16 S rRNA in situ Hybridization Followed by Flow Cytometry for Rapid Identi fi cation of Acetic Acid Bacteria Involved in Submerged Industrial Vinegar Production

Acetic acid bacteria are involved in various biotechnological processes, among them the most traditional being the acetic acid production during making of diff erent types of vinegar (1). In some countries, this process has been traditionally performed in wooden vessels with naturally present microbiota that has been identifi ed as Acetobacter aceti and/or Acetobacter pasteurianus (2). In contrast to this process, a quick submerged bioprocess was developed in 1950s, with two main benefi ts: very short oxidation cycle and the production of vinegar with very high acetic acid volume fraction (10 %) (3). In the extreme conditions of such bioprocesses only very well adapted strains of acetic acid bacteria survive, which according to the present knowledge belong to the following species: Komagataeibacter europaeus, Komagataeibacter intermedius, Komagataeibacter oboediens and Gluconacetobacter entanii (4–10). A typical characteristic of these species is a resistance to high percentage of acetic acid in contrast to the species of Acetobacter genus (7). Species of the genus Acetobacter may cause a delay of the oxidation cycle in submerged bioprocess. Therefore, the ability to follow the highly productive and highly acetic acid-resistant bacteria during industrial vinegar production is of importance.


Introduction
Acetic acid bacteria are involved in various biotechnological processes, among them the most traditional being the acetic acid production during making of diff erent types of vinegar (1).In some countries, this process has been traditionally performed in wooden vessels with naturally present microbiota that has been identifi ed as Acetobacter aceti and/or Acetobacter pasteurianus (2).In contrast to this process, a quick submerged bioprocess was developed in 1950s, with two main benefi ts: very short oxidation cycle and the production of vinegar with very high acetic acid volume fraction (10 %) (3).In the extreme conditions of such bioprocesses only very well adapted strains of acetic acid bacteria survive, which according to the present knowledge belong to the following species: Komagataeibacter europaeus, Komagataeibacter intermedius, Komagataeibacter oboediens and Gluconacetobacter entanii (4)(5)(6)(7)(8)(9)(10).A typical characteristic of these species is a resistance to high percentage of acetic acid in contrast to the species of Acetobacter genus (7).Species of the genus Acetobacter may cause a delay of the oxidation cycle in submerged bioprocess.Therefore, the ability to follow the highly productive and highly acetic acid-resistant bacteria during industrial vinegar production is of importance.plied this technique for enumeration of bacteria in vinegar, the approach which showed that the majority of the acetic acid bacteria in the submerged bioprocess are not in the cultivable state under the presently known in vitro growth conditions (8).In this report we extend the fl ow cytometry approach for monitoring of specifi c bacteria, K. europaeus, K. intermedius and K. oboediens during the production of vinegar with high acetic acid percentage.

Construction of specifi c DNA probe
A specifi c oligonucleotide Komag (5'-GAACCTTTC-GGGGTTAGTG-3', position on 16S rDNA: 70-88 nt, numbered according to Komagataeibacter medellinensis, acc.no.NC_016027, locus GLX_r0010) was constructed by comparing all available 16S rRNA gene sequences of acetic acid bacteria, available through National Center for Biotechnology Information (NCBI), consisting of GenBank/ EMBL/DDBJ databases.The specifi city of the oligonucleotide was tested in a standard PCR reaction in combination with the oligonucleotide EUB338rev (5'-GCTGCCTC-CCGTAGGAGT-3') using the following PCR amplifi cation conditions: initial denaturation of DNA at 94 °C for 3 min, 30 cycles at 94 °C for 30 s, at 68 °C for 30 s and at 72 °C for 30 s, and a fi nal extension at 72 °C for 7 min followed by cooling at 4 °C.The amplifi cation was performed in a TProfessional Basic Cycler (Biometra, Gött ingen, Germany) in 20-μL reaction mixture containing 10 ng of DNA, 2.5 mM of MgCl 2 , 20 pmol of each primer, 0.5 U of Taq DNA polymerase (Thermo Fisher Scientifi c, Waltham, MA, USA), 0.2 mM of dNTP (Thermo Fisher Scientifi c) and 2 L of 10× Taq buff er (Thermo Fisher Scientifi c).The specifi c PCR product is 247 bp in length.Based on the sequence of primer Komag, a DNA probe Komag-fl uorescein isothiocyanate (FITC) (5'-CACTAACCCCGAAAGGTTC-3') was constructed.All primers and a DNA probe were provided by MWG Genomics (Munich, Germany).The specifi city of the probe Komag-FITC was tested in silico using the RDP probe match (11) and also practically with reference strains (Table 1) that were obtained from the BCCM/ LMG (Belgian Coordinated Collections of Microorganisms, Brussels, Belgium) culture collection, DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) culture collection and Sugar Research Institute (SRI, Mackay, Australia).

In situ hybridization
A protocol developed by Lipoglavšek and Avguštin (12) for in situ hybridization of ruminal samples was optimized for acetic acid bacteria as follows.The bacteria were grown in liquid reinforced acetic acid and ethanol (RAE) medium (13) in glass tubes at 30 °C by shaking in 10-litre benchtop shaking incubator (Domel, Železniki, Slovenia) at 15×g until logarithmic growth phase was achieved (A 600 nm =0.6-1.0).Then the biomass was harvested in a centrifuge Centric 400R (Domel) at 2000×g and 4 °C for 10 min.The biomass was washed with phosphate-buff ered saline (PBS, pH=7.4) and fi xed in 900 L of 4 % (by mass per volume) paraformaldehyde at 4 °C for 12 h.Subsequently, the cells were washed with PBS buff er (pH=7.4) and suspended in 500 L of ethanol (96 %) and 500 L of PBS buff er (pH=7.4).If the hybridization did not proceed immediately, the samples were stored at -20 °C.The fi xed cells (50 L) were washed and suspended in 12 L of PBS buff er (pH=7.4).A volume of 100 L of hybridization buf-

Flow cytometry analysis
Cells were stained with 1 g/mL of Trypan Blue (Thermo Fisher Scientifi c) in PBS buff er (pH=8.0)for 10 min.Aft er centrifugation (6000×g, 10 min, 4 °C) the cells were suspended in 700 L of PBS buff er (pH=8.0)and analyzed by FACScan fl ow cytometer (Becton Dickinson, Heidelberg, Germany).Prior to analysis, the samples were diluted to the concentration of approx.1000 cells per mL, which was measured by fl ow cytometer in one second.The measurements were performed by CELLQuest v. 3.1 soft ware at Power Macintosh 7300 (Becton Dickinson).The signals were logarithmically amplifi ed.Cells were diff erentiated from noise according to Trypan Blue staining and gates for all cells were created on red fl uorescence (FL3-H)-side scatt er (SSC-H) dot plot.

Results and Discussion
Monitoring the microbiological status in industrial vinegar production is hampered by diffi culties in growing under laboratory conditions the strains of acetic acid bacteria responsible for the production of vinegar with high percentage of acetic acid.Although a progress in growing these strains has been made with description of a double-layer acetic acid and ethanol (AE) agar medium (14) and also RAE agar medium (13), the major part of the bacterial microbiota for industrial vinegar production remains currently in nonculturable form (15,16).Moreover, if bett er cultivation conditions or techniques were known, a problem of time length in growing of the acetic acid bacteria would remain.On the other hand, identifi cation of acetic acid bacteria has been well established over the past few years by using 16S-23S rRNA gene ITS regions as a target for species identifi cation (8,15).To overcome the critical point of isolation of bacteria from diff erent matrices, a fl uorescence in situ hybridization (FISH) with specifi c DNA probes has been proven to be a suitable approach for identifi cation of diverse microbiota (17)(18)(19)(20)(21)(22).Additionally, a combination of FISH technique with fl ow cytometry has oft en been used for identifi cation of food microbiota, especially those originating from liquid matrices (23,24).Therefore, we aimed in this report to develop a specifi c DNA probe for targeting strains for production of vinegar with high percentage of acetic acid from real industrial matrix.
Since the 16S rRNA gene sequences of the acetic acid bacteria show very high similarity to each other, it was impossible to construct 100 % genus-or species-specifi c probe.We constructed a probe that is partially specifi c for genus Komagataeibacter: the probe Komag-FITC is useful for diff erentiation between the species of acetic acid bacteria with very high acetic acid resistance (genus Komagataeibacter) and the species of the genus Acetobacter, Gluconacetobacter and Gluconobacter on the other hand.The exceptions are species A. lovaniensis, A. syzygii, A. ghanensis, A. fabarum and A. okinawensis, which also bind the probe Komag-FITC but have not been identifi ed in wine or apple cider vinegar, which are the predominant vinegars on the European market.The probe also targets another genus of the acetic acid bacteria, i.e.Saccharibacter, but this genus does not resist as low as 0.35 % of acetic acid and is thus not present in vinegar (25).The species of the genus Acetobacter typically show low acetic acid resistance, the feature which is not appreciated in submerged bioprocess during production of vinegar with high percentage of acetic acid (7).
To improve the fl uorescent signal intensity a helper probe was used.This additional unlabelled oligonucleotide opened the rRNA structure and improved the accessibility of the rRNA molecule for the probe by binding upstream or/and downstream of the binding site of the labelled probe (26).Indeed, concomitant application of the FITC-labelled probe Komag-FITC and helper oligonucleotide Komag-up1 (5'-CGTTACTCACCCGTCCGC-3'), binding just upstream in a close proximity of the labelled probe, approximately doubled the intensity of the signal (green fl uorescence, FL1-H; Fig. 1).As the next step we mixed pure cultures of Acetobacter aceti and Komagataeibacter europaeus and proceeded with the above described protocol in order to show that in situ hybridization in combination with fl ow cytometry successfully diff erentiated both genera of the acetic acid bacteria (Fig. 2).The established protocol was then applied on alcohol and wine vinegar samples from industrial sett ings (Fig. 3).Concomitantly with the FITC analysis in combination with fl ow cytometry, the samples were used for direct identifi cation of microbiota using the restriction analysis of the PCR-amplifi ed 16S-23S rRNA gene as described before (8).This approach proved that in both types of vinegar the microbiota is homogenous and composed of a single species (data not shown).However, as noticed in Fig. 3, the applied protocol did not successfully label the

Conclusions
In this work we established a protocol based on in situ hybridization in a combination with fl ow cytometry for quick following of high acetic acid resistance strains of the acetic acid bacteria responsible for an effi cient industrial bioprocess of vinegar production.Because of high similarities among species of the genera Acetobacter, Gluconacetobacter and Komagataeibacter, a construction of a specifi c probe for a single genus was not possible.Nevertheless, the probe Komag-FITC binds to species Komagataeibacter europaeus, Komagataeibacter oboediens and Komagataeibacter intermedius, all typical species for industrial submerged processes for vinegar production with more than 6 % of acetic acid, but not to Acetobacter aceti and Acetobacter pasteurianus, which are typical species for production of vinegar with less than 6 % of acetic acid.The protocol for in situ hybridization followed by fl ow cytometry analysis enables a convenient microbiological monitoring of vinegar.Because of high 16S rRNA gene sequence similarities among species of the genera Acetobacter, Gluconacetobacter and Komagataeibacter, other target molecules should be tested in the future.

Fig. 1 .
Fig. 1.Improvement of signal intensity of probe Komag-FITC with concomitant binding of the helper oligonucleotide.The analysis was performed with a pure culture of Komagataeibacter europaeus.Light grey line represents signal intensity of the labelled probe Komag-FITC and the dark grey line the signal intensity of the labelled probe Komag-FITC with bound helper oligonucleotide Komag-up1.FL1-H=green fl uorescence

Table 1 .
List of strains used for the analysis of the specifi city of probe Komag-FITC.As a control, a probe EUB338-FITC was applied L of the FITC-labelled probe (25 pmol/L) (MWG Genomics) were added to the cells and hybridized at 54 °C for 12 h.This hybridization temperature was selected because the results showed that it enabled diff erentiation among the highest number of target species of the genus Komagataeibacter and the non-targeted species of genera Acetobacter, Gluconacetobacter and Gluconobacter.Unbound probe was washed away with 500 L of PBS buff er (pH=8.0).