth of the analyzed strain G. oxydans. None of the applied substrate concentrations was inhibiting cellular divisions of G. oxydans bacteria. The initial glycerol concentrations that enabled rapid cellular divisions reached 50 g L in the medium with pH 5.0 (coefficient of specific growth rate μ = 0.0550) and 70 g L in the medium with pH 7.0 ( μ = 0.0556). DHA was shown to inhibit the mitotic activity of G. oxydans bacteria even at low concentrations (20-30 g L), whereas at the concentration of 70 g L, it made cell divisions impossible. The applied pH values of the culture media did not inhibit the growth of G. oxydans strain. [ABSTRACT FROM AUTHOR]

Camponotus is the second largest ant genus and known to harbor the primary endosymbiotic bacteria of the genus Blochmannia. However, little is known about the effect of diet and environment changes on the gut bacterial communities of these ants. We investigated the intestinal bacterial communities in the lab-raised and field-collected ants of Camponotus fragilis which is found in the southwestern United States and northern reaches of Mexico. We determined the difference of gut bacterial composition and distribution among the crop, midgut, and hindgut of the two types of colonies. Number of bacterial species varied with the methods of detection and the source of the ants. Lab-raised ants yielded 12 and 11 species using classical microbial culture methods and small-subunit rRNA genes (16S rRNAs) polymerase chain reaction-restriction fragment-length polymorphism analysis, respectively. Field-collected ants yielded just 4 and 1-3 species using the same methods. Most gut bacterial species from the lab-raised ants were unevenly distributed among the crop, midgut, and hindgut, and each section had its own dominant bacterial species. Acetobacter was the prominent bacteria group in crop, accounting for about 55 % of the crop clone library. Blochmannia was the dominant species in midgut, nearly reaching 90 % of the midgut clone library. Pseudomonas aeruginosa dominated the hindgut, accounting for over 98 % of the hindgut clone library. P. aeruginosa was the only species common to all three sections. A comparison between lab-raised and field-collected ants, and comparison with other species, shows that gut bacterial communities vary with local environment and diet. The bacterial species identified here were most likely commensals with little effect on their hosts or mild pathogens deleterious to colony health. [ABSTRACT FROM AUTHOR]

Nanocomposites of poly(vinyl alcohol) (PVA) reinforced with bacterial cellulose (BC) were bioproduced by Gluconacetobacter genus bacteria. BC was grown from a culture medium modified with water-soluble PVA to allow in situ assembly and production of a novel nanocomposite that displayed synergistic property contributions from the individual components. Chemical crosslinking with glyoxal was performed to avoid the loss of PVA matrix during purification steps and to improve the functional properties of composite films. Reinforcement with BC at 0.6, 6 and 14 wt% content yielded nanocomposites with excellent mechanical, thermal and dimensional properties as well as moisture stability. Young's modulus and strength at break increased markedly with the reinforcing BC: relative to the control sample (in absence of BC), increases of 15, 165 and 680 % were determined for nanocomposites with 0.6, 6 and 14 % BC loading, respectively. The corresponding increase in tensile strengths at yield were 1, 12 and 40 %, respectively. The results indicate an exceptional reinforcing effect by the three-dimensional network structure formed by the BC upon biosynthesis embedded in the PVA matrix and also suggest a large percolation within the matrix. Bonding (mainly hydrogen bonding) and chemical crosslinking between the reinforcing phase and matrix were the main contributions to the properties of the nanocomposite. [ABSTRACT FROM AUTHOR]

Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S. cerevisiae and Lactobacillus bacteria in TKGs. [ABSTRACT FROM AUTHOR]

A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4′-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4′-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity. [ABSTRACT FROM AUTHOR]

Seeking high strength vinegar fermentation by acetic acid bacteria (AAB) is still the mission of vinegar producers. AAB alcohol respiratory chain, located on intracellular membrane, is directly responsible for vinegar fermentation. In the semi-continuous vinegar fermentation by Acetobacter pasteurianus CICIM B7003, acetification rate showed positive correlation with the activity of the enzymes in alcohol respiratory chain. Aiming at achieving high strength fermentation process, a series of trials were designed to raise the activity of AAB alcohol respiratory chain. Finally, acetification was enhanced by adding some precursors (ferrous ions and β-hydroxybenzoic acid) of alcohol respiration associated factors and increasing aeration rate (0.14 vvm). As final result, average acetification rate has been raised to 2.29 ± 0.02 g/L/h, which was 28.7% higher than the original level. Simultaneously, it was found that the oxidization of alcohol into acetic acid in AAB cells was improved by well balancing of three factors: enzyme activity in alcohol respiratory chain, precursor of ubiquinone biosynthesis, and aeration rate. [ABSTRACT FROM AUTHOR]

A simple and novel method to modify the surface of glass material withAcetobacter xylinumbacteria to deposit bacterial cellulose (BC) around glass material during the process of fermentation was described. The modified glass material with more hydrophobic and rougher surface was characterized by FTIR, SEM, XPS, peeling experiments, and water/air contact angle. It was found that heat treatment at 140 ∼ 150°C was able to improve the interaction between BC and glass material owing to the increase in chemical bonds between them. The biological modified glass fibers were compounded with epoxy resin. The influence of incubation time and high temperature on the interfacial shear strength (IFSS) between glass fibers and epoxy resin was identified by Microbond Test. The strongest IFSS could be obtained with incubation time of 1 hour and temperature of 140°C, which then offers a biological approach to improve the interface of silicates materials and resin matrix. [ABSTRACT FROM PUBLISHER]

In this study, typical niches of acetic acid bacteria were screened for isolation of cellulose producer strains. Hestrin Schramm broth was used as enrichment and production media. Only nine out of 329 isolates formed thick biofilms on liquid surface and were identified as potential cellulose producers. Physiological and biochemical tests proved that all cellulose producers belonged to Gluconacetobacter genus. Most productive and mutation-resistant strain was subjected to 16S rRNA sequence analysis and identified as Gluconacetobacter hansenii P2A due to 99.8 % sequence similarity. X-ray diffraction analysis proved that the biofilm conformed to Cellulose I crystal structure, rich in I mass fraction. Static cultivation of G. hansenii P2A in HS medium resulted with 1.89 ± 0.08 g/l of bacterial cellulose production corresponding to 12.0 ± 0.3 % yield in terms of substrate consumption. Shaking and agitation at 120 rpm aided in enhancement of the amount and yield of produced cellulose. Productivity and yield reached up to 3.25 ± 0.11 g/l and 17.20 ± 0.14 % in agitated culture while a slight decrease from 78.7 % to 77.3 % was observed in the crystallinity index. [ABSTRACT FROM AUTHOR]

The production of vinegar depends on an oxidation process that is mainly performed by acetic acid bacteria. Despite the different methods of vinegar production (more or less designated as either "fast" or "traditional"), the use of pure starter cultures remains far frombeing a reality. Uncontrolled mixed cultures are normally used, but this review proposes the use of controlled mixed cultures. The acetic acid bacteria species determine the quality of vinegar, although the final quality is a combined result of technological process, wood contact, and aging.This discussion centers on wine vinegar and evaluates the effects of these different processes on its chemical and sensory properties. [ABSTRACT FROM AUTHOR]

Members of the Acetobacter pasteurianus are popular acetic acid bacteria (AAB) for the production of vinegar. Neither phenotypic nor the most frequently applied genotypic marker (16S ribosomal DNA) provides sufficient resolution for accurate identification of the AAB strains. In this study, the gyrB gene was used for species discrimination by direct DNA sequencing and as marker in a species-specific PCR assay. All examined A. pasteurianus strains were clearly distinguished from the closely related species by comparative sequence analysis of the gyrB gene. The average sequence similarity for the gyrB gene (82.2 %) among type strains was significantly lower than that of the 16S rRNA sequence (98.2 %). Therefore, the gyrB gene can be proposed as an additional molecular marker for A. pasteurianus and related taxa that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the gyrB and 16S rRNA gene sequences, which were then employed for PCR using the template DNA of Acetobacter strains. The PCR primer pairs were shown to be specific for A. pasteurianus, A. peroxydans and papayae. Our data indicate that the phylogenetic relationships in the A. pasteurianus group are easily resolved by direct sequencing of the gyrB gene and combined with species-specific PCR assays. [ABSTRACT FROM AUTHOR]

Why humans evolved a taste for sour foods Of those animals, pigs and primates seem to really like acidic foods. For example, wild pigs (Sus scrofa) are really attracted to fermented corn, and gorillas (Gorilla gorilla) have shown a preference for acidic fruits in the ginger family. [Extracted from the article]

Xanthine oxidase (XO) acts at the end of a catabolic sequence of purine nucleotide metabolism in humans and a few other uricotelic species. The EtOAc layer showed strong xanthine oxidase inhibitory activity (~99% XO inhibition). Fraction 9 (18% XO inhibition) was purified by prep. TLC (CH SB 2 sb Cl SB 2 sb -MeOH, 12:1) to give I p i -hydroxybenzoic acid ( B 3 b ) (3.6 mg) and gallic acid 4-methyl ether ( B 4 b ) (2.5 mg). [Extracted from the article]

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics. [ABSTRACT FROM AUTHOR]

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5–6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions. [ABSTRACT FROM AUTHOR]

Vinegar production requires acetic acid bacteria that produce, tolerate, and conserve high levels of acetic acid. When ethanol is depleted, aerobic acetate overoxidation to carbon dioxide ensues. The resulting diauxic growth pattern has two logarithmic growth phases, the first associated with ethanol oxidation and the second associated with acetate overoxidation. The vinegar factory isolate Acetobacter aceti strain 1023 has a long intermediate stationary phase that persists at elevated acetic acid levels. Strain 1023 conserves acetic acid despite possessing a complete set of citric acid cycle (CAC) enzymes, including succinyl-CoA:acetate CoA-transferase (SCACT), the product of the acetic acid resistance (aar) gene aarC. In this study, cell growth and acid production were correlated with the functional expression of aar genes using reverse transcription-polymerase chain reaction, Western blotting, and enzyme activity assays. Citrate synthase (AarA) and SCACT (AarC) were abundant in A. aceti strain 1023 during both log phases, suggesting the transition to acetate overoxidation was not a simple consequence of CAC enzyme induction. A mutagenized derivative of strain 1023 lacking functional AarC readily oxidized ethanol but was unable to overoxidize acetate, indicating that the CAC is required for acetate overoxidation but not ethanol oxidation. The primary role of the aar genes in the metabolically streamlined industrial strain A. aceti 1023 appears to be to harvest energy via acetate overoxidation in otherwise depleted medium. [ABSTRACT FROM AUTHOR]

Downstream processes have great influences on bacterial starter production. Different modifications occur to cellular compounds during freeze-drying process and storage of bacterial starters. Consequently, viability and culturability (multiplication capacity) undergo some changes. In this study, the effects of freeze-drying process and storage conditions were examined on cell envelope integrity, respiration and culturability of Acetobacter senegalensis. Freezing of cells protected with mannitol (20% w/w) did not affect cell multiplication and respiration considerably; however, 19% of cells showed compromised cell envelope after freezing. After drying, 1.96×1011 CFU/g were enumerated, indicating that about 34% of the cells could survive and keep their culturability. Drying of the cells induced further leakage in cell envelope and finally 81% of cells appeared as injured ones; however, 87% of the dried cells maintained their respiration capacity. Storage temperature had significant effect on cell multiplication ability; higher storage temperature (35°C) caused 8.59-log reduction in cell culturability after nine-month period of storage. Collapse of cell envelop integrity and respiration was observed at 35°C. At lower storage temperature (4°C), the culturability decreased about one-log reduction after nine months. Cell envelope integrity was subjected to minor changes during a period of nine month-storage at 4°C whereas a heterogeneous population of cells with different respiration capacity emerged at 4°C. These results indicate that a major part of cells undergone drying process and storage entered into viable but non-culturable state. In addition, usage of different culture media didn't improve resuscitation. Besides, it seems that sub-lethal damages to cell envelope caused uptake of propidium iodide, however these kinds of injuries could not impress cell multiplications and respiration. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria (AAB) are obligate aerobic microorganisms which have large significance in human life. Traditionally, AAB species have been used to produce fermented food and beverages thanks to their ability to oxidize ethanol to acetic acid. Moreover, in the last decades, they have been extensively investigated for other industrial biotechnology applications as the development of processes for highvalue products or biosensors. The potential exploitation of AAB diversity requires the existence of microbial culture collections, which are able to supply not only strains but essential data for fundamental microbial research. Therefore, microbial collections can be helpful to provide critical insights into AAB physiology and metabolism as well as integrate sequence data with transcriptional and functional studies to better define complex traits and develop new potential microbial processes. This article reviews the significance of microbial collections, with an overview of the well-known European Biological Resources Centers (BRCs) collecting AAB, and provides an insight into their cultivability and metabolic activity. It also discusses appropriate techniques in preserving authentic strains, quality control implications, databases and BRC networking as well as connections among collections and stakeholders. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria (AAB) have undergone continuous taxonomic revision, resulting in an increased number of genera and species ascribed to this group. Thus, the description of the most common AAB in grapes, must and wine has changed dramatically since the initial assessments, which were primarily based on physiological methods. In the present study, we identified the AAB isolated from different grape musts by sequencing the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer region. We had previously performed studies from the same winery. However, in this study we now identified AAB species that have been recently described in wines, as well as others that were identified for first time in this niche. Gluconobacter cerinus, G. japonicus, G. thailandicus and G. oxydans were previously identified as G. oxydans. Kozakia baliensis was also within this group. Acetobacter pasteurianus, A. cerevisiae and A. malorum were formerly grouped as Acetobacter sp. Many isolates previously described as G. oxydans or A. aceti likely correspond to other, newly described species of the same genera. [ABSTRACT FROM AUTHOR]

Gluconacetobacter diazotrophicus as a member of the acetic acid bacteria group, oxidize alcohol to acetic acid through two sequential reactions catalyzed by the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase, both enzymes are membrane-bound and oriented to the periplasmic space. ADH is a quinohemoprotein carrying one pyrroloquinoline quinone moiety, one [2Fe:2S] cluster and four c-type cytochromes, as prosthetic groups. In recent years has been described the presence of the inactive ADH (ADHi) in the acetic acid bacteria. In the present review we make a comparative study of the molecular and catalytic properties of the active and inactive forms of ADH purified from G. diazotrophicus, variation in the redox state of enzymes as purified could explain the notorious differences seen in the activity power of the compared enzymes. [ABSTRACT FROM AUTHOR]

The Kitora tumulus, which is thought to have been built around the late seventh to early eighth century, has beautiful mural paintings which were executed directly on a very thin layer of plaster in the stone chamber. When the paintings were found, the plaster was partly detached from the stone wall and the murals were therefore considered to be likely to fall off the wall with a casual touch or stimulation. Therefore, it was decided to detach the mural paintings and to store them flat and under controlled environmental conditions. This was initiated immediately after the excavation of the tumulus in 2004. However, fungal growth and biofilm development were observed within the stone chamber during the relocation work. In 2005, small holes containing black substances were observed on certain areas of the ceiling plaster, and following investigation an acetic acid bacterium, Gluconacetobacter sp., was isolated from the black substances. The bacterium was also isolated from the ceiling, floor, and east wall in the stone chamber in 2008 after the relocation of most of the paintings had been completed. These bacteria were shown to decompose calcium carbonate (CaCO3), one of the primary components of the plaster, and to produce organic acids such as acetic acid. Additionally, they were observed to decrease the pH of the culture media significantly in the presence of ethanol and glucose. This is the first example of the characterization of acetic acid bacteria isolated from decayed plaster paintings, and it is likely that microbes such as these bacteria have been involved in the deterioration of the plaster. Chemicals to treat microbes in the Kitora tumulus during the relocation work were selected on the basis of their antimicrobial efficacy, low potential to cause adverse effects on the paintings, and low level of toxicity to humans, depending on the condition of the plaster or stone in each area. However, some chemicals, especially ethanol, may act as a carbon source, which could encourage the growth of microbes and thereby the production of acids by the microbes when diluted to a low concentration or in a degraded state. Moreover, prior contamination by other microbial species in the form of a biofilm could also encourage the growth of the acetic acid bacteria by providing low-molecular-weight organic materials as a nutrient source. [ABSTRACT FROM AUTHOR]