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Article

First Microbial Survey of a Submerged Petrified Forest in the Black Sea: Culture-Based and Metagenomic Insights

by
Mihail Iliev
1,
Ralitsa Ilieva
1,*,
Slavil Peykov
2,3,4,
Viktoria Terziyska
1,
Anton Pelkin
1 and
Lyubomir Kenderov
5
1
Department of General and Industrial Microbiology, Sofia University “St. Kliment Ohridski”, 1164 Sofia, Bulgaria
2
Department of Genetics, Sofia University “St. Kliment Ohridski”, 1164 Sofia, Bulgaria
3
BioInfoTech Laboratory, Sofia Tech Park, 111 Tsarigradsko Shose Blvd., 1784 Sofia, Bulgaria
4
Department of Medical Microbiology “Corr. Mem. Prof. Ivan Mitov, MD, DMSc”, Faculty of Medicine, Medical University of Sofia, 2 Zdrave Str., 1431 Sofia, Bulgaria
5
Department of General and Applied Hydrobiology, Sofia University “St. Kliment Ohridski”, 1164 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(8), 583; https://doi.org/10.3390/d17080583
Submission received: 16 July 2025 / Revised: 14 August 2025 / Accepted: 18 August 2025 / Published: 20 August 2025
(This article belongs to the Special Issue Diversity in 2025)
Browse Figures
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Abstract

The submerged petrified forest in Sozopol Bay, located along Bulgaria’s southeastern coast in the Black Sea, is an extraordinarily rare natural phenomenon that has remained unexplored in terms of microbial diversity until now. This study focuses on characterizing the microbial communities associated with this unique habitat. Ancient petrified tree remnants located at depths of 18–20 m were sampled in August–September 2024, targeting four tree trunks from different sites within the bay. The quantitative assessment of selected bacterial groups, essential for nutrient cycling, organic matter degradation, and marine ecosystem health, revealed distinct community profiles. 16S rDNA sequencing of cultivated isolates identified a diverse microbial community predominantly composed of γ-Proteobacteria, with key representatives such as Vibrio aestuarianus, Vibrio orientalis, Pseudoalteromonas, and Cobetia sp. The culture-independent approach confirmed the dominance of Proteobacteria, along with other prevalent phyla like Bacteroidetes, Planctomycetes, and Actinobacteria. The most abundant taxa included Woeseia oceani, Ilumatobacter coccineus, Halioglobus maricola, and Vibrio breoganii. Archaea made up about 3% of classified reads. Fungal sequences accounted for less than 2% of the total reads, indicating a low fungal prevalence. These results provide essential baseline data for future monitoring and the conservation of this unique habitat and its diverse microbial communities.

1. Introduction

Submerged petrified forests are exceptionally rare geological phenomena, primarily owing to the specific combination of geological conditions required for their formation, limited geographic distribution, preservation challenges, and significant scientific interest they evoke. While there are a few well-known examples of submerged petrified forests, such as those in the Gulf of Mexico and off the coast of Nova Scotia, these remain exceptions rather than the norm [1,2,3,4].
An examination of the literature concerning permineralized woods reveals that the majority of documented cases are from the Miocene age and involve fossilized trees described in terrestrial habitats [5,6,7,8,9,10]. Overall, while there are numerous known land petrified forest sites, marine petrified forest sites are comparatively fewer due to the challenges associated with underwater exploration and documentation.
The current study is part of a comprehensive investigation of the natural phenomenon known as the Underwater Petrified Forest, located within the waters of Sozopol Bay on the southeastern coast of Bulgaria. The site is distinguished by the presence of in situ fossils, including ancient tree stumps, branches, and roots, preserved in a petrified state and located at a depth of 18–20 m below sea level. A hypothesis suggests that these trees thrived in a freshwater swamp environment, with a shallow lagoon confined within a volcanic caldera. With the transgression of the Black Sea (7500 years BP), the lagoon was inundated with seawater, ultimately transforming the site into a coastal–marine environment [11].
The absence of related microbiological studies underscores the novelty and significance of this research effort, which aims to deepen our understanding of the microbial communities inhabiting this unique underwater ecosystem. The interest in the submerged petrified trees of Sozopol Bay is driven primarily by two key factors. Firstly, they create a unique habitat that allows diverse microbial life to colonize their surfaces, a phenomenon that has not been previously explored at this specific site. A broad range of aquatic microorganisms can colonize various surfaces, facilitating biofilm formation and the emergence of specialized processes within these structures [12]. Secondly, the potential risk of microbial-related biodeterioration at this location warrants attention. The surfaces of these petrified trees may function as “hot spots” for microbially catalyzed biogeochemical activities, some of which could pose a significant threat to their structural integrity. Although there are no specific studies addressing the microbial degradation of petrified wood in marine environments, existing scientific literature suggests that petrified wood may resist decay due to mineralization, but it is not entirely passive in environments where microbial activity is present [13,14].
In this study, the microbial diversity colonizing the surfaces of petrified trees in Sozopol Bay, Bulgaria, has been investigated using both cultural and culture-independent approaches. By understanding the microbial diversity associated with this unique natural phenomenon, we can gain valuable insights into the complex interactions between ancient geological formations and contemporary marine life. Furthermore, our findings may have implications for the conservation and management of similar underwater habitats worldwide.

2. Materials and Methods

2.1. Site Description

The sampling site is situated in the aquatory of Sozopol Bay, located along the southeastern coast of Bulgaria, in the western region of the Black Sea (Figure 1). The exact dimensions of the whole area where the petrified trees are located are currently unknown, as the documentation process is ongoing. In our study, we are examining only a well-documented portion, which covers an area of 67,344 m2. The coordinates of this portion are 42.42794 latitude and 27.6940 longitude (central point of the transect). In the selected portion of the forest, at the depth from 15.0 to 20.0 m, we discovered and documented numerous petrified trees, totaling more than 30 in count. Among them, some are found upright in a growth position, ranging in height from 40 cm to 2 m, while others have fallen, extending up to 20 m in length. For the purpose of the recent study, four of these petrified trees were selected. These chosen objects are situated in different sites within the area.

2.2. Sampling

The survey took place during August–September (2024) as a component of the research diving expeditions conducted in the area. This sampling timeframe was selected due to the optimal visibility for diving activities in the Black Sea during this period. The sampling points for investigation are four tree trunks, each with distinct characteristics, providing a diverse range of environmental conditions for microbial communities. These trunks represent a range of locations within Sozopol Bay, allowing us to account for potential spatial variations in microbial communities. Two of the trunks (Object 1 and Object 2) are located in the shallow waters (17 m) near the shore and exhibit significant visual colonization by various marine organisms (Figure 2A). In contrast, the other two trunks (Object 3 and Object 4) are found in deeper waters (20–22 m), where visual colonization appears to be less pronounced (Figure 2B).
A total of 20 samples (5 from each sampling point) were collected as follows: biological material was scraped from the external surface of each petrified tree, using sterile spatula blade. Each sample was promptly transferred into a 2.0 mL microcentrifuge tube. Sediment samples were collected by removing the sandy overburden around the surface of petrified tree roots and scraping off the exposed surface to a depth of 20 cm from the seafloor. Preservation of the samples for further manipulation was conducted following standard procedures for microbiological analysis.
Sampling was conducted by members of the scientific team, all of whom were certified divers (CMAS 3***) with a Scientific Diver rank.
Basic hydrochemical parameters of seawater were measured during sampling campaigns, including dissolved oxygen concentration and saturation, temperature, salinity, and electrical conductivity. Field measurements were conducted using portable WTW multiparameter instruments (model series 330i). Data were collected at two depths: at the sea surface (0 m) and near the seabed (18 m). Water transparency was assessed using a standard Secchi disk.

2.3. Quantification Analysis of Cultivable Bacterial Presence

According to presumption of most probable groups, several target groups were subjected to analysis with relevant medias as follows: Zobell Marine Agar (Z–MA) (HIMEDIA, Thane, India), used for cultivation of heterotrophic aerobic bacteria [15]; BG-11 (HIMEDIA, Thane, India), used for cultivation of cyanobacteria; R2A, used for cultivation of oligotrophs (Reasoner & Geldreich, 1985) [16]; Photobacterium Broth 38719 (Sigma-Aldrich, St. Louis, MO, USA), used for cultivation of various species of Photobacterium; Thiosulfate–citrate–bile salts–sucrose agar (TCBS), used for the selective isolation of cholera vibrios and Vibrio parahaemolyticus [17]; Thiobacillus Agar (HIMEDIA, Thane, India), used for cultivation of Thiobacillus species [18]; Sulphate Reducing Medium (HIMEDIA, Thane, India), used for cultivation of sulfate-reducing bacteria [19].
All nutrient media used in this study were prepared using marine water collected directly from the sampling site. For each of the four sampling objects, five individual subsamples—including material scraped from the petrified wood surface and surrounding sediments—were pooled into one composite sample. The composite was vortexed for 10 s to achieve homogenization. Serial ten-fold dilutions (10−1 to 10−7) were prepared in sterile 0.9% NaCl solution. Culturable microorganisms were enumerated using two standard approaches: (i) spread plate method, whereby 0.1 mL of each dilution was inoculated in duplicate onto selective agar media; and (ii) most probable number (MPN) method, involving inoculation of 1 mL of each dilution into three replicate test tubes containing relevant liquid media. All cultures were incubated in the dark at ambient room temperature (24 ± 2 °C) for 3–5 days, depending on medium type and target group. Microbial abundance was calculated as colony-forming units per milliliter (CFU/mL) or as MPN/mL. Since biological replicates were pooled into a single composite sample per site, the error bars shown in the figures (Figure 3 and Figure 4) reflect technical variability only. Where positive growth was detected in liquid cultures, samples were streaked onto solid media to isolate individual colonies. Dominant strains were selected based on recurring colony morphology for subsequent molecular identification. After enumeration, a select group of most dominant isolates were obtained from the microbial communities associated with the underwater petrified trees in Sozopol Bay.

2.4. Molecular Identification of Dominant Bacterial Strains

Total DNA extracts of the selected bacterial isolates were obtained using a Genomic DNA Purification Kit (Promega, Madison, WI, USA). Subsequently, the 16S rRNA gene was amplified through polymerase chain reaction (PCR) using the universal primers 27F (5′ AGA GTT TGA TCM TGG CTC AG 3′) and 1492R (5′ CGG TTA CCT TGT TAC GAC TT 3′). The reaction mixtures consisted of 1 µL of 10X buffer, 0.4 µL of 50 mM MgCl2, 0.5 µL of 2.5 mM dNTPs, 0.5 µL of 5 mM forward and reverse primers, 0.05 µL of 5 U/µL Taq polymerase, and 2 µL of template DNA, with a total volume of 10 µL per reaction. The PCR method involved an initial denaturation step at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 40 s, 72 °C for 60 s, and a final extension at 72 °C for 5 min. Amplified products were then analyzed by electrophoresis on a 1% agarose gel. DNA sequencing was carried out at Macrogen Inc. (Amsterdam, The Netherlands), utilizing an ABI 3730xl genetic analyzer (Applied Biosystems, Foster city, CA, USA). The raw sequencing data were examined for quality and trimmed using the Codon Code Aligner software 12.0.3 (Codon Code Corporation, Boston, MA, USA). Following this, the trimmed sequences were subjected to Basic Local Alignment Search Tool (BLASTn) searches against the GenBank database to identify the closest related sequences in the available reference database.
The obtained 16S rRNA sequences were aligned using Clustal Omega [20]. The resulting multiple sequence alignment was used to infer a maximum likelihood phylogenetic tree with IQ-TREE (Galaxy Version 2.4.0) [21], implemented within the Galaxy platform [22]. Automatic model selection was carried out using ModelFinder (Galaxy Version 2.4.0) [23] with 1000 bootstrap replicates, and the final tree was visualized with iTOL (v7) [24].

2.5. Genomic Analysis of Microbial Diversity

2.5.1. Total DNA Isolation

Total DNA from the submerged petrified forest was obtained by the ZymoBIOMICS DNA Miniprep Kit (Zymo Research Corp., Irvine, CA, USA), according to the manufacturer’s instructions. The extraction was performed from on a composite sample (200 mg), prepared by pooling equal quantities of material derived from four distinct trunks (designated as Objects 1–4).

2.5.2. Shotgun Metagenomic Sequencing

The obtained sample underwent shotgun metagenomic sequencing to enable a comprehensive characterization of the microbial community structure. The extracted DNA was randomly fragmented, size-selected, and ligated to adapters, followed by PCR amplification. Following these steps, the generated libraries were sequenced on an Illumina NovaSeq 6000 platform (Novogene, Cambridge, UK), generating 2 × 150 bp paired-end reads.

2.5.3. Pre-Processing of the Raw Sequencing Reads

All generated raw sequencing reads underwent a standardized quality control and trimming procedure. Quality assessment was performed using FastQC (v0.11.9; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 20 May 2025), while quality filtering was conducted using Trimmomatic (v0.36) with the sliding window parameter set to 4 bases and a minimum average quality threshold of 20 [25]. Both tools were executed through the KBase platform using default settings unless otherwise specified [26].

2.5.4. Taxonomic Classification of the Processed Sequencing Reads

All processed sequencing reads were subjected to taxonomic classification using the Kaiju tool (v1.9.0) as implemented in KBase [27]. Two separate runs were performed, using the RefSeq Genomes (No Euks) (contains protein sequences from completely assembled bacterial, archaeal, and viral genomes from NCBI RefSeq, current as of 23 March 2022) and Fungi Reference (contains protein sequences from a representative set of fungal genomes, current as of 29 March 2022) databases to facilitate the identification of bacterial, archaeal, and fungal taxa, respectively.

2.5.5. Functional Profiling of the Processed Sequencing Reads

Functional annotation of the processed reads was conducted using the HUMAnN 3.6 pipeline [28], with the ChocoPhlAn and UniRef90 databases employed for gene and pathway mapping. After normalization and regrouping, MetaCyc pathway abundances were quantified [29]. The 20 most abundant unstratified reactions were visualized using a custom Python script utilizing the Pandas (v2.2.1), Matplotlib (v3.10.0), and Seaborn (v0.13.2) libraries.

2.5.6. Detection of Genes Involved in the Nitrogen Cycle

Trimmed sequencing reads were mapped against the NCycDB database [30] using the NCyc_95.faa.gz file provided by the authors. Mapping was performed using the DIAMOND tool (v2.0.15) [31], and the resulting data were analyzed and normalized using custom Python scripts.

2.5.7. Identification of Proteins Related to Sulfur Metabolism

A comprehensive list of manually curated proteins with known role in the sulfur metabolism was obtained from the work of Tanabe and Dahl [32]. A Python script was used to download the corresponding sequences from NCBI RefSeq, which were then formatted into a database using the DIAMOND tool (v2.0.15) [31]. The same tool was subsequently employed to map the trimmed sequencing reads against this database, and the resulting data were analyzed in a manner similar to that used for nitrogen-cycle genes.

3. Results

3.1. Hydrochemical Characterization of Seawater in the Underwater Petrified Forest

The assessment of microbial communities in the Underwater Petrified Forest requires careful consideration of abiotic parameters that are instrumental in shaping these ecological systems. Field measurements revealed clear variations in the main hydrochemical parameters of the seawater in the location. Temperature values were typical for the season and regional climatic conditions, ranging from 20.6 °C to 25.5 °C in the near-bottom layer, and increasing slightly at the surface to a maximum of 27.7 °C due to intense solar radiation. Salinity ranged between 10.8 and 11.1 PSU, indicating weak vertical stratification and minimal freshwater input. Electrical conductivity values (18.16–18.57 mS/cm) showed a strong correlation with both salinity and temperature. Dissolved oxygen concentrations in surface waters reached up to 11.3 mg/dm3 (139% saturation), most likely resulting from active phytoplankton photosynthesis. In the near-bottom layer, oxygen levels showed a moderate decrease. Water transparency varied from 4.5 to 10.0 m, with a mean value of 5.84 m. These fluctuations are presumably related to varying concentrations of suspended particles and phytoplankton biomass.

3.2. Quantitative Analysis of Culturable Bacterial Abundance

Quantitative analysis of culturable bacterial abundance in the sampled petrified trees revealed a distinct bacterial community profile depending on the location of the objects. In samples originating from Object 1, representatives of all tested groups were detected, with heterotrophic microorganisms (1.9 × 105 CFU/mL) and oligotrophic microorganisms (2.1 × 103 CFU/mL) dominating the community (Figure 3). Relatively low abundances of cyanobacteria and Photobacterium spp. were observed (2.2 × 102 CFU/mL; 1.9 × 102 CFU/mL). Sulfate-reducing microorganisms and Thiobacillus representatives were present in negligible amounts, while low counts were recorded for representatives of cholera vibrios and V. parahaemolyticus (1.1 × 101 CFU/mL).
Similarly, the microbial community profile in samples from Object 2 showed a predominance of heterotrophic microorganisms and oligotrophic microorganisms, with levels slightly higher but still comparable to those found in sample from Object 1 (2.0 × 106 CFU/mL; 1.0 × 104 CFU/mL). Comparable quantities of cyanobacteria and members of Photobacterium spp. were observed in samples from Object 2, with low levels detected for the remaining tested groups.
The results of the quantitative analysis of the culturable bacterial microflora in samples from Object 3 and Object 4 show a distinct bacterial presence profile. Heterotrophic representatives again dominate quantitatively, but at significantly lower levels than the first group of objects. Concurrently, the quantity of oligotrophs has increased comparatively. Sampling of Object 3 did not detect representatives of cholera vibrios and V. parahaemolyticus. On the other hand, there was an increased number of sulfate-reducing microorganisms and Thiobacillus representatives in samples from Object 4 (Figure 4).

3.3. Bacterial Isolates Identified Through 16S rRNA Sequencing

The bacterial isolates obtained from the cultivated samples were subjected to 16S sequencing to identify the dominant species present in the microbial community. The results are presented in Table 1. Out of the 35 isolates analyzed, only 25 demonstrating high sequence similarity (≥97%) compared to published data were included in this analysis.
A maximum likelihood phylogenetic tree was constructed from the obtained 16S rRNA sequences, as shown in Figure 5.

3.4. Shotgun Metagenomic Analysis

3.4.1. Sequencing Data

Shotgun metagenomic sequencing of the composite sample derived from the submerged petrified forest yielded a total of 23,549,701 paired-end reads, corresponding to approximately 7 Gb of sequencing data with an average GC content of 46%. Following quality filtering and trimming, 98.1% of the reads were retained for downstream analyses.

3.4.2. Microbial Community Composition

Taxonomic profiling of the composite sample using the Kaiju classifier revealed that Proteobacteria was the dominant bacterial phylum, accounting for over 50% of the classified sequencing reads (Figure 6). Other prevalent phyla included Bacteroidetes, Planctomycetes, and Actinobacteria. At the species level, the most abundant taxa identified were Woeseia oceani, Ilumatobacter coccineus, Halioglobus maricola, and Vibrio breoganii.
Archaea constituted approximately 3% of the total classified organisms in the sample. Among these, members of the genus Nitrosopumilus were predominant, comprising 75% of the archaeal reads. Nitrosopumilus ureiphilus was the most abundant archaeon, representing 13% of all archaeal sequences identified.
Fungal taxonomic profiling indicated that Ascomycota was the predominant phylum, accounting for nearly 50% of the classified fungal reads (Figure 7). The most abundant species included Synchytrium microbalum, Batrachochytrium dendrobatidis, and Spizellomyces punctatus, each represented by a similar number of reads. Notably, fungal sequences constituted less than 2% of the total classified reads, suggesting a relatively low fungal presence within the microbiome of the submerged petrified forest.

3.4.3. Functional Analysis

The twenty most abundant unstratified reactions, as identified by MetaCyc pathway analysis, are shown in Figure 8.
The most abundant reaction identified was DNA-directed RNA polymerase (DNA-DIRECTED-RNA-POLYMERASE-RXN; 0.000180), exhibiting approximately twice the abundance of the next most prevalent reactions: ATP synthase (ATPSYN) and peptidyl-prolyl isomerase (PEPTIDYLPROLYL-ISOMERASE-RXN), which is involved in protein folding. SUCCCOASYN-RXN (succinyl-CoA synthetase) and ENOYL-COA-HYDRAT-RXN (enoyl-CoA hydratase), both part of core metabolic pathways, are associated with the tricarboxylic acid (TCA) cycle and fatty acid metabolism, respectively.
Metagenomic profiling of nitrogen-cycling genes, performed by mapping shotgun sequencing data to the NCycDB, yielded numerous hits, with 1.966% of all trimmed reads successfully mapped. Following normalization, the 20 most abundant genes are shown in Figure 9.
The most abundant gene detected, glnA, encodes the enzyme glutamine synthetase, which plays a crucial role in nitrogen metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia.
Mapping of sequencing reads to manually curated proteins with established roles in sulfur compound oxidation, reduction, transport, and intracellular transfer identified the most prevalent elements of this type within the microbial community of the submerged petrified forest. The overall amount of mapped trimmed reads was 0.126%. The 20 most abundant proteins are shown in Figure 10.
The most prevalent protein in our sample was TtrR, a component of the two-component regulatory system TtrR/TtrS, which functions as a positive transcriptional regulator for the synthesis of tetrathionate reductase. TtrS was also found among the top hits.

4. Discussion

The underwater petrified forest site in Sozopol Bay, Black Sea, stands as a testament to the unparalleled uniqueness of both its geological formation and the Black Sea itself. The ecosystem of the Black Sea exhibits a distinctive character shaped by its geological origins. Characterized by its semi-enclosed nature, the Black Sea maintains a salinity level approximately half that of the global ocean, owing to the influx of freshwater from rivers and its limited connection to open waters [33,34,35]. In analyzing the microbial communities inhabiting the site of Underwater Petrified Forest, it is essential to consider the following factors: (1) The location itself is situated in a relatively narrow space between the coastline and the largest island on the Bulgarian Black Sea coast, St. Ivan Island, creating a highly turbulent marine environment with strong underwater currents and waves prevalent throughout much of the year. This proximity to the shore, combined with the depth and dynamics of water masses, contributes to reduced visibility, which has earned the site a notorious reputation among local diving communities. (2) On the other hand, the location implies increased anthropogenic pressure due to its highly urbanized surroundings and active tourism. The waters around Sozopol are close to the entrance of Burgas Bay, which hosts one of the busiest ports in the Black Sea. This leads to heightened traffic of commercial vessels, increasing the likelihood of sporadic pollution incidents in the area, including oil spills, with the most recent one recorded in the spring of the current year.
Historically, investigations in the Underwater Petrified Forest have primarily concentrated on geological and paleontological aspects, with no attention given to microbiological studies [36,37,38]. In this context, microbiological studies of the site are essential, given the critical role that microorganisms play in the intricate interactions between these ancient geological formations and the current marine ecosystem. Marine microflora, specifically culturable bacteria, play significant roles in nutrient cycling, organic matter degradation, and the overall health of marine ecosystems. Furthermore, the cultivation of marine microflora is particularly relevant in the context of environmental monitoring and climate change. Studies like those by Xu et al. delineate the importance of understanding community dynamics to foster pro-environmental behaviors, linking microbial health to broader ecological outcomes in coastal regions [39]. The adaptability of culturable microorganisms to changing environmental conditions can provide insights into the resilience of marine ecosystems.
The quantitative analysis of culturable marine microorganisms from petrified trees provides valuable insights into the microbial community structure and their ecological roles within this unique habitat. Our findings highlight significant variations in cultivable bacterial communities based on investigated petrified trees location, underscoring the impact of environmental factors on microbial assemblages.
The prevalence of heterotrophic microorganisms in all sampled petrified trees aligns with their fundamental role in marine ecosystems. Heterotrophs, which depend on organic carbon for energy, are critical for recycling nutrients, thereby supporting primary production [40]. The highest abundance of heterotrophs (1.9 × 105 CFU/mL in Object 1, and 2.0 × 106 CFU/mL in Object 2) suggests robust microbial activity in these environments. This is further supported by similar observations in the literature, where microbial communities play essential roles in nutrient remineralization and energy flow, affecting overall ecosystem dynamics [41]. In contrast, studies of bacterial isolates from marine habitats cultivated on Z–MA have reported significantly lower numbers compared to our findings. Bernard et al. reported heterotrophic microorganisms from Mediterranean Sea samples at approximately 1.31 × 104 CFU/mL [42]. Similarly, a study by Zweifel and Hagstrom estimated heterotrophs in the Mediterranean at around 1 × 103 CFU/mL [43]. In this investigation, the abundance of heterotrophs was even lower in Baltic Sea samples, averaging 4.8 × 102 CFU/mL, while the North Sea showed an average of 2.3 × 103 CFU/mL, reflecting variability in heterotrophic abundance across different marine environments [44].
Additionally, Sardo et al. examined the Tyrrhenian Sea along the Italian coastline, detecting between 2 and 463 CFU/mL on the SWC culture medium, which was similarly used for cultivating heterotrophic microorganisms [45]. The higher abundance of heterotrophs in the Sozopol region compared to other marine ecosystems may indicate a more favorable nutrient availability or conditions conducive to microbial growth, further supporting the need for continued study of microbial dynamics in unique marine habitats.
The consistent presence of oligotrophic microorganisms, with higher counts in Object 2 (1.0 × 104 CFU/mL), indicates adaptability to nutrient-limited conditions. This finding corresponds well with existing literature that describes the abundance of low-nutrient adapted oligotrophs in various marine environments, typically ranging from less than 103 to more than 105 CFU/mL [46]. Oligotrophs are known to thrive in environments where organic nutrients are sparse, reflecting resilience in shifting ecological conditions [47]. Their presence may suggest nutrient status and potentially inform about ecosystem health. The rise in oligotrophic bacteria in Objects 3 and 4, alongside a decrease in heterotroph numbers, could be indicative of shifts in resource availability or environmental conditions affecting microbial life.
The low abundance of cyanobacteria and Photobacterium spp. (2.2 × 102 CFU/mL and 1.9 × 102 CFU/mL, respectively) in Objects 1 and 2 reflects potential limitations in light availability or nutrient conditions supporting their growth. Despite their low numbers, cyanobacteria play crucial roles in primary production and nitrogen fixation in marine ecosystems, prompting the need for monitoring their abundance to understand ecosystem dynamics and predict changes in phytoplankton productivity [48]. The consistent levels across samples from both objects may suggest stable ecological conditions for these phototrophs, but their low prevalence indicates that primary production may be limited in these environments.
The significant presence of sulfate-reducing bacteria in Object 4 highlights the importance of anaerobic processes in microbial communities associated with organic matter breakdown. These microorganisms play an essential role in the sulfur cycle, impacting sediment biogeochemistry and potential habitat degradation. The variability in their abundance across samples indicates differing ecological functions, potentially linked to sediment composition or organic matter availability. The implications of these findings extend to understanding the preservation and ecological significance of the petrified trees. In a recent study on the mineralogy of the petrified trees at the location, it was established that pyrite, a key mineral involved in the fossilization of organisms, makes up 80% of the composition of the examined samples from the site [49]. The relationship between Thiobacillus spp. and pyrite is particularly notable due to their metabolic capabilities. These bacteria are known sulfur-oxidizing microorganisms that thrive in environments rich in reduced sulfur compounds, such as those associated with pyrite oxidation.
Although petrified wood is highly mineralized and generally resistant to degradation, its surface may still be affected by microbial activity, especially under changing environmental conditions. Factors such as increased organic input, hypoxia, pH shifts, or temperature rise could alter microbial community composition and metabolism, potentially accelerating mineral dissolution or bioerosion over time. While speculative, these processes may influence the long-term stability of submerged petrified wood and merit further investigation.
The detection of cholera vibrios and V. parahaemolyticus at extremely low levels (1.1 × 101 CFU/mL) underlines their potential as health indicators in marine environments. Their relative scarcity in Objects 3 and 4, where no representatives were detected, further emphasizes the importance of ongoing monitoring, particularly in relation to public health and environmental safety.
The results from the 16S rDNA sequencing of marine isolates from the underwater petrified trees in Sozopol reveal a diverse microbial community predominantly consisting of γ-Proteobacteria, with key representatives such as Vibrio aestuarianus, Vibrio orientalis, Pseudoalteromonas, and Cobetia sp. The presence of these bacteria underscores the significance of various ecological roles they perform in marine ecosystems [50]. The γ-Proteobacteria group includes several species that are crucial for nutrient cycling and organic matter degradation in marine environments. For instance, Vibrio species, particularly V. aestuarianus and V. orientalis, are known for their versatility in metabolizing organic compounds and their ecological adaptability in fluctuating environmental conditions [51]. These bacteria can also play a role in phytoplankton dynamics and nutrient availability, contributing to the health of the marine ecosystem surrounding the petrified trees. Pseudoalteromonas, another member of this class, is noted for its production of bioactive compounds that can inhibit the growth of fouling organisms and pathogens [52]. This suggests that they contribute not only to the microbial diversity of the site but also to the biochemical interactions that take place in the underwater ecosystem. The detection of C. amphilecti indicates the presence of bacteria that can thrive in variable salinities and may facilitate organic matter degradation within the sediment. The identification of Micrococcus yunnanensis and Micrococcus luteus from the Actinobacteria phylum enhances the documented microbial diversity in this environment. These bacteria are often associated with bioactive compound production, which can be beneficial for ecological interactions and potential biotechnological applications [53]. The presence of Staphylococcus saprophyticus within the Firmicutes phylum raises considerations regarding the interactions between pathogenic and beneficial microbial communities. While S. saprophyticus is primarily regarded as a pathogen, its presence in this ecological context could have implications for the health of the surrounding marine ecosystem. The coexistence of pathogenic bacteria alongside beneficial species may reflect complex ecological dynamics, including competition and symbiosis within the microbial community.
In addition to the culture-dependent approaches utilized to investigate microbial presence in the target location, we have also applied culture-independent methods to further enrich our understanding of microbial dynamics in the environment. While there have been substantial contributions from recent studies utilizing metagenomic techniques to explore microbial diversity in the Black Sea, the overall body of work remains comparatively limited [54,55,56]. The uneven distribution of studies employing metagenomics in the Black Sea contrasts sharply with the extensive research available for other marine environments, such as the Mediterranean or the Caribbean Sea.
Shotgun metagenomics surpasses traditional amplicon-based sequencing by providing insights into the entire metagenome, thereby enhancing the resolution of microbial community structures. The predominance of Proteobacteria aligns with findings from several research endeavors, where this phylum has often been highlighted as a major component of marine microbial communities as well it corresponds with our culture-dependent quantification analysis [57,58]. Additionally, the presence of Bacteroidetes and Actinobacteria among the dominant phyla is consistent with other metagenomic studies that frequently identify these groups as critical members of marine microbial communities, contributing to various biogeochemical processes [59,60]. At the species level, the identification of W. oceani, I. coccineus, H. maricola, and V. breoganii as the most abundant taxa in our metagenomic study aligns with recent research on microbial diversity in marine environments. W. oceani has received attention in studies focused on microbial communities associated with organic matter degradation and hydrocarbon transformation as well as its participation in nitrogen cycling [61,62]. I. coccineus is noted for its biodegradative capabilities, although it is less frequently discussed compared to other genera [63]. The latter research also suggests that taxa such as H. maricola are critical in nutrient cycling within marine ecosystems, affirming their significance in the microbial food webs. The identification of taxa such as V. breoganii in our results warrants further attention, as Vibrio species are known to exhibit dynamic populations in marine environments influenced by seasonal fluctuations and anthropogenic impacts [64]. The results from our metagenomic study indicate that Archaea constituted approximately 3% of the total classified organisms, with Nitrosopumilus being the predominant genus, specifically N. ureiphilus, which represented 13% of all archaeal sequences identified. The predominance of Nitrosopumilus in our dataset is particularly noteworthy, as this genus is known for its contribution to nitrogen cycling through ammonia oxidation, which is crucial for maintaining biogeochemical balance in marine ecosystems. Moreover, research indicates that environmental factors, such as chemical pollution, can influence the abundance and activity of archaea, including N. ureiphilus, which may adapt in response to these pressures [65]. The results from our metagenomic analysis indicated that Ascomycota was the predominant fungal phylum, accounting for nearly 50% of classified fungal reads. It is consistent with findings from similar investigations in marine environments, where Ascomycota often constitutes a substantial portion of the fungal community [66,67]. Overall, the limited representation of fungi in our findings echoes the results from studies by Chrismas et al., who reported a low overall abundance of fungal taxa in coastal marine ecosystems, despite the presence of certain abundant species [68]. The low proportion of fungal sequences (<2%) may reflect the limited availability of suitable substrates in the mineralized wood, combined with the high salinity and reduced oxygen conditions typical of the site. Moreover, the identified fungal taxa, primarily chytrids such as Synchytrium microbalum and Spizellomyces punctatus, are not typical marine species and are more commonly associated with soil or freshwater environments, suggesting they may be transient or non-active members of the microbial community.
The results of the functional analysis illuminate the metabolic capabilities of the microorganisms associated with the petrified wood and reflect the broader ecological dynamics at play in marine environments enriched by organic matter input. These findings suggest a highly integrated metabolic framework where energy production and protein biosynthesis are closely tied to the overall functionality of the microbial community thriving on the petrified wood surfaces. In our metagenomic dataset, glnA emerged as the most abundant nitrogen-cycle gene. This gene encodes glutamine synthetase, a key enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia to glutamine, thereby playing a central role in nitrogen assimilation and homeostasis. Its high prevalence suggests a dominant role of ammonium assimilation in the microbial community. Even more noteworthy is the high prevalence of both components of the two-component regulatory system TtrR/TtrS, which acts as a positive regulator of the operon-encoding tetrathionate reductase [69]. This enzyme catalyzes the reduction of tetrathionate to thiosulfate and is typically found in bacteria that use tetrathionate as a terminal electron acceptor during anaerobic respiration.

5. Conclusions

This research represents the first comprehensive microbiological investigation of the unique phenomenon of an underwater petrified forest, which is exceptionally rare worldwide. Employing both culture-dependent and culture-independent methodologies, our study reveals a stable microbial component persisting in an extremely turbulent environment under significant anthropogenic pressure. These petrified trees, remnants of a long-lost ancient ecosystem, themselves form the basis of a distinct marine habitat today, supporting diverse life forms intricately linked to the engaged microbiota. Given its uniqueness, this natural wonder holds substantial potential for scientific interest and could become a world-class destination for diving tourism. Therefore, its conservation should be a high priority. Despite their age dating back to the Miocene epoch, it is important to note that the petrified trees have been preserved in a relatively protected environment, buried under sandy sediments. However, in contemporary times, some of these trees, such as those examined in this study, are now exposed to a highly turbulent and variable marine environment, which may lead to their degradation potentially exacerbated by microbially catalyzed biogeochemical activities.

6. Limitations of the Study

Both cultivation-based and sequencing-based approaches have inherent limitations that may affect microbial diversity assessments. Culture-based methods typically recover only a small fraction of the total community, favoring fast-growing or media-adapted organisms, and may overlook slow-growing or fastidious taxa. In contrast, amplicon sequencing is subject to biases arising from DNA extraction efficiency, primer specificity, PCR amplification, and errors in database annotation. The shotgun metagenomic approach in this study was limited by a relatively low sequencing depth of approximately 7 Gb, which constrains the resolution of functional analyses. Additionally, taxonomic abundance estimates derived from Kaiju, like those from other metagenomic classifiers, depend heavily on the completeness and composition of the reference database used. These limitations should be carefully considered when interpreting the microbial community composition and relative abundances in the studied environment.

Author Contributions

Conceptualization, M.I.; methodology, R.I., S.P. and A.P.; software, S.P.; validation, M.I., R.I. and V.T.; formal analysis, A.P.; investigation, V.T.; resources, L.K.; data curation, R.I.; writing—original draft preparation, R.I.; writing—review and editing, M.I.; visualization, S.P. and L.K.; supervision, M.I.; project administration, L.K.; funding acquisition, L.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Science Fund, Ministry of Education and Science of Bulgaria, Project No. KP-06-N61/11-15.12.2022, “Complex ecosystem study of the water area of the natural phenomenon Underwater Petrified Forest, Sozopol Bay”.

Data Availability Statement

The raw sequencing reads from the shotgun have been deposited in SRA under BioProject number PRJNA1303121. The obtained 16S rRNA sequences have been uploaded to GenBank under numbers PX121709-PX121733.

Acknowledgments

We extend our heartfelt gratitude to the Port Authorities of Chernomorets for their invaluable assistance in providing technical support for the diving expeditions in their waters. Additionally, we express our sincere appreciation to the members of the University Underwater Club “South Bay” for their enthusiastic participation.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Z–MAZobell Marine Agar
TCBSThiosulfate–citrate–bile salts–sucrose agar
PCRPolymerase chain reaction
SWCSeawater complete
BLASTBasic Local Alignment Search Tool
CFUColony Forming Units

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Figure 1. Location of the Underwater Petrified Forest in the Black Sea near Sozopol, Bulgaria (Image generated by Map data © 2025 GeoBasis-DE/BKG).
Figure 1. Location of the Underwater Petrified Forest in the Black Sea near Sozopol, Bulgaria (Image generated by Map data © 2025 GeoBasis-DE/BKG).
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Figure 2. Petrified tree trunks. (A) Objects 1 and 2, general view. (B) Objects 3 and 4, general view.
Figure 2. Petrified tree trunks. (A) Objects 1 and 2, general view. (B) Objects 3 and 4, general view.
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Figure 3. Quantitative assessment of bacterial colonization on petrified tree remnants at depth of 17 m.
Figure 3. Quantitative assessment of bacterial colonization on petrified tree remnants at depth of 17 m.
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Figure 4. Quantitative assessment of bacterial colonization on petrified tree remnants at depths of 20–22 m.
Figure 4. Quantitative assessment of bacterial colonization on petrified tree remnants at depths of 20–22 m.
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Figure 5. Maximum likelihood phylogenetic tree inferred from 16S rRNA sequences of 25 isolates exhibiting high sequence similarity to reference sequences in public databases.
Figure 5. Maximum likelihood phylogenetic tree inferred from 16S rRNA sequences of 25 isolates exhibiting high sequence similarity to reference sequences in public databases.
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Figure 6. All bacterial taxa identified at various taxonomic levels in the combined sample: (A) phylum; (B) genus; (C) species.
Figure 6. All bacterial taxa identified at various taxonomic levels in the combined sample: (A) phylum; (B) genus; (C) species.
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Figure 7. All fungal taxa identified at various taxonomic levels in the combined sample: (A) phylum; (B) genus; (C) species.
Figure 7. All fungal taxa identified at various taxonomic levels in the combined sample: (A) phylum; (B) genus; (C) species.
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Figure 8. Most abundant unstratified reactions based on MetaCyc annotations.
Figure 8. Most abundant unstratified reactions based on MetaCyc annotations.
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Figure 9. Most abundant nitrogen-cycling genes identified based on NCycDB annotations. Normalized values are expressed as RPKM (Reads Per Kilobase Million). *—distinct database entries differing in sequence length.
Figure 9. Most abundant nitrogen-cycling genes identified based on NCycDB annotations. Normalized values are expressed as RPKM (Reads Per Kilobase Million). *—distinct database entries differing in sequence length.
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Figure 10. Most abundant sulfur metabolism-related proteins within the microbial community of the submerged petrified forest. Normalized values are expressed as RPKM (Reads Per Kilobase Million).
Figure 10. Most abundant sulfur metabolism-related proteins within the microbial community of the submerged petrified forest. Normalized values are expressed as RPKM (Reads Per Kilobase Million).
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Table 1. Identification of bacterial taxa within the microbial community based on 16S rRNA gene sequencing of cultivated isolates.
Table 1. Identification of bacterial taxa within the microbial community based on 16S rRNA gene sequencing of cultivated isolates.
IsolateBLASTnPhylumIdentity, %Accession Number
BlS_10Vibrio sp. strain NCIS28γ-Proteobacteria99PP267458.1
BlS_15Vibrio aestuarianus strain 6-25γ-Proteobacteria99MW041305.1
BlS_17Pseudoalteromonas undina strain VRG_S9γ-Proteobacteria99OL979359.1
BlS_18Vibrio parahaemolyticus strain BF-1γ-Proteobacteria100KR137715.1
BlS_23Vibrio orientalis strain S-17γ-Proteobacteria99JF412251.1
BlS_26Pseudoalteromonas espejiana strain ATCC 29659γ-Proteobacteria98CP011028.1
BlS_29Cobetia sp. strain INV PRT183γ-Proteobacteria99MZ015175.1
BlS_31Pseudoalteromonas sp. strain S1-4-6γ-Proteobacteria99MK743964.1
BlS_33Pseudoalteromonas sp. strain S1-4-6γ-Proteobacteria99MK743964.1
BlS_40Pseudoalteromonas espejiana strain ATCC 29659γ-Proteobacteria99CP011028.1
BlS_43Vibrio owensii strain F77142γ-Proteobacteria99HQ908717.1
BlS_47Pseudoalteromonas sp. strain S1-4-6γ-Proteobacteria99MK743964.1
BlS_48Pseudoalteromonas sp. B-1054γ-Proteobacteria100DQ347554.1
BlS_50Vibrio breoganii strain FF50γ-Proteobacteria100CP016177.1
BlS_51Micrococcus yunnanensis strain Y19Actinobacteria99PP892572.1
BlS_52Rheinheimera sp. Gbf-Ret-3γ-Proteobacteria99AM117933.1
BlS_53Pseudoalteromonas sp. AB291dγ-Proteobacteria99FR821211.1
BlS_63Pseudarthrobacter sulfonivorans strain NC756Actinobacteria100MW741513.1
BlS_64Vibrio celticus 96-414γ-Proteobacteria99LN832940.1
BlS_65Vibrio orientalis strain S-17γ-Proteobacteria100JF412251.1
BlS_74Vibrio sp. VibC-Oc-051γ-Proteobacteria99KF577060.1
BlS_75Pseudoalteromonas sp. strain S1-4-6γ-Proteobacteria99MK743964.1
BlS_76Marinomonas lutimarisγ-Proteobacteria99MZ725944.1
BlS_78Micrococcus luteus strain KUDC1784Actinobacteria100KC355291.1
BlS_86Staphylococcus saprophyticus strain AA78Firmicutes100MW255282.1
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MDPI and ACS Style

Iliev, M.; Ilieva, R.; Peykov, S.; Terziyska, V.; Pelkin, A.; Kenderov, L. First Microbial Survey of a Submerged Petrified Forest in the Black Sea: Culture-Based and Metagenomic Insights. Diversity 2025, 17, 583. https://doi.org/10.3390/d17080583

AMA Style

Iliev M, Ilieva R, Peykov S, Terziyska V, Pelkin A, Kenderov L. First Microbial Survey of a Submerged Petrified Forest in the Black Sea: Culture-Based and Metagenomic Insights. Diversity. 2025; 17(8):583. https://doi.org/10.3390/d17080583

Chicago/Turabian Style

Iliev, Mihail, Ralitsa Ilieva, Slavil Peykov, Viktoria Terziyska, Anton Pelkin, and Lyubomir Kenderov. 2025. "First Microbial Survey of a Submerged Petrified Forest in the Black Sea: Culture-Based and Metagenomic Insights" Diversity 17, no. 8: 583. https://doi.org/10.3390/d17080583

APA Style

Iliev, M., Ilieva, R., Peykov, S., Terziyska, V., Pelkin, A., & Kenderov, L. (2025). First Microbial Survey of a Submerged Petrified Forest in the Black Sea: Culture-Based and Metagenomic Insights. Diversity, 17(8), 583. https://doi.org/10.3390/d17080583

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