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Acetobacter tropicalis in spontaneously

fermented wines with vinegar fermentation in
Austria

Article in Mitteilungen Klosterneuburg · January 2006

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Mitteilungen Klosterneuburg 56 (2006): 102-107

Acetobacter tropicalis in spontaneously fermented

wines with vinegar fermentation in Austria

KARIN SILHAVY and KARIN MANDL

HoÈhere Bundeslehranstalt und Bundesamt fuÈr Wein- und Obstbau
A-3400 Klosterneuburg, Wiener Strasse 74
E-mail: Karin.Silhavy@hblawo.bmlfuw.gv.at

Acetic acid bacteria are known for their ability to spoil wines irreversibly. To get to know the species diversity in Au-
stria, 84 bacterial strains were isolated from spontaneously fermented wines with following acetic fermentation. The
strains were examined with biochemical and molecular-biological methods such as RFLP analysis of PCR-amplified
DNA fragments, for their affiliation to the family of acetic acid bacteria. Furthermore, the DNA of some of the
strains were sequenced. One of the isolated strains showed 99% similarity in the sequenced 16S rDNA region of
the type strain of Acetobacter tropicalis.
Keywords: Wine, acetic acid bacteria, DNA analysis, 16S rDNA, Acetobacter tropicalis

Acetobacter tropicalis in spontan vergorenem oÈsterreichischen Wein mit anschlieûender EssigsaÈuregaÈrung. Essig-
È ster-
saÈurebakterien sind bekannt dafuÈr, dass sie Wein irreversibel verderben koÈnnen. Um die ArtendiversitaÈt in O
reich kennen zu lernen, wurden 84 BakterienstaÈmme aus spontan vergorenem Wein mit anschlieûender EssigsaÈure-
fermentation isoliert. Die StaÈmme sind mit biochemischen und molekularbiologischen Methoden, wie RFLP-Ana-
lyse von PCR-amplifizierten DNA-Fragmenten, auf ihre ZugehoÈrigkeit zur Familie der EssigsaÈurebakterien unter-
sucht worden. Weiters wurde die DNA von einigen StaÈmmen sequenziert. Einer dieser StaÈmme zeigte eine 99%
ige UÈ bereinstimmung der sequenzierten 16S rDNA Region mit der des Typstammes von Acetobacter tropicalis
SchlagwoÈrter: Wein, EssigsaÈurebakterien, DNA-Analyse, 16S rDNA, Acetobacter tropicalis

La deÂtection en Autriche d'Acetobacter tropicalis dans du vin fermente spontaneÂment et transforme en vinaigre.
Afin d'eÂtudier la population de bacteÂries aceÂtiques dans des vins autrichiens, 84 souches bacteÂriennes ont eÂte isoleÂes
de vins fermenteÂs spontaneÂment puis soumis aÁ une fermentation aceÂtique. L'appartenance des souches aÁ la famille
des bacteÂries aceÂtiques a eÂte eÂtudieÂe par des meÂthodes biochimiques et biomoleÂculaires, telles que l'analyse RFLP
de fragments d'ADN amplifieÂs par PCR. En outre, l'ADN de quelques souches ont eÂte seÂquenceÂes. La reÂgion seÂ-
quenceÂe 16S rADN d'une de ces souches preÂsentait une concordance de plus de 99 % de avec celle de la souche
type d'Acetobacter tropicalis.
Mots cleÂs : vin, bacteÂries aceÂtiques, analyse d'ADN, 16S rADN, Acetobacter tropicalis

Acetic acid bacteria taxonomically belong to the family ginning from the later stage of alcoholic fermentation
Acetobacteraceae. This family consists of 18 genera because they prefer alcohol as a carbon source (DE
(GARRITY et al., 2004). The three genera Acetobacter, LEY et al., 1984).
Gluconacetobacter and Gluconobacter with the strains Acetic acid bacteria are aerobic, therefore their growth
Acetobacter aceti, A. pasteurianus, Gluconacetobacter is inhibited during fermentation because of lack of oxy-
hansenii, Ga. liquefaciens, Ga. xylinus and Gluconobac- gen and presence of free sulphur dioxide. But there are
ter oxydans are those which are mostly responsible for studies (DRYSDALE and FLEET, 1989) which report that
wine spoilage. G. oxydans is mainly found on sound, they are able to survive under such conditions. If there
undamaged grapes and in juice, because it prefers su- is a small amount of oxygen, then they will start with
gar-rich substrates. Acetobacter and Gluconacetobacter, the production of acetic acid. They are also known to
however, are found on damaged grapes or in wines be- produce other compounds besides acetic acid, for ex-

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Mitteilungen Klosterneuburg 56 (2006): 102-107 Silhavy et al.

ample dihydroxyaceton from glycerol, which may in- The activity of catalase was determined by observing
fluence the quality of wine (DRYSDALE and FLEET, 1989). the production of oxygen after addition of 5% hydro-
The traditional characterisation procedure with bioche- gen peroxide. Ketogenesis from glycerol was tested on
mical tests is not completely trustworthy for the un- glycerol-agar (2% glycerol, 2% agar) using the method
equivocal identification of acetic acid bacteria. There- of BACK (2000). Over-oxidation from acetic acid to car-
fore nowadays these methods are complemented by bondioxide and water was conducted on Frageur-me-
different molecularbiological techniques such as dium (1% yeast extract, 2% CaCO3, 2% (w/v) ethanol
DNA-DNA hybridization (SIEVERS et al., 1992), se- 96%, 2% agar) (DE LEY et al., 1984) and on Carr-me-
quence analysis (YAMADA et al., 1997), RFLP of PCR dium (3% yeast extract, 2% (w/v) ethanol (96%),
amplified 16S rDNA (POBLET et al., 2000; RUIZ, 2000), 0.0022% bromocresol green, 2% agar). The ability of
SDS-PAGE numerical analysis of total cell proteins using mannitol as carbon source was tested on YPM
(DU TOIT and LAMBRECHTS, 2002), and RAPD-PCR medium (0.5% yeast extract, 0.3% peptone, 2.5% man-
(BARTOWSKY et al., 2003). nitol, 1.5% agar) (DE LEY et al., 1984). The oxidation of
For this study phenotypic tests as well as PCR-RFLP glucose to gluconic acid was detected on modified
(Restriction fragment length polymorphism) of the 16S GYC medium. The capability to produce acid from D-
and the 16S-23S ITS (Internal Transcribed Spacer) re- fructose was tested with an Acetobacter medium (1%
gion and sequence analysis of the 16S region were used. yeast extract, 1% D-fructose, 0.004% bromocresol pur-
ple) (BACK, 2000).

Material and Methods Extraction of DNA

The genomic DNA of the bacterial strains was isolated
with the ¹GFX Genomic Blood DNA Purification
Bacterial strains and cultivation Kitª from Amersham Biosciences. The quantification
Additionally the new isolates the following type strains of the extracted DNA was done by electrophoresis in
from DSMZ (Deutsche Sammlung von Mikroorganis- a 1% (w/v) agarose gel in TBE-buffer. The ethidium
men und Zellkulturen GmbH, Braunschweig, Ger- bromide stained DNA was visualized under UV light,
many) were used: photographed and compared with commercial available
Gluconobacter sp. (DSM 3504) length standards (SAMBROOK and RUSSELL, 2001).
Gluconobacter oxydans subsp. suboxydans (DSM 50049)
Acetobacter aceti (DSM 3508) Primer Design
Acetobacter pasteurianus (DSM 3509) The primers were designed with the help of the pro-
Gluconacetobacter hansenii (DSM 5602) gram Primer3 (ROZEN and SKALETSKY, 2000). The origi-
Gluconacetobacter liquefaciens (DSM 5603) nal sequence (S000380829) was that of the 16S rDNA
Gluconacetobacter xylinus (DSM 2325) of E. coli (BROSIUS et al., 1978). Two new primers
(615R and 1358F) were chosen, after they had been ve-
Isolation of strains rified in the database of the RDP (Ribosomal Database
After finalisation of the alcoholic fermentation samples Project-II, Probe Match) (COLE et al., 2005).
were taken and incubated at 288C. During the acetic
fermentation samples were drawn and plated on diffe- PCR-RFLP-analysis of the 16S rDNA and
rent culture media (see ¹Phenotypic analysisª). The the ITS between 16S and 23S
plates also were incubated at 288C for ten days. Repre- The PCR-RFLP analysis of the 16S rRNA gene was
sentative colonies were purified by repeated streaking done as published by POBLET et al. (2000).
on AAB-medium (1.5% malt extract, 0.5% yeast ex- The ITS region between 16S and 23S rRNA was ampli-
tract, 1.5% agar, 3% ethanol) (BACK, 2000) and modi- fied by PCR with the following primer: Its1Ac (RUIZ,
fied GYC-medium (1% yeast extract, 2% D-glucose, 2000) and 488R (TRCÏEK, 2002). The amplification of an
2% CaCO3, 2% agar) (TREK, 2002). aliquot of DNA was performed in a 50 ml reaction mix-
ture containing 10x PCR buffer, 20 pmol forward pri-
Phenotypic analysis mer, 20 pmol reverse primer, 100 mM of each dNTP
The Gram behaviour was tested by applying the potas- (Sigma, Steinheim, Germany) and 2.5 units Taq DNA
sium-hydroxide method as described by BACK (2000). polymerase (Eppendorf, Germany) in a MasterCycler1

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Mitteilungen Klosterneuburg 56 (2006): 102-107 Silhavy et al.

gradient (Eppendorf, Germany) and the following tem- CGGGGATTTCACATCTGACT-3', position 615
perature program: 948C for 5min, following sequence through 596), Ac3 (POBLET et al., 2000), 785F (SEE-
which is repeated 30 times: 948C for 30s, 658C for ARUNRUANGCHAI et al., 2004), 1358F (5'- TCAG(A/
1min and 728C for 2min and finally 728C for 8min. Af- C)ATGCC(A/G)CGGTGAATA-3', position 1358
terwards the PCR products were digested with the en- through 1377), 1541R (SEEARUNRUANGCHAI et al.,
zyme TaqI and loaded on a 2.5% (w/v) agarose gel for 2004).
fragment size analysis. As size marker a 100 bp DNA
ladder (New England Biolabs, Frankfurt, Germany) Results
was used.
From the five wines 84 strains have been isolated, and
Amplification and sequencing of the were incubated at 288C. They were examined with bio-
16S rRNA gene chemical and molecular-biological methods for their af-
An aliquot of the rDNA was amplified in a 50 ml filiation to the family of the acetic acid bacteria. After
reaction mixture containing 10x PCR buffer, 20 pmol completion of the biochemical tests the results showed,
forward primer, 20 pmol reverse primer, 100 mM of that all strains were Gram negative and catalase-posi-
each dNTP (Sigma, Steinheim, Germany) and 2.5 tive rods, which belong to the family of the acetic acid
units Taq DNA Polymerase (Eppendorf, Hamburg, bacteria. All strains had the ability to oxidize ethanol
Germany). The following two primers were used for to acetic acid and further to carbon dioxide and water.
the amplification of the 16S rDNA: 1F (TRCÏEK, 2002) Therefore the strains belong either to the genus Aceto-
and 1541R (SEEARUNRUANGCHAI et al., 2004). The bacter or Gluconacetobacter.
reactions were performed in a MasterCycler1 Gra- Due to the results of the other tests described in Mate-
dient (Eppendorf, Hamburg, Germany) and the samp- rial and Methods, 37 strains belonged to Acetobacter
les were incubated at 948C for 5min to denature the aceti, Gluconacetobacter liquefaciens or Gluconaceto-
target DNA and then cycled 30 times at 948C for bacter xylinus. A more precise statement was not possi-
30sec, 628C for 1min and 728C for 2min. For final ble, because these three species could not be distinguis-
extension the samples were incubated for 8min at hed with the applied phenotypic tests made. Thirty two
728C. The PCR products were purified using the Se- strains belonged to Acetobacter pasteurianus, five to
phaglasTM BandPrep Kit (Amersham Biosciences), ac- Gluconacetobacter hansenii and ten could not be assi-
cording to the manufacturers instructions. The puri- gned to a certain species (Tab.1).
fied DNA was quantified after electrophoresis on an The results of the PCR with the primer combination
ethidium bromide stained 1% (w/v) agarose gel with Ac1/Ac3 showed with all samples a unique PCR frag-
the UVP Labworks software (UVP, Cambridge, UK). ment of 869 bp as described by POBLET et al. (2000).
The sequencing was done by the company Ibl (Vi- This is the characteristic fragment length for acetic
enna, Austria) on an ABI PRISMTM model 3100 Ge- acid bacteria. After digest with the enzyme TaqI most
netic Analyzer with the ABI PRISMTM Big Dye Ter- of the strains could be assigned to Gluconacetobacter
minator Cycle Sequencing Kit. The following seven hansenii (results not shown).
primers were used: 9F (SEEARUNRUANGCHAI et al., The PCR-RFLP-analysis of the ITS region showed a
2004), 520F (YUKPHAN et al., 2004), 615R (5'- completely different result. None of the patterns corre-

Tab. 1: Phenotypic characteristics used for the identification of the acetic acid bacterial isolates

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Mitteilungen Klosterneuburg 56 (2006): 102-107 Silhavy et al.

Fig.1: PCR-RFLP of the ITS-region

sponded with those of the reference strains (Fig.1). Discussion

Therefore the samples with different restriction pat- The result of the biochemical tests showed that the iso-
terns were sequenced. The sequences were aligned lated strains from wines with acetic acid fermentation
with the BioEdit Sequence Alignment Editor (HALL, only belonged to the genus Acetobacter or Gluconace-
1999) and the obtained sequences were put into Mega- tobacter. This result was expected, since Gluconobacter
BLAST program (http://www.ncbi.nlm.nih.gov/blast/ occurs mainly on sound grapes and in juice and is rarely
megablast.shtml) for comparison with the known bac- found in wine, because wine does not belong to its pre-
teria sequences. One of these strains could be identified ferred nutritive solutions due to the low sugar content.
as Acetobacter tropicalis. Acetobacter tropicalis is an until quite recently un-
known species of the genus Acetobacter. This species

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Mitteilungen Klosterneuburg 56 (2006): 102-107 Silhavy et al.

Tab. 2: Results of genetic comparisons of the new strains References

with the MegaBLAST program
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