Nitrogen-fixing and cellulose-producing Gluconacetobacter kombuchae sp. nov., isolated from Kombucha tea

Endophytic bacteria colonize the internal tissues of the host plant for mutual benefit. The nitrogen-fixing endophyte Gluconacetobacter diazotrophicus was found to be associated with sugar cane, pineapple, Cameroon grass, sweet potato, mango and banana (Muthukumarasamy et al., 2002), while Gluconacetobacter azotocaptans and Gluconacetobacter johannae inhabit coffee plants (Fuentes-Ramírez et al., 2001); these organisms might provide host plants with useful fixed nitrogen and growth stimulants (Lee et al., 2000; Muthukumarasamy et al., 2002). Based on the strikingly similar high-sugar and low-pH environments presented by the habitats of sugar cane and Kombucha tea (Blanc, 1996), we explored the bacterial flora of Kombucha tea and isolated a nitrogen-fixing strain, Acetobacter nitrogenifigens RG1^T (Dutta & Gachhui, 2006), and another nitrogen-fixing and cellulose-producing strain, RG3^T, belonging to the family Acetobacteraceae.

The family Acetobacteraceae has been divided into 10 genera: Acetobacter, Gluconacetobacter, Gluconobacter, Acidomonas (Yamada et al., 1997), Asaia (Yamada et al., 2000), Kozakia (Lisdiyanti et al., 2002), Saccharibacter (Jojima et al., 2004), Swaminathania (Loganathan & Nair, 2004), Neoasaia (Yukphan et al., 2005) and Granulibacter (Greenberg et al., 2006). Only six members of the family, G. diazotrophicus (Gillis et al., 1989), G. johannae (Fuentes-Ramírez et al., 2001), G. azotocaptans (Fuentes-Ramírez et al., 2001), Acetobacter peroxydans (Muthukumarasamy et al., 2005), Swaminathania salitolerans (Loganathan & Nair, 2004) and Acetobacter nitrogenifigens (Dutta & Gachhui, 2006), are known to fix nitrogen. The genus Gluconacetobacter comprises 15 species with validly published names at present, differentiated on the basis of DNA–DNA relatedness, phylogenetic relationships and morphological characteristics. Isolation of strain RG3^T from Kombucha tea is the first instance of a strain within the family having both nitrogen-fixing and cellulose-producing activity. We present morphological, biochemical and genetic evidence that indicates that RG3^T represents a novel nitrogen-fixing species within the genus Gluconacetobacter.

Kombucha tea is a fermented tea that contains an association of yeast and bacteria. A jelly-like membrane floats in the nutrient solution of tea and sugar exposed to oxygen. At the right temperature, it multiplies continuously. It first spreads over the entire surface of the tea, and then thickens. Kombucha tea was subcultured every 7–10 days by mixing 10 % of old soup with 10 % sucrose dissolved in brewed black tea. After teasing the mat apart in the soup, aliquots of Kombucha mat suspension were spread on LGI agar plates (0.06 % KH₂PO₄, 0.02 % K₂HPO₄, 0.02 % MgSO₄, 0.002 % CaCl₂, 0.001 % FeCl₃, 0.0002 % Na₂MoO₄, 10 % sucrose, pH 4.5; Cavalcante & Döbereiner, 1988) containing 150 mg cycloheximide l⁻¹ (Jimenez-Salgado et al., 1997) and 150 mg nystatin l⁻¹. Plates were incubated at 30 °C for 5 days. Repeated streaking on LGI plates, which contain no combined nitrogen, purified the bacterial isolate. Gas-tight vials of LGI medium inoculated with bacteria (under a microaerophilic environment, without shaking) were assayed for acetylene reduction activity (Stal, 1988). Nitrogenase-positive isolates were selected for further characterization. Strain RG3^T, a nitrogen-fixing bacterial strain that exhibited cellulose-producing ability even in nitrogen-free LGI broth, was isolated.

Strain RG3^T produced cellulose when grown in HS medium (Hestrin & Schramm, 1954) under stationary as well as shaking culture conditions at 30 °C after incubation for 3 days. The cellulosic character of the pellicle was confirmed by boiling the pellicles with a dilute NaOH solution (Forng et al., 1989; Navarro & Komagata, 1999). The polymer was a simple carbohydrate in nature, as indicated by the greenish-blue colour of the supernatant with o-toluidine. In acid-hydrolysed bacterial pellicle samples, the amount of reducing sugars released was found to be comparable to the amount of glucose residues estimated by using glucose oxidase. Thus, the pellicle had repetitive glucose units. Liberation of glucose units upon enzymic digestion of the pellicle with cellulase confirmed it to be composed of cellulose.

Reference strains (Gluconacetobacter hansenii JCM 11196^T, a gift of Y. Nakagawa and Y. Yamada, Gluconacetobacter intermedius LMG 18909^T, Gluconacetobacter europeaus LMG 1518^T, Gluconacetobacter oboediens LMG 18849^T and Gluconacetobacter swingsii LMG 22125^T, from the BCCM/LMG, and Gluconacetobacter xylinus JCM 7644^T from the JCM) were grown in different media according to the instructions of the culture collections. Colony morphology was examined on LGI agar plates and on potato agar plates containing 10 % sucrose. Various phenotypic and morphological characters were tested using standard techniques described previously (Franke et al., 1999; Schüller et al., 2000; Dellaglio et al., 2005; Fuentes-Ramírez et al., 2001). Isoprenoid quinones of the isolate were extracted with chloroform/methanol (2 : 1, v/v) and purified by TLC on silica gel 60 F₂₅₄ plates (20×20 cm; Merck) by using benzene as the developing solvent. Quinones recovered from the TLC plates were dissolved in acetone and analysed by HPLC (Lu et al., 1999). The HPLC system was equipped with a reversed-phase column [Luna 5U C18 (2) 100A, 250×4.6 mm; Phenomenex] and a mixture of methanol and isopropanol (2 : 1, v/v) was used as the mobile phase at a flow rate of 1 ml min⁻¹. Types of quinone were identified by absorption at 275 nm and compared with coenzyme Q-9 and coenzyme Q-10 standards from Sigma-Aldrich. Ubiquinone Q-10 was present in strain RG3^T, in agreement with previous observations of the presence of this ubiquinone type in the genus Gluconacetobacter (Cleenwerck et al., 2002).

A 1442 bp fragment of the 16S rRNA gene was amplified by PCR with bacteria-specific primers fD1 and rD1 (Weisburg et al., 1991) using Taq polymerase and genomic DNA from RG3^T as the template. The nucleotide sequence showed the following levels of similarity to sequences from strains of the genus Gluconacetobacter after performing similarity searches with fasta (ungapped): 99.1 % with G. hansenii LMG 1527^T, 99.0 % with G. entanii LTH 4560^T, 98.6 % with G. rhaeticus DST GL02^T, 98.5 % with G. swingsii DST GL01^T, 98.3 % with G. xylinus JCM 7644^T and G. europeaus JK2, 98.2 % with G. saccharivorans LMG 1582^T, 98.1 % with G. oboediens LTH 2460^T, G. intermedius TF2^T and G. nataicola LMG 1536^T, 96.7 % with G. diazotrophicus LMG 7603^T, 96.6 % with G. azotocaptans CFN-Ca54^T, 96.5 % with G. johannae CFN-Cf55^T and G. liquefaciens LMG 1382^T and 96.4 % with G. sacchari IF 2-6. The phylogenetic tree was deduced using mega version 3.1 (Kumar et al., 2004) software after multiple alignment with 16S rRNA gene sequences of other acetic acid bacteria with clustal w (Thompson et al., 1994). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining method were determined by using bootstrap values (Felsenstein, 1985) based on 100 replications. As evident from the tree (Fig. 1⇓), the species of this genus are subgrouped phylogenetically into two clusters, comprising nitrogen-fixers such as G. diazotrophicus and cellulose-producers such as G. xylinus, G. swingsii, G. rhaeticus and G. nataicola. The novel strain RG3^T, which exhibits both these characteristics, clustered with the ‘cellulose-producing’ group, along the subbranch formed by G. hansenii and G. entanii.

Strain RG3^T differed biochemically from the phylogenetically closely related species of the genus Gluconacetobacter (Table 1⇓). It could be differentiated from G. hansenii and G. entanii, its phylogenetically closest neighbours, in utilizing sorbitol as a sole source of carbon. Additionally, RG3^T could be differentiated from G. hansenii by the non-utilization of ethanol, mannitol and sucrose as carbon sources and from G. entanii by growth in the absence of acetic acid and utilization of d-mannitol as a carbon source. The major difference between RG3^T and the related ‘cellulose-producing’ gluconacetobacters is the absence of nitrogen-fixing behaviour in the remaining species. Of the genes responsible for nitrogen fixation, the structural genes nifHDK of the nitrogenase enzyme complex are the most important. A 536 bp region encoding dinitrogenase reductase, nifH, was amplified from RG3^T using degenerate primers 19F and 407R (Franke et al., 1998) and sequenced, confirming the presence of the nifH gene in RG3^T.

To determine the genomic relatedness of the new isolate, dot-blot hybridization experiments were carried out with DIG-labelled DNA as described previously (Labrenz et al., 2000) using the detection kit from Roche Applied Sciences following the manufacturer's instructions. Colorimetric quantification of dot intensities was done using the Molecular Analyst software (Bio-Rad) by determining mean pixel densities in circles of equal size. The genomic DNA probe was prepared from strain RG3^T; digested with EcoRI and separated on a 0.7 % agarose gel. Total DNA digests were transferred from gels to nylon membrane by Southern blotting. Hybridization was performed at 75 °C for 16 h and the membrane was washed under high-stringency conditions (twice with 2× SSC/0.1 % SDS at room temperature for 10 min, once with 0.1× SSC/0.1 % SDS at 75 °C for 15 min). Low levels of genomic DNA relatedness (DNA–DNA hybridization values of less than 30 %) were observed between strain RG3^T and its phylogenetically closest relatives; DNA–DNA relatedness values of 24.8 % (with G. hansenii JCM 7643^T), 21.8 % (G. swingsii LMG 22125^T), 27.7 % (G. xylinus JCM 7644^T), 24.6 % (G. europeaus LMG 1518^T), 5.2 % (G. oboediens LMG 18849^T) and 23.4 % (G. intermedius LMG 18909^T) were obtained.

The limitations of 16S rRNA gene sequencing for the differentiation of closely related species have been documented (Fox et al., 1992), but a DNA–DNA reassociation level below 70 % indicates a distinct species (Stackebrandt & Goebel, 1994). Although the 16S rRNA gene sequence similarity levels were greater than 97 %, low levels (below 30 %) of DNA relatedness were found among the closely related Gluconacetobacter species studied. In view of the low physiological, biochemical, phylogenetic and genetic similarities among members of the genus Gluconacetobacter, we recommend that strain RG3^T should be assigned to a novel species of the genus Gluconacetobacter. We propose the name Gluconacetobacter kombuchae sp. nov. for strain RG3^T isolated from Kombucha tea.