Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria

In this study, we compared the growth properties and molecular characteristics of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) among highly acetic acid-resistant strains of acetic acid bacteria. Ga. europaeus exhibited the highest resistance to acetic acid (10%), whereas Ga. intermedius and Acetobacter pasteurianus resisted up to 6% of acetic acid. In media with different concentrations of acetic acid, the maximal acetic acid production rate of Ga. europaeus slowly increased, but specific growth rates decreased concomitant with increased concentration of acetic acid in medium. The lag phase of A. pasteurianus was twice and four times longer in comparison to the lag phases of Ga. europaeus and Ga. intermedius, respectively. PQQ-dependent ADH activity was twice as high in Ga. europaeus and Ga. intermedius as in A. pasteurinus. The purified enzymes showed almost the same specific activity to each other, but in the presence of acetic acid, the enzyme activity decreased faster in A. pasteurianus and Ga. intermedius than in Ga. europaeus. These results suggest that high ADH activity in the Ga. europaeus cells and high acetic acid stability of the purified enzyme represent two of the unique features that enable this species to grow and stay metabolically active at extremely high concentrations of acetic acid.     

Extraction of cellulose-synthesizing activity of Gluconacetobacter xylinus by alkylmaltoside

This study reinvestigated the synthesis of cellulose in vitro with a well-known cellulose-producing bacterium, Gluconacetobacter xylinus. Alkylmaltoside detergents, which are more frequently used in recent structural biological researches, are uniquely used in this study to solubilize cellulose-synthesizing activity from the cell membrane of G. xylinus. Activity comparable to that previously reported is obtained, while the synthesized cellulose is crystallized into a non-native polymorph of cellulose (cellulose II) as well as the previous studies. In spite of this failure to recover the native activity to synthesize cellulose I microfibril in vitro, the product is a polymer with a degree of polymerization greater than 45 as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was thus concluded that the established protocol can solubilize cellulose-synthesizing activity of G. xylinus with polymerizing activity.     

Metabolic flux analysis of Gluconacetobacter xylinus for bacterial cellulose production

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.     

Characterization of Cellulose Production by a Gluconacetobacter xylinus Strain from Kombucha

The aims of this work were to characterize and improve cellulose production by a Gluconoacetobacter xylinus strain isolated from Kombucha and determine the purity and some structural features of the cellulose from this strain. Cellulose yield in tea medium with both black tea and green tea and in Hestrin and Schramm (HS) medium under both static and agitated cultures was compared. In the tea medium, the highest cellulose yield was obtained with green tea (~0.20 g/L) rather than black tea (~0.14 g/L). Yield in HS was higher (~0.28 g/L) but did not differ between static and agitated incubation. ¹H-NMR and ¹³C-NMR spectroscopy indicated that the cellulose is pure (free of acetan) and has high crystallinity, respectively. Cellulose yield was improved by changing the type and level of carbon and nitrogen source in the HS medium. A high yield of ~2.64 g/L was obtained with mannitol at 20 g/L and corn steep liquor at 40 g/L in combination. In the tea medium, tea at a level of 3 g/L gave the highest cellulose yield and the addition of 3 g/L of tea to the HS medium increased cellulose yield to 3.34 g/L. In conclusion, the G. xylinus strain from Kombucha had different cellulose-producing characteristics than previous strains isolated from fruit. Cellulose was produced in a pure form and showed high potential applicability. Our studies extensively characterized cellulose production from a G. xylinus strain from Kombucha for the first time, indicating both similarities and differences to strains from different sources.     

Biotechnological applications of acetic acid bacteria

The acetic acid bacteria (AAB) have important roles in food and beverage production, as well as in the bioproduction of industrial chemicals. In recent years, there have been major advances in understanding their taxonomy, molecular biology, and physiology, and in methods for their isolation and identification. AAB are obligate aerobes that oxidize sugars, sugar alcohols, and ethanol with the production of acetic acid as the major end product. This special type of metabolism differentiates them from all other bacteria. Recently, the AAB taxonomy has been strongly rearranged as new techniques using 16S rRNA sequence analysis have been introduced. Currently, the AAB are classified in ten genera in the family Acetobacteriaceae. AAB can not only play a positive role in the production of selected foods and beverages, but they can also spoil other foods and beverages. AAB occur in sugar- and alcohol-enriched environments. The difficulty of cultivation of AAB on semisolid media in the past resulted in poor knowledge of the species present in industrial processes. The first step of acetic acid production is the conversion of ethanol from a carbohydrate carried out by yeasts, and the second step is the oxidation of ethanol to acetic acid carried out by AAB. Vinegar is traditionally the product of acetous fermentation of natural alcoholic substrates. Depending on the substrate, vinegars can be classified as fruit, starch, or spirit substrate vinegars. Although a variety of bacteria can produce acetic acid, mostly members of Acetobacter, Gluconacetobacter, and Gluconobacter are used commercially. Industrial vinegar manufacturing processes fall into three main categories: slow processes, quick processes, and submerged processes. AAB also play an important role in cocoa production, which represents a significant means of income for some countries. Microbial cellulose, produced by AAB, possesses some excellent physical properties and has potential for many applications. Other products of biotransformations by AAB or their enzymes include 2-keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium was described, representing the newest and tenth genus of AAB.     

Viability and cellulose synthesizing ability of Gluconacetobacter xylinus cells under high-hydrostatic pressure

The effect of pressure on viability and the synthesis of bacterial cellulose (BC) by Gluconacetobacter xylinus ATCC53582 were investigated. G. xylinus was statically cultivated in a pressurized vessel under 0.1, 30, 60, and 100 MPa at 25°C for 6 days. G. xylinus cells remained viable and retained cellulose producing ability under all the conditions tested, though the production of cellulose decreased with increasing the pressure. The BCs produced at each pressure condition were analyzed by field emission scanning electron microscopy (FE-SEM) and Fourier Transform Infrared (FT-IR). FE-SEM revealed that the widths of BC fibers produced under high pressure decreased as compared with those produced under the atmospheric pressure. By FT-IR, all the BCs were found to be of Cellulose type I, as the same as typical native cellulose. Our findings evidently showed that G. xylinus possessed a piezotolerant (barotolerant) feature adapting to 100 MPa without losing its BC producing ability. This was the first attempt in synthesizing BC with G. xylinus under elevated pressure of 100 MPa, which corresponded to the deep sea at 10,000 m.     

Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.     

Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I 2281

The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidation of ethanol to acetate and carbon dioxide. In contrast, bacterial growth initiated without delay on 35 g/l glucose and 5 g/l acetate. It was found that acetyl CoA was activated by the acetyl coenzyme A synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway. The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active. A carbon balance calculation showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis liked to ethanol catabolism. This calculation showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate. This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethanol.     

Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell–cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.The nucleotide sequences of 16S rDNA that are reported in this paper were submitted to GenBank/EMBL/DDBJ under the accession numbers AB016864 (st-20) and AB016865 (st-60-12).     

Growth behavior of immobilized acetic acid bacteria

Summary Released free cells of immobilized growing acetic acid bacteria during 460 days of long run in a bioreactor were increased at a high dilution rate. This was interpreted by the facts that newly released cells from the carrier showed a synchronized growth and had extremely high growth rates and a respiratory activity. The suitable leakage is effective for high productivity of the immobilized growing cell culture.     

"Nanocellulose" as a single nanofiber prepared from pellicle secreted by Gluconacetobacter xylinus using aqueous counter collision

This study attempted to prepare a single cellulose nanofiber, "nanocellulose", dispersed in water from 3D networks of nanofibers in microbial cellulose pellicle using aqueous counter collision (ACC), which allows biobased materials to be down-sized into nano-objects only using water jets without chemical modification. The nanocellulose thus prepared exhibited unique morphological properties. In particular, the width of the nanocellulose, which could be controlled as desired on nanoscales, was smaller than that of just secreted cellulose nanofiber, resulting in larger specific surface areas. Moreover, ACC treatment transformed cellulose I(α) crystalline phase into cellulose I(β) phase with the crystallinity kept >70%. In this way, ACC method depending on the treatment condition could provide the desired fiber width at the nanoscale and the different ratios of the two crystalline allomorphs between cellulose I(α) versus I(β), which thus opens further pathways into versatile applications as biodegradable single nanofibers.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.