Species Composition and Phylogenetic Diversity of Acetic Acid Bacteria Communities in Homemade Vinegars

Abstract

Despite their significance, the diversity of acetic acid bacteria (AAB) in homemade vinegars remains understudied. This study aimed to explore the species-level diversity of AAB in homemade vinegars and to assess their community structure to better understand this microbial niche. To investigate the diversity of these bacteria, we employed recently established customized 16S-23S rDNA internal transcribed spacer (ITS) amplicon metagenomics to identify AAB at the species level. By applying Hill numbers, we calculated species richness, relative abundance, and dominance, providing a clearer understanding of the community structure of AAB in 11 homemade vinegars. Although species richness was relatively high, both relative abundance and dominance were considerably lower, suggesting a community structure dominated by a few highly abundant taxa, with most species being rare or low in abundance. The most dominant genera across most samples were Komagataeibacter and Acetobacter, both of which are known for their roles in oxidative fermentation. Several previously unreported, potentially novel species of AAB were identified, along with two potentially novel genera. This is one of the first studies to examine the diversity of AAB in homemade vinegars using a culture-independent amplicon metagenomic approach. Our findings suggest that the microbiota of homemade vinegars remains an underexplored niche and a source for novel species with biotechnological potential. The results provide valuable baseline data for future microbial studies and may help in the development of novel, customized starter cultures for the improvement and standardization of vinegar production.

1. Introduction

Acetic acid bacteria (AAB) have a variety of functions and are used in many industrial processes, including the production of bacterial nanocellulose, vitamin C, kombucha, kefir, acetans, and acetan-like polysaccharides. However, their most well-known and traditional application remains the production of vinegar, which relies on their unique oxidative fermentation that transforms a variety of substrates into valuable products [1,2,3,4,5]. Vinegars are known for their antimicrobial, antidiabetic, antioxidant, anti-obesity, and antihypertensive properties, which they owe to their polyphenols, micronutrients, and other bioactive compounds. As well as being used in the human diet, they have traditionally been used as natural remedies in various cultures [6].

Both homemade and industrial-scale production processes can use vinegar oxidative fermentation, which can proceed spontaneously or be initiated through back-slopping (using a bit of the previous batch to start a new fermentation) or the use of starter cultures [7,8]. The quality of vinegar depends on several factors, including the raw material and production method [9], technological process, contact with wood, and aging, with AAB species also playing an important role [10]. The composition of the microbial community in vinegar is influenced by both the fermentation stage [11] and the season in which the sample is collected [12]. It also depends on other environmental factors, such as ethanol concentration, acidity, temperature, microbial interactions, and oxygen availability [13]. The distinct flavors of vinegar are the outcome of the unique metabolism of specific microorganisms [13,14]. Understanding which microorganisms are present in vinegar is therefore essential [15].

Advances in microbiology over the past decades have been driven by a shift from culture-based methods to culture-independent ones, such as 16S rRNA gene sequencing. These approaches enable large-scale analysis of microbial communities [16]. When studying AAB, it is common to encounter the challenge of the high similarity of 16S rRNA gene sequences among species [17,18,19]. This limits the ability to distinguish them reliably at the species level, with identification typically only possible at the genus level. To overcome this limitation, a customized 16S-23S rDNA ITS amplicon metagenomics was developed and validated for species-level identification of AAB in vinegars and kombuchas. This method focuses on the ITS regions between the 16S and 23S rRNA genes, which serve as high-resolution markers for accurately distinguishing AAB species [14].

Understanding microbial diversity has become essential, yet the selection of diversity indices remains context-dependent, with no single approach universally superior. Among the available methods, Hill-based indices are well-established and provide a concise and consistent framework for measuring dissimilarity between microbial communities [20,21]. Nevertheless, Hill numbers also have certain limitations: they do not account for functional traits, and their sensitivity to species abundance depends on the choice of the parameter q, which influences the interpretation of diversity. In our study, we did not apply a functional diversity approach, as functional traits for AAB are not yet systematically defined or standardized in the context of vinegar microbiota. While some traits, such as cellulose production or acid tolerance, are known for specific strains, a comprehensive trait-based classification is currently lacking. Therefore, Hill numbers were selected as the most appropriate tool for assessing taxonomic and phylogenetic diversity in this context [20,22,23,24].

Hill numbers offer a unified and adaptable framework for measuring biodiversity, integrating species richness and evenness into a single, parametrized system. Unlike traditional indices that usually focus on just one aspect, Hill numbers use the parameter q to adjust the sensitivity of the measure to species abundances [25]. The parameter q determines how the index responds to differences in species abundances: when q = 0 (q0), all species are weighted equally, and the Hill number corresponds to species richness; when q = 1 (q1), species are weighted proportionally to their abundance, yielding the exponential of Shannon entropy; and when q = 2 (q2), dominant species are emphasized, corresponding to the inverse Simpson’s concentration index [21,26,27].

Biodiversity can be assessed through different dimensions. Taxonomic diversity reflects both species richness and relative abundances, whereas phylogenetic diversity considers species abundance and their evolutionary relationships [28]. In this study, Hill numbers were applied to both the taxonomic and the phylogenetic dimensions of AAB diversity, providing a more comprehensive understanding of AAB within vinegar communities. Phylogenetic Hill numbers, in particular, help determine whether bacterial species originate from a broad evolutionary spectrum or are clustered within a few lineages.

Studying microbial communities in vinegars is important because these products harbor AAB species that play a vital role in vinegar production and contribute to the synthesis of valuable bioactive compounds. A recent study isolated novel species from homemade vinegar, including members of the genera Komagataeibacter and Novacetimonas, both of which demonstrated the ability to produce bacterial nanocellulose. In addition, metagenomic analyses have revealed several previously undescribed AAB species that may have significant industrial potential [29], while also contributing to the preservation of microbial heritage [30].

Here we have investigated the alpha diversity of AAB in homemade fruit vinegars using a customized 16S-23S rDNA ITS amplicon metagenomic approach. We hypothesized that homemade vinegars, due to their spontaneous or semi-controlled fermentation processes, harbor more diverse and potentially novel AAB communities compared to commercial vinegars. By applying Hill numbers, we sought to quantify species richness, evenness, and dominance and to assess whether the microbial communities are composed of closely related taxa or span broader evolutionary lineages. This approach provides insight into the ecological complexity of vinegar microbiota and identifies candidate species with potential biotechnological relevance.

2. Materials and Methods

2.1. Samples and 16S-23S rDNA ITS Amplicon Metagenomic Analysis

Table 1. Overview of vinegar samples, their location of sampling, and their characteristics.

No.	Sample	Origin	Raw Material	Type of Production	Type of Vessel	pH
1	Mixed1	Lovrenc na Pohorju	Mixed fruit	Spontaneously	Plastic barrel	3.6
2	Blackcurrant1	Lovrenc na Pohorju	Blackcurrant	Back-slopping	Glass vessel	2.9
3	Apple1	Lovrenc na Pohorju	Apple	Spontaneously	Plastic barrel	3.4
4	Apple2	Vurberk	Apple	Back-slopping	Plastic barrel	3.2
5	Apple3	Lovrenc na Pohorju	Apple	Spontaneously	Wooden barrel	2.9
6	Honey1	Videm pri Ptuju	Honey	Added pre-culture	Stainless steel barrel	2.8
7	Mixed2	Pesnica pri Mariboru	Mixed fruit	Back-slopping	Plastic barrel	3.6
8	Apple4	Lovrenc na Pohorju	Apple	Back-slopping	Wooden barrel	3.2
9	Blackberry1	Lovrenc na Pohorju	Blackberry	Back-slopping	Glass vessel	2.8
10	Apple5	Lovrenc na Pohorju	Apple	Back-slopping	Wooden barrel	3.3
11	Vine1	Lovrenc na Pohorju	Wine	Back-slopping	Plastic barrel	3.2

lists the vinegar samples used in the metagenomic analysis of the amplified 16S-23S rDNA ITS intergenic regions, as well as the other characteristics of the samples (origin, raw material used for production, type of production, vessel type, and pH of the vinegar). The samples were collected in north-eastern Slovenia. While the geographic distribution is centered around Lovrenc na Pohorju, additional sampling sites (i.e., Vurberk, Videm pri Ptuju, Pesnica pri Mariboru) were included to introduce spatial diversity. All samples were provided by individuals who produce vinegar on a small scale for household use, except for the sample Honey1, which is also sold commercially. Samples Blackcurrant1, Blackberry1, and Apple3 all originated from the same vinegar producer. The microbial culture from Apple3 was used as a starter culture to produce the other two samples. Similarly, the Apple4 and Vine1 vinegars were obtained from the same producer. The microbial culture from the Apple4 vinegar was transferred to the substrate used to produce the Vine1 vinegar. All samples were collected at the end of the vinegar production process, during the aging stage. The samples were transferred into sterile flasks with 0.2 µm three-hole membrane screw caps (DWK Life Sciences, Mainz, Germany) before being taken to the laboratory. This setup allowed for oxygen exchange while preventing external contamination.

DNA extraction was performed as described by Ribič and Trček [14]. In brief, the bacterial cells were pelleted at 10,000 rpm for 10 min, washed with 0.85% NaCl, and the DNA was extracted using a NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) according to the protocol for Gram-negative bacteria. The sequencing was performed at Novogene (Cambridge, UK). The ITS region, located between the 16S and 23S rDNA, was targeted using custom-designed primers based on the conserved tRNA^Ala coding region. Primer pair SpaFw/tAla2Rev was used. The Blast algorithm at NCBI was then used to analyze the sequences with a relative abundance of ≥0.01%.

2.2. Taxonomic Diversity of AAB

At Novogene, raw reads were demultiplexed, barcodes and primers were removed, and paired-end reads were merged. Quality filtering was performed in QIIME2 (v2020.6) using VSEARCH (v2.30.1.). Chimeras were detected with UCHIME (v4.2.40) against the Gold database and removed. ITS sequences were clustered into OTUs at 97% similarity using UPARSE (v7.0.1001).

Taxonomic assignments based on BLAST (v2.15.0) analysis of 16S-23S rDNA ITS sequences were used to assess species and phylogenetic diversity. AAB species showing less than 94% sequence identity in a sequence length of 230–357 bp were considered potential novel species. This threshold has been previously validated for distinguishing AAB species, as all additional taxonomic parameters, such as average nucleotide identity (ANI) and in silico DNA-DNA hybridization, confirmed the novel species status [29,31]. The ITS percentage identity lower than 75% in a sequence length of 350 to 337 bp was considered as a potentially novel genus of AAB. The sequences are presented in Tables S1–S11.

To assess taxonomic diversity, Hill numbers were calculated using the hillR package [27] in RStudio (v2025.05.0). A rectangular species-by-sample matrix was constructed, with each cell representing the relative abundance of a given AAB species in a specific vinegar sample. This matrix served as the input for the hill_taxa() function to compute Hill numbers of orders q = 0, 1, and 2, corresponding to species richness (S), the exponential of Shannon entropy (exp(H’)), and inverse Simpson’s concentration index (1/λ), respectively. This approach enabled the evaluation of species richness, evenness, and dominance within AAB communities across samples.

To incorporate evolutionary relationships into diversity analysis, the hill_phylo() function from the hillR package was used. In addition to the abundance matrix, a phylogenetic tree in Newick format, representing the evolutionary distances among the identified AAB species, was provided as input. Phylogenetic Hill numbers (q0, q1, q2) were then calculated, capturing both the diversity of lineages present and their relative abundances, thereby providing insights into the evolutionary breadth of the microbial communities.

The phylogenetic tree was reconstructed using MEGA11 [32], applying the maximum likelihood method based on the generalized time-reversible (GTR) model with gamma-distributed rates among sites. The tree was exported in Newick format and used to calculate phylogenetic diversity metrics.

Graphical representations of diversity metrics and species composition were generated using R packages ggplot2 [33], pheatmap [34], and tidyverse [35] in RStudio [36]. Bar plots were used to display Hill numbers across samples, while heatmaps illustrated the relative abundance of AAB species and their distribution among samples. Dendrograms based on Bray–Curtis dissimilarity were included to visualize clustering patterns in microbial community composition. These visualizations facilitated the interpretation of diversity patterns and highlighted differences among vinegar samples.

3. Results and Discussion

3.1. Species Diversity Detected with 16S-23S rRNA ITS Sequencing

As shown in

Table 2. Species diversity across samples based on Hill numbers. The table presents the calculated diversity values for each sample: species richness (q0), the exponential of Shannon entropy (q1), and the inverse Simpson’s concentration index (q2).

Sample	q0	q1	q2
Mixed2	21	11.49	8.91
Apple1	13	6.73	5.18
Apple4	16	3.60	2.03
Vine1	15	5.58	3.65
Apple3	9	4.65	3.42
Apple5	9	4.10	3.20
Honey1	7	2.65	1.87
Blackberry1	6	1.77	1.59
Mixed1	4	2.42	1.97
Blackcurrant1	4	2.08	2.02
Apple2	2	1.01	1.00

and Figure 1, the highest species richness (q0) was observed in Mixed2 vinegar, with 21 species detected. This was followed by Apple4 vinegar, with 16 species detected. There was a relatively large range between the highest and the lowest species richness, with only 2 species present in Apple2 vinegar and 4 in Mixed1 and Blackcurrant1. Previous studies using the same method identified 9 species in commercial elderflower vinegar, 8 in commercial apple cider vinegar, and 5 in commercial wine vinegar [14], as well as 17 species in homemade pear vinegar and 12 species in homemade apple-grape vinegar [29]. Previously reported richness values ranged from 5 species in commercial wine vinegar to 17 in homemade pear vinegar, demonstrating significant variability between different types of vinegar. As data are only available for three commercial vinegar samples, containing between 5 and 9 species, it is not possible to conclude that species richness is more consistent in commercial vinegars than in homemade vinegars. These observations are descriptive and should be interpreted with caution, as they are based on limited sampling and heterogeneous production conditions. Nevertheless, the expectation of greater consistency in commercial processes is supported by standardized fermentation protocols and the frequent use of defined starter cultures. Further research in this field is needed, as studies focusing on commercial vinegars and comparing microbial communities across different fermentation methods remain extremely limited. Using defined starter cultures enables faster, more efficient, and consistent fermentation [37], which allows for the standardization of the taste and quality of the final product. It also enables control over the diversity of the microbial community [38] and improves process control and hygiene [39]. Therefore, this research may support the future development of starter cultures, enabling more controlled and consistent fermentation processes across different types of vinegar production.

Figure 2 shows a heatmap illustrating the relative abundance of AAB species detected in the analyzed vinegar samples. Darker colors indicate higher relative abundances, while gray denotes the absence of a species in a sample. A total of 59 different AAB species were identified across all samples. The heatmap shows that one species, Komagataeibacter melaceti, stands out due to its exceptionally high abundance. This species was only detected in the Apple2 sample, accounting for 99.7% of the bacterial community. Although such dominance is unusual in spontaneous fermentations, in this particular sample K. melaceti may have encountered a highly favorable substrate and fermentation environment, allowing it to outcompete other strains. Other AAB species that were more commonly shared across samples included Komagataeibacter xylinus, found in 6 out of 11, Komagataeibacter saccharivorans, detected in 7 out of 11 samples, and Novacetimonas maltaceti, present in 5 out of 11 vinegars. Despite some species being present in multiple samples, no single species was found in all samples, indicating a high degree of variability in microbial composition among the different vinegars.

As shown in Figure 2, there are also numerous potentially novel species from the AAB group that have yet to be isolated and characterized. Altogether, there are 11 novel species from the Acetobacter genus, 13 from the Komagataeibacter genus, 1 from the Novacetimonas genus, and 1 from the Gluconacetobacter genus. This means that 44.1% of all AAB species identified across the samples represent novel species, which is a remarkably high proportion. The high number of species suggests that homemade vinegars are a rich and largely unexplored source of microbial diversity. These species may possess unique metabolic or technological properties. In a previous study, we isolated four novel species from two homemade vinegar samples, further confirming the high occurrence of previously undescribed taxa in homemade vinegars and the possible implications of newly described species [29]. This finding highlights the need for further cultivation-based studies, as well as phenotypic and genotypic characterization, to fully describe and understand the usefulness of these AAB species.

Some novel species were found in more than one vinegar sample. For example, Acetobacter sp. nov. 5 was identified in Apple4, Apple5, Vine1, and Mixed1. Its presence in both Vine1 and Apple4 is expected, given that Vine1 was produced using the Apple4 culture, which already contained this strain. This species is likely to have entered the fermentation process through the apples used to make Apple4, Apple5, and Mixed1. In the case of Vine1, the strain was introduced through the inoculated culture from Apple4, as previously mentioned. Vine1 and Apple4 share 7 species, accounting for 43.7% of the species common to Apple4 and 46.7% of those common to Vine1. The same was observed for Komagataeibacter sp. nov. 6, which was present in samples Apple1, Apple4, and Vine1. However, Acetobacter sp. nov. 6 was present in Apple1, Apple4, and Apple5, but appears to thrive only in apple-based substrates, as it was also absent from sample Vine1. Komagataeibacter sp. nov. 1, Komagataeibacter sp. nov. 4 and Komagataeibacter sp. nov. 5 were each present in two samples. All the other novel species were present in only one of our samples, suggesting that they are specialized for the specific environment provided by vinegar. Vinegar is a substrate known for its high acidity, which creates a stressful environment for microbial life [40]. Under such harsh conditions, both speciation and environmental filtering can occur at an accelerated pace. This makes vinegar an ideal habitat for the development of novel bacterial species. Several studies have demonstrated that environmental stress can drive adaptive divergence and increase genetic variability, key mechanisms in the formation of new species [41].

Species richness (q0) is highly sensitive to rare species and does not take species abundance into account. Therefore, we also calculated q1, which incorporates species richness and evenness, and is sensitive to rare species, as well as q2, which emphasizes the most abundant species and reflects community dominance [26]. Together, the three-diversity metrics provide a more balanced and comprehensive assessment by capturing the presence and distribution of species within a sample [42]. As shown in Table 2, the sample Mixed2 has the highest species richness (q0 = 21) and a more even species distribution than the other samples (q1 = 11.49). At the same time, it also harbors a considerable number of rare species (q2 = 8.91). In contrast, samples such as Apple2 (q0 = 2) and Blackcurrant1 (q0 = 4) exhibit very low richness, with q1 and q2 values close to 1 for Apple2 and values close to 2 for Blackcurrant1, indicating highly uneven communities dominated by one or two species. This domination can also be seen in Figure 2. Apple1, Apple3, and Apple5 exhibit intermediate diversity, while Apple4 has relatively high richness (q0 = 16) but lower q1 (3.6) and q2 (2.03) values, meaning that the community is dominated by a few species and contains many rare ones.

3.2. Phylogenetic Diversity Detected with 16S-23S rRNA ITS Sequencing

The composition of microbial species in the AAB of vinegar samples was analyzed using 16S-23S rDNA ITS metagenomic methodological approach. The most prevalent genus in the vinegars was Komagataeibacter, which was present in all samples (Figure 3). One of the samples, Apple2, contained only this genus. Acetobacter was another genus that was present at high abundance in all vinegars except for the Apple2. The next most prevalent genus was Novacetimonas, which was present in all samples except Honey1, Mixed1, and Apple2. The AAB genera Novacetimonas, Gluconobacter, Swaminathania, Gluconacetobacter, and two potentially novel genera, Acetobacteraceae gen. nov. 1 and Acetobacteraceae gen. nov. 2, were also present. Swaminathania is a monotypic genus, with Swaminathania salitolerans as its only known species. It has been isolated in association with wild rice plants [43]. This study is the first to confirm its presence in vinegar.

Phylogenetic diversity takes both species abundance and evolutionary history [25,44]. We used phylogenetic Hill numbers (q0, q1, q2) to evaluate the evolutionary diversity of the microbial communities present in the vinegar samples (see

Table 3. Phylogenetic diversity across samples based on phylogenetic Hill numbers. The table presents calculated diversity numbers for each sample.

Sample	q0	q1	q2
Mixed2	2.82	1.23	0.82
Apple1	2.42	1.24	0.94
Apple4	2.31	0.62	0.50
Vine1	1.95	0.62	0.51
Apple3	3.47	0.89	0.65
Apple5	2.68	0.98	0.80
Honey1	1.57	0.60	0.49
Blackberry1	1.51	0.50	0.47
Mixed1	0.86	0.61	0.54
Blackcurrant1	0.80	0.53	0.50
Apple2	0.44	0.41	0.41

and Figure 4). To support the interpretation of phylogenetic diversity, we provide a Supplementary Figure S1 showing the phylogenetic tree of all AAB species (framed in red) detected across the vinegar samples. This tree was reconstructed using the maximum likelihood method and reflects the evolutionary relationships and branch lengths used in the calculation of phylogenetic Hill numbers. The tree represents the gamma diversity of the dataset and serves as the basis for sample-specific diversity metrics, including phylogenetic richness (q0), evenness (q1), and dominance (q2). Its inclusion allows for a clearer understanding of how evolutionary distances contribute to the diversity structure observed across samples.

The phylogenetic q0 value was the highest for the sample Apple3 at a value of 3.47, indicating that the total branch length of the phylogenetic tree corresponds to approximately 3.47 distinct evolutionary lineages. However, q1 was lower (0.89), suggesting that although multiple lineages are present in this sample, a few dominate in terms of their relative abundance. These were Komagataeibacter saccharivorans (35.9%), Acetobacter ghanensis (35.9%), Gluconobacter cerinus (8.6%), Novacetimonas hansenii (7.4%), and Acetobacter papayae (3.6%). Other species were present in quantities of less than 1%. The q2 value is even lower (0.65), as the abundant and evolutionarily related genera Komagataeibacter and Acetobacter influence the community. Apple3 is followed by Mixed2 (q0 = 2.82) and Apple5 (q0 = 2.68), both of which also exhibit relatively high diversity across all Hill numbers. Apple1 stands out with the highest q1 (1.24) and q2 (0.94) values, indicating an even distribution among phylogenetically diverse species. In contrast, Apple2 and Blackcurrant1 demonstrate the lowest phylogenetic diversity across all Hill numbers, suggesting that they contain closely related and uneven communities. The low numbers could be primarily due to the fermentation stage. All samples were taken during the final fermentation stage, after vinegar had already been produced. While microbial communities generally remain stable throughout fermentation, microbial diversity undergoes regular changes [45]. During the acetic acid fermentation process, which involves decreased sugar concentrations and high acidity conditions, a well-defined succession pattern of AAB was observed in previous studies [46]. Few organisms can survive in these conditions, but there are many species of AAB adapted to such environments. Despite the considerable number of different species, most of them belong to the Komagataeibacter and Acetobacter genera, with a few belonging to the genera Novacetimonas, Gluconobacter, and Gluconacetobacter and Swamminathania, which was present at an exceptionally low level in one sample only. The chemical composition shapes the dominant species involved throughout the vinegar formation process [15]. Phylogeny and functional traits reflect different aspects of ecological variation between species, often complementing one another [47]. Therefore, we would expect to isolate species with more diverse traits from vinegars with higher phylogenetic diversity.

The samples in the heatmap (Figure 2) are clustered based on Bray–Curtis dissimilarity, which is calculated from the relative abundance of each species present in the vinegars. This clustering represents similarities in species composition between samples. There are three obvious clusters shown. One contains samples Blackcurrant1, Blackberry1, and Apple3. The same culture from Apple3 was used for Blackcurrant1 and Blackberry1; therefore, the similarity between them is expected. Vine1 and Apple4 were also made from the same culture, but here we see that, given the similarity of the microbial community, these two samples are not the closest. This is most likely due to the specificity of the wine vinegar, where the conditions were not favorable for a large proportion of the culture transferred from Apple4. However, Apple4 is in a different cluster and shows close similarity with Apple5. This may be a perfectly expected link, given that they are made from the same raw material.

3.3. Non-Acetic Acid Bacteria

Previous studies have shown that the primers used in this methodological approach also bind to lactic acid bacteria (LAB), though with lower specificity than those targeting the 16S rRNA gene [14]. To improve transparency, LAB and other non-AAB are grouped under the category “Other” in Figure 3. Three genera of the group of LAB have been detected: Oenococcus, Secundilactobacillus, and Pediococcus. Oenococcus was found in samples Mixed2, Apple1, Apple4, and Apple5, while the genus Secundilactobacillus was found in Apple1. Pediococcus was found in samples Apple4 and Apple5.

As well as binding to AAB and LAB, the primers also bound to several other genera, including Metabacillus, Tatumella, Pediococcus, Kosakonia, Gibbsiella, Pseudomonas, and Rahnella. Due to the lower specificity, it is not possible to ensure reliable species-level identification for the genera listed above. This limitation could be avoided by sequencing the entire 16S-23S rDNA ITS region, using customized long-read amplicon approaches, once they are commercially available [14].

The genus Pseudomonas has previously been detected in Zhenjiang aromatic vinegar [45], despite some sources reporting that vinegar may inhibit the growth of certain species in this genus [48,49]. However, as previously mentioned, it is not possible to reliably identify non-AAB species present in the samples, as the method used enables species-level resolution only for AAB. Gibbsiella, Rahnella, Kosakonia, and Tatumella were found only in sample Apple3. As this vinegar was produced in a wooden barrel, it is hypothesized that the genera Rahnella [50,51,52,53], Gibbsiella [54,55], Kosakonia [56,57,58], and Metabacillus [59,60], which are known as plant pathogens or plant growth-promoting bacteria, could be connected to the wood from which the barrel was made. Alternatively, they could have been introduced to the barrel with the apples used as substrate. However, some species from the genera Tatumella, Rahnella, and Kosakonia have been recognized as human pathogens [61,62,63,64]. These OTUs were present in less than 1.3%, and their presence may also result from unclean tools or containers.

A key limitation of metagenomic methods is that they cannot distinguish between DNA derived from viable microorganisms and residual DNA from non-viable or lysed cells [65]. Temperature and pH are both key factors affecting DNA stability. Acidic and basic conditions can accelerate DNA hydrolysis by increasing either the reactivity of the DNA or the nucleophilicity of the water [66]. However, no studies have yet examined exactly how vinegar affects DNA and its stability. This is particularly relevant in acidic environments like vinegar, where low pH may compromise bacterial viability and the quality of DNA, yet extracellular or intracellular DNA may still be detectable. The presence of Pseudomonas, known for its high antibiotic resistance [67], could therefore reflect residual DNA. It is plausible that horizontal gene transfer, including the integration of DNA from lysed cells into the genomes of surviving AAB, could contribute to the observed levels of antibiotic resistance in these bacteria, which was previously demonstrated by Cepec and Trček [68]. Future studies should aim to clarify this possibility, as it has important implications for understanding microbial adaptation and resistance dynamics in acidic fermentation environments.

The differences in species richness and diversity across vinegar samples were assessed using Hill numbers, which provide a consistent and interpretable framework for comparing alpha diversity. However, due to the limited number of samples, we did not apply formal statistical tests or modeling approaches to account for potential confounding factors. While our results offer descriptive insights into microbial community structure, future studies with larger datasets could benefit from hypothesis-driven statistical modeling, including permutation tests, mixed-effects models, and corrections for multiple comparisons, to strengthen the reliability and generalizability of findings.

4. Conclusions

This study shows that homemade vinegars exhibit relatively high species richness, yet low phylogenetic diversity and low evenness, as indicated by Hill numbers q1 and q2. This suggests that while many species are present, the communities are dominated by a few closely related taxa in the final stages of vinegar production. The application of Hill numbers provided a nuanced view of community structure, highlighting both the presence of rare species and the dominance of specific lineages. Results confirm that home-prepared vinegars provide an environment in which many new and unexplored species can be found, primarily belonging to the Komagataeibacter genus, which is known for its capacity to synthesize bacterial nanocellulose, and the Acetobacter genus, which, like Komagataeibacter, is already widely used in vinegar production. The new strains may possess unique characteristics that could enhance the flavor of vinegar and other fermented products, as well as optimize the production process. The identification of potentially pathogenic bacterial genera in vinegar suggests that future research should focus on the importance and impact of these species on the bacterial community in which they are present.