Acetobacter Strains Research Articles

Acetic acid bacteria as enantioselective biocatalysts

Acetic acid bacteria (five strains of Acetobacter and five strains of Gluconobacter) were used for the biotransformation of different primary alcohols (2-chloropropanol and 2-phenylpropanol) and diols (1,3-butandiol, 1,4-nonandiol and 2,3-butandiol). Most of the tested strains efficiently oxidized the substrates. 2-Chloropropanol and 1,3-butandiol were oxidized with good rates and low enantioselectivity (enantiomeric excess=18–46% of the S-acid), while microbial oxidation of 2-phenylpropanol furnished ( S)-2-phenyl-1-propionic acid with enantiomeric excess (e.e.) >90% with 10 strains. The dehydrogenation of 2,3-butandiol was strongly dependent on the stereochemistry of the substrate; the meso form gave S-acetoin with all the tested strains, the only exception being a Gluconobacter strain. The formation of diacetyl was observed only by using R,R-2,3-butandiol with Acetobacter strains. Oxidation of 1,4-nonandiol gave γ-nonanoic lactone in one step, although with moderate enantioselectivity.

Purification and Characterization of a Novel Polysaccharide Involved in the Pellicle Produced by a ThermotolerantAcetobacterStrain

Acetobacter strains able to produce a thick pellicle at 37 degrees C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37 degrees C or 40 degrees C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30 degrees C than at 37 degrees C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.

Identification of Acetobacter strains isolated from Indonesian sources, and proposals of Acetobacter syzygii sp. nov., Acetobacter cibinongensis sp. nov., and Acetobacter orientalis sp. nov.

Forty-six strains of acetic acid bacteria newly isolated from flowers, fruits, and fermented foods collected in Indonesia were taxonomically studied. They were Gram-negative rods, produced acetic acid from ethanol, oxidized acetate and lactate to CO(2) and H(2)O, and had Q-9 as the major ubiquinone system. On the basis of DNA-DNA similarity, all strains studied, including type strains and reference strains of the genus Acetobacter, were separated into eleven groups (Groups I to XI). Of the 46 isolates, two isolates were included in Group II and identified as Acetobacter pasteurianus, five in Group IV as A. orleanensis, 16 in Group V as A. lovaniensis, five in Group VII as A. indonesiensis, and three in Group VIII as A. tropicalis. The remaining 15 isolates constituted three new groups based on DNA-DNA similarity; four isolates were included in Group IX, two in Group X, and nine in Group XI. No isolates were identified as A. aceti (Group I), A. peroxydans (Group III), and A. estunensis (Group VI). Phylogenetic analysis based on 16S rDNA sequences of representative strains of the Groups indicated belonging to the strains of the genus Acetobacter. On the basis of DNA base composition, DNA-DNA similarity, and 16S rDNA sequences, three new species of the genus Acetobacter are proposed: Acetobacter syzygii sp. nov. for Group IX, Acetobacter cibinongensis sp. nov. for Group X, and Acetobacter orientalis sp. nov. for Group XI. The distribution of Acetobacter strains in Indonesia is discussed in light of isolation sources.

Molecular identification of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector.

Three new Acetobacter strains were isolated from vinegar. By plasmid profiling they were recognized as genotypically different from each other. Sequencing of the genes for 16S and 23S rRNA and DNA-DNA hybridization of total DNA against DNA of all type strains of Acetobacter identified Acetobacter strains JK2 and V3 as A. europaeus, and Acetobacter strain JK3 as A. intermedius. In contrast to the type strain of A. europaeus (DSM 6160), A. europaeus JK2 and V3 do not require acetic acid for growth and can be successfully transferred between media with and without acetic acid. This phenotypic characteristic enables convenient handling of both strains in genetic studies. Plasmid pJK2-1 from A. europaeus JK2 was used as the basis for shuttle plasmid construction with the aim of developing an efficient vector system for these strains. The entire nucleotide sequence of pJK2-1 was determined. High amino acid identities were found for three open reading frames: Rep (replication protein); Dinjl (DNA damage inducible enzyme); and Dinj2 proteins. A recombinant plasmid pUCJK2-1 (5.6 kb) consisting of the entire plasmid pJK2-1 and the entire plasmid pUC18 was successfully used in transformation experiments. Plasmid pJT2 (5.8 kb) was constructed from pUCJK2-1 with the aim of reactivating the lacZ' gene.

Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov.

Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of DNA-DNA relatedness. Of 31 reference strains, six showed the presence of ubiquinone 10 (Q-10). These strains were eliminated from the genus Acetobacter. The other 25 reference strains and 45 Indonesian isolates were subjected to a systematic study and separated into 8 distinct groups on the basis of DNA-DNA relatedness. The known species, Acetobacter aceti, A. pasteurianus, and A. peroxydans are retained for three of these groups. New combinations, A. orleanensis (Henneberg 1906) comb. nov., A. lovaniensis (Frateur 1950) comb. nov., and A. estunensis (Carr 1958) comb. nov. are proposed for three other groups. Two new species, A. indonesiensis sp. nov. and A. tropicalis sp. nov. are proposed for the remaining two. No Indonesian isolates were identified as A. aceti, A. estunensis, and A. peroxydans. Phylogenetic analysis on the basis of 16S rDNA sequences was carried out for representative strains from each of the groups. This supported that the eight species belonged to the genus Acetobacter. Several strains previously assigned to the species of A. aceti and A. pasteurianus were scattered over the different species. It is evident that the value of DNA-DNA relatedness between strains comprising a new species should be determined for the establishment of the species. Thus current bacterial species without data of DNA-DNA relatedness should be reexamined for the stability of bacterial nomenclature.

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures.

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.

Biotransformations in two-liquid-phase systems: Production of phenylacetaldehyde by oxidation of 2-phenylethanol with acetic acid bacteria

Phenylacetaldehyde can be obtained by oxidation of 2-phenylethanol with acetic acid bacteria in two-liquid-phase systems where the aldehyde is removed into the organic phase before its further conversion to acid. Two Acetobacter strains (ALEF and ALEG) were able to accumulate aldehyde when aliphatic hydrocarbons were used. A two-liquid-phase system, composed of water and isooctane (v/v, 1/1), was particularly suited for a significant accumulation of the aldehyde: Acetobacter sp. ALEG furnished 9 g/l of phenylacetaldehyde within 4 h starting from 10 g/l of alcohol and still 8 g/l were recovered after 24 h in the organic phase, whereas strain ALEF gave 3.5 g/l of aldehyde from 5.0 g/l of substrate. Acetobacter sp. ALEG also showed satisfactory long-term stability, being able to perform the transformation with 80% of the original activity after 3 days of contact with the solvent.

Isolation and characterization of two genetically distant groups of Acetobacter diazotrophicus from a new host plant Eleusine coracana L.

Acetobacter diazotrophicus strains were isolated from Eleusine coracana, a new host plant cultivated along the coast of Tamil Nadu in India. Using a species-specific oligonucleotide primer and PCR amplification, the presence of this bacterium was demonstrated directly in plant tissues, proving its endophytic nature, and it was absent in non-rhizosphere soils. The isolates were also characterized on the basis of typical morphology, electron microscopy and biochemical tests, including nitrogen-fixing efficiency, to assess their diversity. When RAPD analysis was performed on the isolates, they fell into two distinct genetically related groups when compared with the type strain PA1 5 (ATCC 49037). In view of the importance of E. coracana to this region, associated nitrogen-fixing Acetobacter strains may be agronomically important because they could supply part of the nitrogen that the crop requires.

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum.

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.

Identification of acetic acid bacteria isolated from Indonesian sources, especially of isolates classified in the genus Gluconobacter.

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.

Characterization, Differentiation and Identification of Wild-type Cellulose-synthesizing Acetobacter strains Involved in Nata de Coco Production

Summary Cellulose-producing bacteria used for Nata de Coco production in the Philippines were isolated and characterized. Two distinct wild-type strains (ITDI 2.1 and PA 2.2) were selected from thirty-eight isolates obtained. The two strains, and the rest of the cellulose producers, were established to be members of the genus Acetobacter based on rapid phenotypic characterization. They were characterized and differentiated based on pellicle type and colony morphology, carbon-utilization pattern (Biolog™ assay), amount of cellulose production and 16S rDNA sequence. The thick pellicle cellulose-producing strain (ITDI 2.1) has a stable and consistent production of a very thick pellicle in any sugar-based, acidic medium, with a dry weight up to twenty-six times that of the thin pellicle cellulose-producing strain (PA 2.2) after 7–8 days static batch culture. Species identification using 16S rDNA sequencing revealed that the two strains belong to two different species of Acetobacter, Acetobacter xylinus and A. hansenii and may be new subspecies under these species designation.

Description of Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov., two new species isolated from industrial vinegar fermentations.

Two strains of Acetobacter sp., LTH 2460T and LTH 2458T, have been isolated from running red wine and cider vinegar fermentations, respectively. Taxonomic characteristics of the isolates were investigated. Comparative analysis of the 165 rRNA sequences revealed > 99% similarity between strain LTH 2460T and the type strains of the related species Acetobacter europaeus and Acetobacter xylinus and between strain LTH 2458T and Acetobacter pasteurianus. On the other hand, low levels of DNA relatedness (< 34%) were determined in DNA-DNA similarity studies. This relatedness below the species level was consistent with specific physiological characteristics permitting clear identification of these strains within established species of acetic acid bacteria. Based on these results, the names Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov. are proposed for strains LTH 2460T and LTH 2458T, respectively. The phylogenetic positions of the new species are reflected by a 16S rRNA-based tree. Furthermore, a 16S rRNA-targeted oligonucleotide probe specific for A. oboediens was constructed.

Increased cellulose production from sucrose with reduced levan accumulation by an Acetobacter strain harboring a recombinant plasmid.

Cellulose production from sucrose by Acetobacter strains is accompanied by the accumulation of a water-soluble polysaccharide, called levan. To improve cellulose productivity, a levansucrase-deficient mutant, LD-2, was derived from Acetobacter strain 757 and used as a host for the construction of recombinant strains. An LD-2 mutant harboring a plasmid containing the sucrase gene, sucZE3, from Zymomonas mobilis together with zliS, a gene that encodes a secretion-activating factor under the control of the Escherichia coli lac promoter, had sucrase activity and produced much cellulose and little levan in a medium containing sucrose. In addition, a mutant levansucrase gene, mutant sacB, from Bacillus subtilis, which encodes a protein with little levan-forming activity, was generated by site-directed mutagenesis and introduced into the LD-2 mutant. This introduction also resulted in the higher cellulose productivity and little levan.

Production and application of microbial cellulose

Cellulose, the basic material of all plant substance, is also produced by green algae ( Valonia) and some bacteria, principally of the genera Acetobacter, Sarcina and Agrobacterium. Special attention has been given to strains from Acetobacter, specially Acetobacter xylinum. Acetobacter strains are well known for oxidizing alcohols to acids and ketones, especially for the production of vinegars using ethanol, wine or cider as carbon sources. The formation of the cellulose pellicle occurs on the upper surface of the supernatant film. Cellulose production was reported to be stimulated by addition of lactic acid, methionine, tea infusion and corn steep liquor. Although for the production process new non-conventional bioreactors have been developed, static cultures are still preferred. A large surface area is important for a good productivity. Relatively low glucose concentrations also gave better productivity and yields than higher ones. Bacterial cellulose can be applied in areas where plant cellulose can hardly be used. New applications were described as thickener to maintain viscosity in food, cosmetics, etc., as nonwoven fabric or paper for old document repair, as food additives and others. We could make use of cellulose films as a temporary substitute for human skin in the case of burns, ulcers, decubitus and others. Biofill ®, Bioprocess ® and Gengiflex ® are products of microbial cellulose that now have wide applications in surgery and dental implants.

Increased Cellulose Production from Sucrose by Acetobacter after Introducing the Sucrose Phosphorylase Gene.

A sucrose phosphorylase (SPase) gene derived from Leuconostoc mesenteroides was introduced into a cellulose-producing Acetobacter strain and expressed under the lac promoter. The activity of the SPase was detected in extracts of the transformed cells and cellulose production from sucrose by the cells was found to have increased, which strongly suggests that the increase was the result of the new metabolizing pathway. Furthermore, the level of SPase expression was increased by altering the length of the lac promoter.

Occurrence of polypeptides in other organisms cross-reacting with antibodies against A. xylinum cellulose synthase

Antibodies (anti-83 and anti-93) against the cellulose synthase complex from A. xylinum ATCC 53582 have been employed to study the evolutionary conservation of this enzyme complex among various A. xylinum strains, selected species of other cellulose- producing bacteria, algae, and vascular plants. Of the 18 A. xylinum strains examined, the 83 Kd polypeptide clearly is detected only in 4 strains while the 93 Kd polypeptide is observed in all 18 strains. Assuming that the revised acsAB gene (Saxena et al., 1994) encoding the 83 and 93 Kd polypeptides as a single polypeptide holds true for all A. xylinum strains, it is proposed that the cellulose synthase is conserved in A. xylinum but with varying degrees of homology. An unknown regulatory mechanism causing the degradation of the 83 Kd polypeptide in response to agitated culturing conditions has been suggested to explain the absence of the 83 Kd polypeptide in most of the Acetobacter strains examined. A. xylinum cellulose synthase appears to be conserved in phylogenetically related Rhizobium and Agrobacterium species, but not in algae and plants.

Biological nitrogen fixation in non-leguminous field crops: Facilitating the evolution of an effective association between Azospirillum and wheat

Recent advances towards achieving significant nitrogen fixation by diazotrophs in symbioses with cereals are reviewed, referring to the literature on the evolution of effective symbioses involving rhizobia and Frankia as microsymbionts. Data indicating that strains of Acetobacter and Herbaspirillum colonizing specific cultivars of sugarcane as endophytes make a significant contribution to the nitrogen economy of this crop improves the prospects that similar associative systems may be developed for other gramineous species such as rice and wheat. By contrast, the transfer of nodulation genes similar to those in legumes or Parasponia to achieve nodulation in crops like rice and wheat is considered to be a more ambitious and distant goal. Progress in developing an effective associative system for cereals has been materially assisted by the development of genetic tools based on the application of lacZ and gusA fusions with the promoters of genes associated with nitrogen fixation. These reporter genes have provided clear evidence that ‘crack-entry’ at the points of emergence of lateral roots or of 2,4-D induced para-nodules is the most significant route of endophytic colonization. Furthermore, using the laboratory model of para-nodulated wheat, there is now evidence that the ability of azospirilla and other nitrogen fixing bacteria to colonize extensively as endophytes can be genetically controlled. The most successful strain of Azospirillum brasilense (Sp7-S) for endophytic colonization and nitrogen fixation in wheat seedlings is a mutant with reduced exopolysaccharide production. Most other strains of azospirilla do not colonize as endophytes and it is concluded that though these are poorly adapted to providing nitrogen for the host plant, they are well adapted for survival and persistence in soil. A research program combining the study of endophytic colonization by azospirilla with an examination of the factors controlling the effectiveness of association (oxygen tolerance and nitrogen transfer) is now being pursued. It is proposed that a process of facilitated evolution of para-nodulated wheat involving the stepwise genetic improvement of both the prospective microsymbionts and the cereal host will eventually lead to effective nitrogen-fixing associations. In the attempt to achieve this goal, continued study of the endophytes occurring naturally in sugar cane and other grasses (e.g. Azoarcus sp.) should be of assistance.

High Rate Production in Static Culture of Bacterial Cellulose from Sucrose by a Newly IsolatedAcetobacterStrain

For the industrial production of bacterial cellulose from sucrose in static cultures, the possibility of a high rate of cellulose production was investigated. An Acetobacter strain, S-35, which had been isolated from a grape, was selected from 1500 isolates. This strain was found to produce a large amount of cellulose from either glucose or fructose. Using this strain, high cellulose production rates of 3.3g/liter/d or 40g/m2/d from sucrose were seen in static culture.

Production of bacterial cellulose by agitation culture systems

Abstract

Two types of cellulase activity produced by a cellulose-producing Acetobacter strain

Cellulase activities were detected in a culture supernatant concentrate (designated EX) and a cell extract (designated IN) of Acetobacter xylinum subsp. sucrofermentans BPR2001. IN and EX each hydrolyzed carboxymethylcellulose (CMC) strongly, but neither hydrolyzed bacterial cellulose (BC), Avicel or cellobiose. However, they did decrease the degree of polymerization of BC. By contrast, IN hydrolyzed cellotriose (G3). From the results of TLC assays and pH stability tests, it is suggested that this organism produces not only CMCase but also another cellulase, G3ase.