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1 BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
2 Research Group of Industrial Microbiology and Food Biotechnology, Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
3 Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
Ilse Cleenwerck
Ilse.Cleenwerck{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains R-29337T, 444B and 384 are EF030713, DQ887337 and DQ887338, respectively.
A maximum-parsimony tree based on 16S rRNA gene sequences of Acetobacter ghanensis sp. nov. and related species of the family Acetobacteraceae is available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Loganathan & Nair, 2004; Jojima et al., 2004; Yukphan et al., 2005; Greenberg et al., 2006). A common feature of AAB (with the exception of the genus Asaia) is the ability to oxidize ethanol to acetic acid under neutral and acidic (pH 4.5) conditions. Because of this characteristic, AAB are often involved in the production of fermented foods, either in a beneficial (chocolate products, coffee, vinegar, nata de coco and speciality beers) or detrimental (spoilage of beers, wines and ciders) manner (Kersters et al., 2006).
AAB contribute to the development of high-quality cocoa since they play a critical role in the spontaneous fermentation of cocoa beans, regarded as the first stage in chocolate production (Schwan & Wheals, 2004). They oxidize ethanol, initially produced by yeasts, to acetic acid, which then diffuses into the beans, and this – in combination with heat produced by this exothermic bioconversion – causes the death of the seed embryo as well as the end of fermentation. In turn, biochemical changes in the beans are initiated, leading to the formation of precursor molecules that are necessary for the development of the characteristic aroma, flavour and colour of the beans. These properties are further developed during drying, roasting and final processing of well-fermented cocoa beans to produce cocoa powder and chocolate (Hansen et al., 1998; Thompson et al., 2001).
Today, little is known about AAB associated with cocoa bean fermentation, although strains of the genera Acetobacter and Gluconobacter are most commonly found (Schwan & Wheals, 2004). During a biodiversity study on micro-organisms involved in the spontaneous fermentation of Ghanaian cocoa beans, 132 AAB strains were isolated. These strains, together with 64 AAB reference strains, including the type strains of most recognized AAB species, were investigated by rep-PCR fingerprinting using the (GTG)5-primer, a genotypic technique that has proven to be useful for rapid and reliable species recognition and typing of lactobacilli (Gevers et al., 2001) and enterococci (
vec et al., 2005). (GTG)5-PCR fingerprinting revealed a cluster of 23 AAB isolates that did not contain any AAB type strain, suggesting that they could represent a novel AAB species (L. De Vuyst, N. Camu, T. De Winter, K. Vandemeulebroecke, V. Van de Perre, M. Vancanneyt, P. De Vos and I. Cleenwerck, unpublished results). Genotypic and phenotypic data demonstrate that these 23 AAB isolates indeed represent a novel species of the genus Acetobacter, for which the name Acetobacter ghanensis sp. nov. is proposed.
The 23 AAB strains (430AT, 131, 134A, 141B, 153A, 384, 429, 415, 421, 130B, 422, 118A, 440, 112A, 444B, 110, 140A, 118B, 444A, 428A, 435, 430B1 and 387A) were isolated from seven traditional heap fermentations of Ghanaian cocoa beans, performed during the main- and mid-crop of 2004. Deoxycholate-mannitol-sorbitol (DMS) medium was used and incubation was carried out at 42 °C for 1–4 days. The isolates were preserved at – 80 °C in MYP medium [2.5 % (w/v) D-mannitol, 0.5 % (w/v) yeast extract and 0.3 % (w/v) bacteriological peptone (Oxoid)], supplemented with 25 % (v/v) glycerol as cryoprotectant. Isolates were recovered by incubation at 30 °C in MYP broth under aerobic conditions for 1–4 days. Isolates 444B, 430AT and 415 were deposited in the Research Collection of the Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium, as strains R-29336, R-29337T and R-29338, respectively. On solidified MYP medium [1.5 % (w/v) agar], the novel strains appeared as beige, round, convex, smooth, shiny colonies with a diameter of approximately 0.8 mm after 4 days incubation. The type strains of the Acetobacter species used in this study were obtained from the BCCM/LMG Bacteria Collection (http://www.belspo.be/bccm/). They were grown according to the provider's specifications, unless indicated otherwise.
Cells of the 23 isolates were tested for their Gram reaction, cell shape and cell size. Catalase activity was tested by adding young cells from MYP agar to a drop of 20 % (v/v) hydrogen peroxide solution. Oxidase activity was tested using the Oxidase DrySlide testkit (Becton Dickinson). Lactobacillus plantarum LMG 6907T (a catalase-negative, oxidase-negative lactic acid bacterium) and Acetobacter aceti LMG 1504T (a catalase-positive, oxidase-negative AAB) were used as controls. Cells of all isolates were Gram-negative rods, approximately 0.8 µm wide and 1.5–2.5 µm long. Cells occurred singly or in pairs and were oxidase-negative and catalase-positive.
Production of acetic acid from ethanol and gluconic acid from glucose were tested following growth of the strains at 30 °C for 48 h in basal medium [0.05 % (w/v) yeast extract and 0.3 % (w/v) vitamin-free Casamino acids (Difco)] plus ethanol (0.3 %, w/v) and GY medium [5 % (w/v) D-glucose and 0.5 % (w/v) yeast extract], respectively, using high pressure anion-exchange chromatography-conductivity following the protocol of Van der Meulen et al. (2006). The production of 2- and 5-ketogluconic acids and 2,5-diketogluconic acid from glucose were determined by the methods described by Gosselé et al. (1980). All isolates oxidized ethanol to acetic acid and produced gluconic acid from glucose. They did not produce 2-ketogluconic acid, 5-ketogluconic acid or 2,5-diketogluconic acid from glucose. These characteristics indicated that the strains probably belong to the genus Acetobacter that, at the time of writing, comprises 16 species with validly published names (Lisdiyanti et al., 2000, 2001; Cleenwerck et al., 2002; Silva et al., 2006; Dutta & Gachhui, 2006).
Fatty acids were extracted and analysed according to the recommendations of the commercial identification system MIDI (Microbial Identification System) from reference strains (A. aceti LMG 1261T, Acetobacter lovaniensis LMG 1579T, Acetobacter orleanensis LMG 1583T and Acetobacter pasteurianus LMG 1262T) and six of the isolates (430AT, 444B, 415, 131, 153A and 110) from cells grown on MYP agar for 24 h at 28 °C under aerobic conditions. Stenotrophomonas maltophilia LMG 958T was used as the control. The predominant fatty acid was the straight-chain, unsaturated C18 : 1
7c fatty acid, which accounted for approximately 50 % total fatty acid content. This is in agreement with results obtained previously for AAB (Urakami et al., 1989; Franke et al., 1999; Dellaglio et al., 2005; Kersters et al., 2006). Other fatty acids found in the isolates in minor but still significant amounts were C16 : 0 (6–16 %), C16 : 0 2-OH (9–12 %), C14 : 0 2-OH (5–7 %), C14 : 0 (3–5 %) and C19 : 0 cyclo 8c (5–15 %). Compared with reported data for species of the genus Acetobacter (Urakami et al., 1989; Franke et al., 1999), the novel isolates contained high levels of C19 : 0 cyclo 8c.
16S rRNA gene sequences of strains R-29337T, 444B and 384 were determined as described by Franz et al. (2006) with the following modifications. DNA for 16S rRNA gene sequencing was extracted from strain R-29337T by the method of Wilson (1987) with minor modifications (Cleenwerck et al., 2002) and from strains 444B and 384 by the method of Gevers et al. (2001), except that mutanolysin was substituted by proteinase K [0.0025 g proteinase K ml–1 Tris/HCl (10 mM)+EDTA (1 mM) buffer (pH 8.0)]. Nearly complete sequences were determined using the eight sequencing primers listed by Coenye et al. (1999), except for strain R-29337T where primer BKL1 (position 536–516 according to the Escherichia coli 16S rRNA gene sequence numbering; 5'-GTATTACCGCGGCTGCTGGCA-3') was used instead of primer PD. Pairwise similarities between the obtained consensus sequences and 16S rRNA gene sequences from the EMBL database were calculated with the BioNumerics 4.5 software package (Applied Maths) using an open gap penalty of 100 % and a unit gap penalty of 0 %. Phylogenetic trees were constructed with the BioNumerics 4.5 software package using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Felsenstein, 1985) methods. Bootstrapping analysis (Felsenstein, 1985) was undertaken to test the statistical reliability of the topology of the neighbour-joining tree using 1000 bootstrap resamplings of the data.
The neighbour-joining tree (Fig. 1) was slightly different to the maximum-parsimony tree (see Supplementary Fig. S1 in IJSEM Online); however, both trees clearly showed that strains R-29337T, 444B and 384 belonged to the genus Acetobacter and, more precisely, to the subcluster containing A. lovaniensis, Acetobacter syzygii, Acetobacter peroxydans, A. pasteurianus and Acetobacter pomorum (Lisdiyanti et al., 2001; Cleenwerck et al., 2002; Dutta & Gachhui, 2006). The 16S rRNA gene sequence similarities obtained by pairwise alignment showed that isolates R-29337T, 444B and 384 were most closely related to each other (100 %) and to A. syzygii (99.7 %) and A. lovaniensis (99.5 %); lower similarities were observed with A. pomorum (98.1 %), A. peroxydans (97.9 %), Acetobacter orientalis (97.8 %), A. pasteurianus (97.7), Acetobacter cibinongensis (97.7 %), Acetobacter tropicalis (97.7 %), Acetobacter indonesiensis (97.7 %), A. aceti (97.6 %), Acetobacter estunensis (97.4 %), Acetobacter oeni (97.3 %), Acetobacter cerevisiae (97.2 %), Acetobacter malorum (97.1 %), A. orleanensis (96.8 %) and Acetobacter nitrogenifigens (96.4 %).
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with minor modifications (Cleenwerck et al., 2002). Only high-molecular-mass DNA with A260/A280 and A234/A260 absorption ratios of 1.8–2.0 and 0.40–0.60, respectively, was used. DNA–DNA hybridizations were performed according to a modification (Goris et al., 1998; Cleenwerck et al., 2002) of the microplate method described by Ezaki et al. (1989). The hybridization temperature was 46 °C. Reciprocal reactions (e.g. AxB and BxA) were performed and their variation was within the limits of this method (Goris et al., 1998). The DNA binding values reported are mean values of at least four hybridization experiments, including the reciprocal reactions. Strain R-29336 showed high DNA–DNA hybridization values with strains R-29337T and R-29338 (99 and 88 %, respectively) and intermediate DNA binding values with the type strains of A. syzygii LMG 21419T (46 %) and A. lovaniensis LMG 1617T (47 %), phylogenetically the most closely related Acetobacter species. Low DNA–DNA hybridization values were found with A. pomorum LMG 18848T (18 %), A. peroxydans LMG 1635T (18 %), A. orientalis LMG 21417T (11 %), A. pasteurianus LMG 1262T (20 %), A. cibinongensis LMG 21418T (11 %), A. tropicalis LMG 19825T (11 %), A. indonesiensis LMG 19824T (14 %), A. aceti LMG 1504T (5 %), A. estunensis LMG 1626T (10 %), A. oeni LMG 21952T (6 %), A. cerevisiae LMG 1625T (15 %), A. malorum LMG 1746T (9 %), A. orleanensis LMG 1583T (13 %) and A. nitrogenifigens LMG 23498T (4 %). These DNA–DNA hybridization data prove that the cocoa isolates represent a novel genospecies within the genus Acetobacter (Wayne et al., 1987; Stackebrandt et al., 2002).
The DNA base composition of strains R-29336, R-29337T and R-29338 was determined by HPLC according to the method of Mesbah et al. (1989). Non-methylated phage 
DNA (Sigma) was used as the calibration reference. The G+C content ranged from 56.9 to 57.3 mol%, which is consistent with DNA G+C contents of members of the genus Acetobacter (Lisdiyanti et al., 2000, 2001; Cleenwerck et al., 2002; Silva et al., 2006).
Isolates R-29336, R-29337T and R-29338 were tested for their ability to grow on the carbon sources glycerol (0.3 %), maltose (0.3 %), methanol (0.3 %) and ethanol (10 %), and on yeast extract with 30 % glucose as described previously (Cleenwerck et al., 2002). The utilization of ammonium as the sole nitrogen source with ethanol as carbon source was also tested as described previously (Cleenwerck et al., 2002). The three novel isolates were able to grow on glycerol (weakly), but not on maltose or methanol as carbon source. Growth on ethanol was variable (strain R-29337T was negative). All isolates grew on yeast extract with 30 % glucose, but were unable to grow with ammonium as sole nitrogen source and ethanol as carbon source. Phenotypic differentiation from phylogenetically related Acetobacter species is summarized in Table 1. The isolates differed from A. syzygii mainly by their growth on yeast extract with 30 % glucose, but not on maltose. They differed from A. lovaniensis by their inability to produce 2-ketogluconic acid from glucose, and their growth on yeast extract with 30 % glucose, but not on methanol or on ethanol with ammonium as nitrogen source.
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Description of Acetobacter ghanensis sp. nov.
Acetobacter ghanensis (gha'nen.sis. N.L. masc. adj. ghanensis pertaining to Ghana, referring to the country where the first isolates of this species were obtained).
Cells are Gram-negative rods, approximately 0.8 µm wide and 1.5–2.5 µm long. Cells occur singly or in pairs. Oxidase-negative. Catalase-positive. Strictly aerobic. On MYP agar, colonies are beige, round, convex, smooth and shiny and approximately 0.8 mm in diameter after incubation at 28 °C for 4 days. Ethanol is oxidized to acetic acid. Gluconic acid is produced from glucose. Unable to produce 2-ketogluconic acid, 5-ketogluconic acid or 2,5-diketogluconic acid from glucose. C18 : 17c is the predominant fatty acid (approx. 50 %); other fatty acids in significant amounts are C16 : 0 (10–16 %), C16 : 0 2-OH (9–11 %), C14 : 0 2-OH (5–7 %), C14 : 0 (4–5 %) and C19 : 0 cyclo 8c (5–10 %). Able to grow on yeast extract with 30 % glucose, on glycerol (although weakly), but not on maltose or methanol. Unable to grow with ammonium as sole nitrogen source and ethanol as carbon source. The G+C content of the DNA varies from 56.9 to 57.3 mol%. The DNA G+C content of the type strain is 57.3 mol%. Can be differentiated genotypically from other species of the genus Acetobacter by DNA–DNA hybridization and (GTG5)-PCR fingerprinting.
The type strain, strain R-29337T (=430AT=LMG 23848T=DSM 18895T), was isolated from a traditional heap fermentation of Ghanaian cocoa beans.
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ACKNOWLEDGEMENTS |
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