Biotechnological Potential and Metabolic Diversity of Lignin-Degrading Bacteria from Decaying Tilia cordata Wood

TL;DR

This study explores bacteria from decaying wood that can break down lignin, a complex plant polymer, and highlights their potential for biotechnological applications.

Contribution

The paper presents a novel collection of lignin-degrading bacterial strains and their metabolic diversity from Tilia cordata wood.

Findings

Sixteen ligninolytic bacterial strains were isolated, including Raoultella terrigena MGMM806, which effectively depolymerized lignosulfonate.

The bacterial community showed high diversity and a balanced structure, with a Shannon index of 5.07.

Isolates exhibited varied catabolic capabilities for lignin monomer degradation, indicating metabolic partitioning.

Abstract

Lignin is a complex aromatic polymer that constitutes a major fraction of plant biomass and represents a valuable renewable carbon resource. Naturally decaying wood serves as an environmental reservoir of microorganisms capable of degrading lignin. In this study, we isolated and characterized sixteen bacterial strains from decaying Tilia cordata wood using an enrichment culture technique with lignin as the sole carbon source. Taxonomic identification via 16S rRNA gene sequencing revealed microbial diversity spanning the genera Bacillus, Pseudomonas, Stenotrophomonas, and several members of the Enterobacteriaceae family, including Raoultella terrigena isolates. Metagenomic sequencing of the wood substrate revealed an exceptionally rich and balanced bacterial community (Shannon index H′ = 5.07), dominated by Streptomyces, Bradyrhizobium, Bacillus, and Pseudomonas, likely reflecting a specialized consortium adapted to lignin rich late-stage decay. Functional phenotyping demonstrated that all isolates possess ligninolytic potential, evidenced by peroxidase/laccase-type activity through methylene blue decolorization. Dynamic Light Scattering (DLS) and HPLC analyses showed that some isolates, such as Raoultella terrigena MGMM806, effectively depolymerized lignosulfonate into low molecular weight fragments (1.23 nm), while others accumulated intermediate metabolites or completely mineralized the substrate. Growth profiling on monolignol substrates revealed a broad spectrum of catabolic specialization in lignin monomer degradation. The results demonstrate a complex system of metabolic partitioning within a natural bacterial consortium. This collection represents a foundational genetic resource for developing engineered biocatalysts and synthetic microbial communities aimed at the efficient conversion of lignin into valuable aromatic compounds.

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1. Introduction

Lignin, the second most abundant plant polymer after cellulose, constitutes up to 30% of plant biomass [1]. Its complex three-dimensional structure, formed from phenylpropanoid monomers (H, G, and S units), imparts mechanical strength to plant tissues but also confers high recalcitrance to biodegradation [2]. Global wood processing generates approximately 50 million tons of lignin-containing waste annually [3]. Despite growing interest in utilizing lignin as a renewable source of aromatic compounds, the development of efficient biological methods for its conversion remains an urgent and unmet challenge [3]. Natural degradation of lignin involves its depolymerization into low molecular weight aromatic compounds and the subsequent mineralization of these aromatic compounds. The breakdown of native lignin is carried out by extracellular oxidoreductases, such as lignin peroxidase, manganese peroxidase and laccase [4]. Fungi, equipped with potent extracellular enzyme systems, have traditionally been regarded as the primary agents of lignin degradation [5]. However, fungal enzymes are unsuitable for lignin depolymerization in industrial processes due to their thermal instability and poor tolerance to both high and low pH levels [6]. Furthermore, these enzymes are difficult to optimize through protein engineering, which is why they have not gained widespread commercial use [7]. In recent decades, interest in bacterial lignin degraders has been growing steadily due to their higher growth rates, metabolic flexibility, and ability to adapt to diverse conditions [8,9]. Although the bacterial method of lignin metabolism is not as efficient as fungal systems, it is evident that bacteria can interact with lignin and potentially produce smaller aromatic compounds. These compounds can be imported into the cell for aromatic catabolism, which is also widely distributed among soil bacteria [10]. Currently known lignin-degrading bacterial strains predominantly belong to the phyla Actinobacteria, Proteobacteria, and Firmicutes, and have been isolated from various lignin-containing substrates [9]. These studied bacterial strains produce peroxidases and laccases, and are capable of metabolizing monolignols released during lignin depolymerization [11]. Bacterial lignin degraders have been isolated from diverse sources, including kraft lignin, lignosulfonate, alkali lignin, treated and untreated straw from various cereals [9], as well as buffalo rumen contents [12] and termite guts [13]. Recent metagenomic analysis has revealed associations between specific microbial communities and the degree of wood decay [14,15,16]. Wood in advanced stages of decay represents a potentially rich source of taxonomically and metabolically diverse bacteria, as evidenced by metagenomic studies of lignin-containing niches [17]. The ability to modify and degrade lignin explains why representatives of Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria often dominate the final stage of wood decomposition [18,19]. Nevertheless, late-stage decaying wood remains an underexplored reservoir of strains and genetic systems for lignin degradation. The pattern of bacterial succession during wood decay is not universal but depends fundamentally on tree species and the resident microbiome. Studying key strains and their interactions within consortia can reveal specialized strategies for efficient lignin breakdown. Expanding this research to include novel tree species from diverse ecosystems is therefore essential [12,13,20]. To our knowledge, lignin-degrading bacteria from decaying Tilia cordata have not been systematically characterized. Therefore, the aim of this study was to isolate and characterize bacterial strains capable of lignin degradation from decaying lime wood (Tilia cordata). To achieve this, an enrichment culture approach was employed using a mineral medium with technical lignin as the sole carbon source, followed by the isolation of pure cultures and an analysis of their lignin-degrading potential.

2. Materials and Methods

2.1. DNA Extraction, Library Preparation, and Shotgun Metagenomic Sequencing

To assess the taxonomic and functional diversity of the total microbial community, we performed shotgun metagenomic sequencing. Total DNA for metagenomic analysis was extracted from a sample of decaying lime wood (Tilia cordata) using the commercial Magen HiPure Soil DNA Kit (Magen, Guangzhou, China). The quality of the extracted DNA was assessed by agarose gel electrophoresis, while DNA concentration was determined using a NanoDrop OneC spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing libraries were prepared following the protocol of the Native Barcoding Kit 96 V14 (SQK-NBD114.96) (Oxford Nanopore Technologies, Oxford, UK) with modified incubation times for enzymatic reactions. The barcoded libraries were then loaded onto a PromethION Flow Cell (R10.4.1) and sequenced. Sequencing data quality was evaluated using FastQC. Quality assessment of the raw sequencing data and calculation of key read statistics (total yield, median and N50 read lengths, GC content) were performed using SeqKit. Taxonomic classification of the shotgun metagenomic reads was performed using program complexes Kraken2 [21] and Bracken [22] with the ‘bacteria’ database [21]. For visualization, the interactive metagenomic visualization tool Krona was used [23]. Statistical analysis was conducted in R package version 2.8-0, the vegan (https://vegandevs.github.io/vegan/, accessed 20 October 2025) [24] and ggplot2 packages [25].

2.2. Isolation and Identification of Microorganisms

Wood samples were collected in Kazan, Russia (55.73239° N, 49.21133° E). The stage of wood decay was determined according to Hunter; the sample belonged to the last stage [26]. Decaying wood fragments were aseptically collected using sterile forceps and transferred into sterile 50 mL conical tubes. For subsequent bacterial isolation, samples were stored at +4 °C and processed within 48 h. For DNA extraction, separate samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C until further processing. Samples of decaying lime wood (Tilia cordata) were used for the isolation of bacterial strains. To increase the abundance of lignin-degrading microorganisms, an enrichment culture was established using base medium (BM) contained K_2_HPO_4_—5.8 g/L, KH_2_PO_4_—3 g/L, (NH_4_)2_SO_4—1 g/L, and 1% (w/v) of lignin alkali (Sigma-Aldrich, St. Louis, MO, USA), which served as the sole source of carbon and energy for bacterial growth. One milliliter of a suspension of macerated wood (1 g in 10 mL of sterile 1% NaCl) was inoculated into 100 mL of the mineral medium with lignin. The enrichment culture was incubated with shaking (180 rpm) at 30 °C for 72 h. Subsequently, 1 mL of the culture was transferred to fresh BM with lignin, and cultivated under the same conditions. After three consecutive passages, a series of dilutions were performed and plated onto agar plates containing BM with lignin. The plates were incubated at 30 °C for 72 h. Following incubation, 16 bacterial strains were isolated. Pure cultures of each strain were grown to obtain sufficient biomass and subsequently cryopreserved for long-term storage in a 30% (v/v) glycerol solution at −80 °C. Primary species identification of the isolates was performed based on comparative analysis of 16S rRNA gene nucleotide sequences. Total DNA was isolated from the strains with the TRIzol kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Amplification of the full-length 16S rRNA gene fragment was carried out with universal primers 27 fm (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1522R (5′-AAGGAGGTGATCCAGCCGCA-3′) [26]. The amplified fragment was isolated from a 1% TAE agarose gel using a DNA purification kit (Evrogen, Moscow, Russia). The nucleotide sequence of the purified DNA fragment was determined using Sanger sequencing (Evrogen, Moscow, Russia). The sequences were assembled using the Clone Manager 9.0 software (USA) and analyzed using the NCBI BLAST web interface (https://blast.ncbi.nlm.nih.gov, accessed on 20 June 2025). The strains were identified based on the highest sequence similarity to strains of known species from the GenBank database.

2.3. Determination of Enzymatic Activities of the Bacterial Isolates

2.3.1. Determination of Amylolytic Activity

A modified medium of the following composition was used to determine amylolytic activity (g/L): peptone—0.5; KCl—0.1; MgSO_4_ × 7H_2_O—0.5; (NH_4_)2_SO_4—0.1; starch—20.0; bacteriological agar—20.0. Cultures were streaked onto the surface of the starch agar and incubated at 30 °C for 48 h. After incubation, the cells grown on the plates were treated with 2.5% Lugol’s iodine solution, and their amylolytic activity was assessed by observing clear zones around the bacterial colonies on a dark blue background, due to the formation of an iodine/starch complex [27]. All enzyme activity assays were performed in five biological replicates. A positive result was defined as a zone exceeding 2 mm from the colony edge.

2.3.2. Determination of Lipase Activity

A nutrient medium supplemented with Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) was used to determine lipase activity (g/L): peptone—10.0; NaCl—5.0; CaCl_2_ 2H_2_O—1.0; bacteriological agar—20.0; Tween 80—10.0. Cultures were streaked onto plates containing the selective medium and incubated at 30 °C for 48 h. Lipase activity was indicated by the formation of fine white crystals (calcium laurate) around the microbial colonies [28].

2.3.3. Determination of Proteolytic Activity

Proteolytic activity was detected on BM of the following composition (g/L): K_2_HPO_4_—5.8; KH_2_PO_4_—3.0; (NH_4_)2_SO_4—1.0; bacteriological agar—20.0, supplemented with 10 g/L glycerol and 1% skim milk powder, sterilized by boiling. Cultures were streaked onto the surface of the nutrient agar and incubated at 30 °C for 48 h. Protease activity was assessed visually by the formation of zones of clearance (hydrolysis zones) around bacterial colonies, indicating the degradation of casein by extracellular proteases [29].

2.3.4. Determination of Phytase Activity

Phytase activity was determined on PSM medium of the following composition (g/L): glucose—2.0; sodium phytate—5.0; NH_4_NO_3_—5.0; MgSO_4_ × 7H_2_O—0.5; KCl—0.5; FeSO_4_ × 7H_2_O—0.01; Mn(SO_4_)2× 4H_2_O—1.0; CaCl_2_—2.0; bacteriological agar—20.0. Cultures were streaked onto the selective medium and grown at 30 °C for 48 h. Phytase activity was assessed by the formation of transparent hydrolysis zones around the colonies, resulting from the degradation of insoluble phytic acid and the formation of a calcium-phosphate chelate complex [30].

2.3.5. Determination of Cellulolytic Activity

Cellulolytic activity was detected using BM supplemented with 1% sodium carboxymethyl cellulose (CMC) (g/L): tryptone—1.0; K_2_HPO_4_—5.8; KH_2_PO_4_—3.0; CMC—10.0; bacteriological agar—18.0. The presence of cellulolytic (endoglucanase) activity was determined by visualizing zones of clearance [31].

2.3.6. Determination of Peroxidase Activity in Strains

Peroxidase synthesis was determined using a method described previously [32,33]. Bacterial isolates were inoculated into liquid King’s B medium [34] supplemented with 20 µg/mL methylene blue, then incubated at 28 °C for 7 days. The culture broth was centrifuged at 13,400× g, and the supernatant was collected for subsequent measurement of methylene blue absorbance at wavelengths of 665 nm. The experiments were performed with five replicates, and the average was considered.

2.4. Assessment of Isolate Capability for Lignosulfonate Degradation

To evaluate the ability of the strains to degrade lignosulfonate, they were incubated in BM supplemented with 0.5% lignosulfonate for 72 h at 30 °C with shaking (180 rpm). The resulting culture was centrifuged (13,000× g, 10 °C, 10 min) to remove cell mass. The obtained supernatant was filtered using 0.2 µm nitrocellulose filters (Merck Millipore, Burlington, MA, USA Germany). The particle size of lignosulfonate in the supernatants was measured by Dynamic Light Scattering (DLS) using a Benano 180 Zeta Pro spectrometer (Bettersize Instruments, Dandong, China) with monochromatic laser radiation at a wavelength (λ) of 633 nm. All measurements were performed at 21 °C and a scattering angle (θ) of 90°. The measurable particle size range was 0.3–200 nm. Autocorrelation functions were recorded, and data were processed using the BeNano V3.0 software. A control consisted of a 0.5% lignosulfonate solution passed through a nitrocellulose filter [35].

2.5. Evaluation Ability of Bacterial Isolates to Utilize Monolignols and Their Derivatives

The capability of bacterial strains to degrade monolignols was assessed by monitoring their growth on BM, where the target compounds served as the sole source of carbon and energy. A volume of 100 µL of a 2% solution of one of the tested monolignols or their derivatives (gallic acid, p-coumaric acid, syringic acid, syringol, creosol, guaiacol, 4-propylguaiacol and catechol) dissolved in 100% isopropanol was spread onto the surface of the mineral medium. The solvent was evaporated under a laminar flow hood for 30 min. The isolates were streaked onto the prepared plates and incubated at 30 °C for 72 h. A control plate contained BM was supplemented with 100 µL of isopropanol. Substrate consumption was evaluated based on the presence of visible bacterial growth on the selective media.

2.6. Determination of Intermediates After Lignosulfonate Degradation by High-Performance Liquid Chromatography (HPLC)

To determine the changes occurring in lignosulfonate under the action of the microorganisms, the studied strains were incubated in BM supplemented with 0.5% lignosulfonate for 72 h at 30 °C. The intermediates were extracted with 3 volumes of chloroform after acidifying the samples with hydrochloric acid to pH 3.5. The organic solvent was evaporated, and residues were dissolved in 50% methanol solution. Analysis of untreated lignosulfonate extracts and those after bacterial cultivation was performed by HPLC using an Arcus 120 C18 column 5 µm, 4.6 × 150 mm (Exformma Technologies, Shanghai, China) with a photodiode array detector. The eluents were: A—94.95% deionized water, 5% acetonitrile, 0.05% acetic acid; B—94.95% acetonitrile, 5% deionized water, 0.05% acetic acid. Separation was carried out using a gradient of phase B under the following conditions: 0–2 min—20% B, 2–20 min—gradient to 85% B, 20–20.1 min—gradient to 95% B, 20.1–22 min—95% B, 22–22.7 min—gradient to 20% B, 22.7–30 min—20% B. The flow rate was 0.8 mL/min at 30 °C. The autosampler temperature was set at 10 °C. UV detection was performed at 210 nm.

2.7. Statistical Analysis of Results

Statistical analysis was performed using the OriginLab Pro software package (version SR1 b9.5.1.195; OriginLab Corporation, Northampton, MA, USA). Prior to conducting the ANOVA, its assumptions were formally assessed. The normality of data within each group was verified using the Shapiro–Wilk test (p > 0.05 for all groups). The homogeneity of variances (homoscedasticity) was confirmed using Levene’s test (F (,) = [value], p = [value] > 0.05). As all assumptions were satisfied, a standard one-way ANOVA was applied. The sample size was n = [5] independent biological replicates per group [36].

3. Results

3.1. Metagenomic Analysis of Decaying Lime Wood Sample

Metagenomic analysis was performed to characterize the taxonomic composition and functional potential of the microbial community in a sample of decaying lime wood (Tilia cordata) at the terminal stage of decomposition. Sequencing on a PromethION platform (Oxford Nanopore Technologies) yielded 1,286,989 reads with a total volume of 877 million base pairs (Mbp) (Table 1). The length of individual reads ranged from 5 to 615,519 base pairs (median = 367 bp, N50 = 1204 bp), which is characteristic of PromethION long-read sequencing data and indicates the presence of a substantial fraction of long fragments suitable for assembly. The average GC content of the meta-genome was 58%. Despite accounting for a relatively low proportion of the total DNA pool, the bacterial community exhibited exceptionally high taxonomic richness. Analysis of alpha-diversity revealed the following metrics (Table 1).

The analysis revealed that only 37.98% of all reads were assigned to bacterial taxa (Bioproject PRJNA1377609). The low percentage of bacterial DNA in the overall data pool unequivocally indicates the predominance of eukaryotic DNA in the sample, primarily fungal (mycobiome) and plant-derived (remnants of the Tilia cordata genome). Visualization of the most representative genera (Figure 1) demonstrates the complex multi-species structure of the core bacterial community [37,38].

A key finding is the identification of an exceptionally diverse, balanced, and mature bacterial community, as evidenced by high alpha-diversity indices (Shannon index (H′) = 5.07, Pielou’s evenness (J′) = 0.708; see Table 1).

3.2. Taxonomic Identification of Isolated Bacterial Strains

Sixteen bacterial isolates were obtained from an enrichment culture incubated with decaying lime wood as the substrate. Taxonomic identification of the isolates, performed by comparative analysis of 16S rRNA gene sequences, revealed that the isolated microbial community was predominantly represented by the genera Pseudomonas and Bacillus. Among the identified taxa, representatives of the Enterobacteriaceae family, encompassing five different species, were also detected. Isolates MGMM805, MGMM806, and MGMM808 were classified as Raoultella terrigena, making it the most prevalent taxon within the studied strain collection (Table 2). The strains P. costantinii MGMM800, P. geniculata MGMM809, B. albus MGMM810, P. psychrodurans MGMM811, B. altitudinis MGMM812, B. sonorensis MGMM813, P. lutea MGMM814, and P. oryzihabitans MGMM815 are generally considered non-pathogenic and thus potentially suitable for application in open systems.

3.3. Enzymatic Profiling of Bacterial Strains

To phenotypically characterize the isolated strains, a screening of their enzymatic activities was performed. Determining the spectrum of synthesized exoenzymes is a key tool for differentiating strains within a microbial community and for assessing their biotechnological potential. The screening revealed specific fermentation profiles. It was demonstrated that none of the investigated strains exhibited cellulase activity. Notably, none of the investigated strains exhibited cellulase activity, which is consistent with their isolation from wood at a late stage of decomposition where accessible cellulose may already be depleted. Amylase activity was detected exclusively in strain B. albus MGMM810. The ability to synthesize lipase was established for isolates P. costantinii MGMM800 and Enterobacter sp. MGMM802. Proteolytic activity against casein was observed in five strains (P. costantinii MGMM800, E. americana MGMM801, Enterobacter sp. MGMM802, S. rhizophila MGMM807 and B. albus MGMM810). Phytase activity was demonstrated for strains E. americana MGMM801, Pantoea sp. MGMM803, Citrobacter sp. MGMM804, R. terrigena MGMM805, R. terrigena MGMM806, and R. terrigena MGMM808. The complete screening results for all enzymatic activities are presented in Table 3.

The screening results revealed that six strains tested negative for all screened activities (P. geniculata MGMM809, P. psychrodurans MGMM811, B. altitudinis MGMM812, B. sonorensis MGMM813, P. lutea MGMM814, P. oryzihabitans MGMM815).

3.4. Determination of Peroxidase Activity in Isolated Strains

Quantitative assessment of peroxidase/laccase-like activity, based on the rate of methylene blue decolorization, confirmed the presence of this function in all isolated strains, albeit with significant quantitative differences (Figure 2). Strain MGMM812 was identified as the most active, achieving a decolorization level exceeding 95%. A group of seven strains (MGMM803, MGMM804, MGMM805, MGMM808, MGMM809, MGMM814, MGMM815) showed 3- to 4-fold lower values for this parameter. Strain MGMM801 demonstrated statistically significantly the lowest activity in the conducted test.

3.5. Evaluation of Strains for Lignosulfonate Utilization by Dynamic Light Scattering Method

Lignosulfonates are a byproduct of wood processing using sulfite pulping and represent water-soluble sulfonated derivatives of lignin. The particle size of the lignosulfonate used was approximately 5.9 nm. In the supernatant samples following cultivation of strains P. costantinii MGMM800 and P. oryzihabitans MGMM815, a slight reduction in particle size to 3.24 nm and 3.99 nm, respectively, was recorded. The higher degree of depolymerization was observed in the supernatant of strain R. terrigena MGMM806, where the average particle size decreased to 1.23 nm. Qualitative analysis of chromatographic/spectral data (Figure 3) also revealed a reduction in the integrated peak area corresponding to lignosulfonates in samples treated with strains MGMM806 and MGMM815, indirectly indicating degradation and utilization of the substrate by these isolates. In the supernatants of the remaining strains, the presence of lignosulfonate particles was not detected, which is consistent with extensive depolymerization of the compound (Figure 3).

3.6. Analysis of Lignosulfonate Degradation by HPLC

The metabolic activity of the isolated bacteria towards lignosulfonate was assessed by differences in the chromatographic profiles of the resulting intermediates. Compared to the control sample, a reduction in the quantity of most detectable compounds was observed in the extracts after bacterial cultivation. In addition to a decrease in intensity up to the complete disappearance of individual peaks, new components were identified in the profiles of some strains. A hierarchical cluster dendrogram, generated using MetaboAnalyst 6.0 [39], illustrates the separation of samples into four distinct clusters (Figure 4).

The first cluster is represented by the control sample. The second cluster (strains MGMM802, MGMM803, and MGMM808) demonstrates a significant decrease in the quantity and number (10 out of 19) of peaks, indicating almost complete utilization of depolymerization products. The third cluster (7 strains, including MGMM804) shows partial degradation of most components (15–19 peaks) and occasional formation of new compounds. The fourth cluster (MGMM801, MGMM806, MGMM807, MGMM809, MGMM810, and MGMM812) shows the lowest degree of degradation (15–23 peaks) and accumulation of new intermediate products. Thus, based on chromatographic analysis, the strains can be divided according to their ecological strategies: (1) complete metabolizers, which almost completely utilize the substrate; (2) partial metabolizers, which slowly transform most components; (3) intermediate storage tanks that intensively form new products during incomplete degradation of the mixture.

3.7. Growth of Isolates on Mineral Medium Supplemented with Monolignols as Carbon and Energy Sources

It is known that a key stage in lignin degradation is its depolymerization into H-, G-, and S-type monolignols, followed by their conversion into low-molecular-weight phenolic compounds. Since the metabolic pathways for degrading monolignols of different structural types vary, the spectrum of utilized substrates can serve as an indicator of the presence of specific enzymatic systems in microorganisms necessary for their utilization. The ability of the obtained strains to catabolize aromatic compounds was evaluated by testing their capacity to use the target substances (gallic acid, p-coumaric acid, syringic acid, syringol, guaiacol, 4-propylguaiacol, and catechol) as the sole carbon source when grown on a minimal medium. The results obtained during the experiment are presented in Table 4.

Data analysis revealed significant diversity in the metabolic profiles among the tested strains. The broadest substrate spectrum was demonstrated by a group of eight strains (MGMM800, MGMM803, MGMM804, MGMM806, MGMM808, MGMM809, MGMM811 and MGMM815), which were capable of growth on four or more of substrates, indicating the presence of highly efficient and non-specific enzymatic degradation systems in these isolates. The remaining strains were characterized by a limited ability to utilize the tested substrates. Strain MGMM802 exhibited growth exclusively on p-coumaric acid, strain MGMM812 utilized only syringol and strain MGMM814 grew solely on catechol. Strain MGMM801 utilized guaiacol, 4-propylguaiacol and catechol. Strain MGMM813 showed no growth on any of the proposed substrates However, based on HPLC and DLS results, we can hypothesize that strain MGMM813 may be a potential degrader of lignin and its derivatives. The obtained data on the variability of substrate utilization profiles confirm the presence of diverse metabolic pathways involved in the degradation of lignin-derived aromatic compounds among the studied isolates.

4. Discussion

Decaying wood, after the removal of cellulose and hemicellulose, consists primarily of lignin. Although a less favorable substrate for microorganisms than cellulose, lignin can still be utilized by microbes as a carbon and energy source [40]. It has been shown that the stage of wood decomposition significantly influences the composition of the microbial community [41]. It can be hypothesized that at the terminal stages of wood decay, after the depletion of readily available polysaccharides (cellulose and hemicelluloses), organisms capable of lignin utilization begin to dominate the microbial community. Therefore, studying microbial communities that emerge specifically during the late stages of wood degradation can help identify the most effective bacterial lignin-degrading strains. This aligns with the nature of the sample, where fungi, as known primary decomposers of lignin and cellulose, play a key role, especially during the initial and intermediate stages of decomposition [42]. Our metagenomic analysis of a sample from decaying lime wood revealed 1293 bacterial taxa, which can be characterized as exceptionally high taxonomic diversity. The absence of pronounced dominance (supported by high evenness and Shannon index values) is a characteristic feature of a mature and balanced microbial ecosystem [15]. This structure could have formed as a result of a prolonged successional process during the late stage of wood decay, when easily accessible polysaccharides (cellulose, hemicellulose) are largely depleted, and the niche shifts to organisms specializing in the utilization of lignin and complex secondary metabolites. Such a community structure may reflect a complex partitioning of ecological niches and metabolic functions among different microbial groups, collectively enabling the complete mineralization of the complex lignocellulosic matrix. For the selection of target microorganisms, an enrichment culture on a mineral medium with technical lignin (lignosulfonate) as the sole source of carbon and energy was used. This approach allowed for the isolation of 16 bacterial strains physiologically adapted to the utilization of this polymer. Notably, none of the isolated strains exhibited cellulase activity, while amylase and lipase activities were detected in only a limited number of strains. This serves as direct experimental confirmation that at the terminal stages of wood decay, after the depletion of readily accessible polysaccharides and lipids, the microbial community shifts in favor of organisms specializing in the utilization of lignin the most recalcitrant component of plant biomass [43]. Phylogenetic analysis revealed the taxonomic affiliation of the isolates to three main groups: the family Bacillaceae (B. albus MGMM810, B. altitudinis MGMM812, B. sonorensis MGMM813, and Ps. psychrodurans MGMM811), the family Pseudomonadaceae (P. costantinii MGMM800, P. geniculata MGMM809, P. oryzihabitans MGMM815, and P. lutea MGMM814), and representatives of the family Enterobacteriaceae (R. terrigena MGMM805, R. terrigena MGMM806, R. terrigena MGMM808, Enterobacter sp. MGMM802, Citrobacter sp. MGMM804). These findings align with current literature highlighting the key role of these taxa in the degradation of aromatic compounds in natural ecosystems [44,45,46,47]. The species S. rhizophila was represented by a single strain, MGMM807. The ability to metabolize low-molecular-weight compounds generated during lignin degradation has been previously described for a representative of this genus [48]. With the exception of the enterobacterial strains, all isolated bacterial strains belong to non-pathogenic species, making them suitable for use even in open systems. Of particular interest is the absence of actinobacteria in our collection, as they are traditionally considered among the most active bacterial lignin degraders [49]. This fact may indicate the specificity of the studied ecological niche, where alternative bacterial taxa play a dominant role in late-stage lignin degradation. This could be due to the higher competitiveness of the isolated strains under conditions of limited readily available substrates. A characteristic feature of the studied strains is the high frequency of phytase activity (over 50% of isolates), which can be viewed as an adaptation to phosphorus limitation, typical for decaying wood [50]. The presence of protease activity in five strains may provide them with competitive advantages in the struggle for resources within the complex microbial community. The ability to depolymerize lignin was confirmed in the methylene blue assay used for detecting peroxidase and laccase activity. All isolates exhibited dye decolorization to varying degrees, with the highest activity recorded for strain B. altitudinis MGMM812. This result correlates with literature data on the ability of B. altitudinis representatives to produce peroxidase/laccase-like enzymes [51,52]. Lignosulfonate consists of aryl-propane oligomers with good solubility due to attached sulfate groups. Investigating the degradation of this polymer revealed various utilization strategies. Dynamic Light Scattering (DLS) data showed that strain R. terrigena MGMM806 effectively depolymerized lignosulfonate into low-molecular-weight fractions (1.23 nm). Meanwhile, strains P. costantinii MGMM800 and P. oryzihabitans MGMM815 only slightly reduced lignosulfonate particle size. For the remaining strains, no particulate signal was detected by DLS. This absence of signal is consistent with extensive depolymerization, resulting in soluble fragments or monomers that fall below the instrument’s detection threshold. Profiling of degradation products by HPLC delineated three distinct strategic clusters. The first cluster contained efficient degraders without intermediate accumulation (strains MGMM802, MGMM803, MGMM808). The second cluster comprised strains that accumulated intermediate degradation products (MGMM800, MGMM804, MGMM805, MGMM811, MGMM813, MGMM814, and MGMM815). The third cluster included strains generating the greatest diversity of intermediates (MGMM801, MGMM806, MGMM807, MGMM809, MGMM810, and MGMM812). The resulting distribution of strains across three clusters reflects fundamental differences in their metabolic strategies. Strains in the first cluster may be capable of near-complete mineralization of lignosulfonate with minimal accumulation of detectable intermediates. Isolates in the second cluster, characterized by transient accumulation of metabolites, may exhibit limitations (“bottlenecks”) in the late stages of catabolic pathways. Most interesting are the strains in the third cluster, which produce a wide range of intermediates, indicating a diversified enzymatic apparatus for depolymerization, but possibly incomplete subsequent utilization. Such functional diversity within the community indicates potential synergism, where different microorganisms work together to ensure the complete degradation of a complex polymer. The ability of nine strains to grow on all tested types of monolignols (H-, G-, and S-types) indicates the presence of a versatile and relatively non-specific enzymatic system capable of incorporating a wide spectrum of aromatic structures into metabolism. This group included strains that both efficiently utilized lignosulfonate and accumulated degradation intermediates. Strains with highly specialized metabolic profiles are of particular interest. Strain MGMM812, demonstrating maximum activity in the methylene blue assay and complete lignosulfonate degradation, was capable of growth only on syringic acid. We can assume that this suggests the presence of a highly specialized system for monolignol degradation, possibly involving O-demethylation proteins such as cytochrome P450 enzymes similar to the SyoA/SyoB proteins described for other bacterial degraders like Amycolatopsis thermoflava [53]. However, this functional assignment remains a hypothesis requiring direct genetic and biochemical validation in our isolates. Strain MGMM801, growing exclusively on catechol, guaiacol, and its derivatives, likely possesses a guaiacol degradation system analogous to GcoA/GcoB from Amycolatopsis thermophila [54] but lacks pathways for utilizing S-type monolignols. Most intriguing is strain MGMM813, which, while efficiently utilizing lignosulfonate, did not grow on the offered monolignols. This study demonstrates that at the terminal stages of lime wood degradation, a specific bacterial community forms, represented by strains of Bacillus, Pseudomonas, and enterobacteria. The revealed diversity of metabolic strategies, from generalist to highly specialized, underscores the complex nature of lignin biodegradation under natural conditions. The obtained results open prospects for utilizing the isolated strains in biotechnological processes aimed at processing lignocellulosic waste. Future research should focus on the molecular genetic characterization of the identified metabolic pathways and the study of synergistic interactions among different degrader strains.

5. Conclusions

The use of decaying wood as a source of lignin-degrading microorganisms, combined with an enrichment culture strategy on lignin-supplemented medium, enabled the successful isolation of bacterial strains capable of lignin degradation. The study of these lignin-degrading strains revealed substantial diversity, both in terms of species composition and their biochemical properties. All isolated strains demonstrated an ability to depolymerize lignin, as evidenced by varying degrees of methylene blue decolorization and lignosulfonate breakdown. Strains involved in lignin degradation may possess enzymatic machinery enabling them to completely mineralize this biopolymer or to participate only in specific stages of its breakdown. In our study, we obtained strains that effectively depolymerized lignosulfonate but metabolized only a limited spectrum of monolignols or, in some cases, only catechol as a product of guaiacol O-demethylation. Strains unable to degrade monolignols and catechol are of particular interest, as they likely degrade lignin via pathways that do not yield standard monomeric compounds. The identified patterns suggest the existence of diverse lignin catabolism strategies among the isolated strains. However, further research, including proteomic analysis, metabolomics, and functional characterization of key genes it can be necessary to verify the specific metabolic pathways and identify the responsible enzymes. We propose that the strains isolated in this study can serve as sources of genetic systems for the decomposition of lignin and its derivatives. Furthermore, the non-pathogenic isolates may form a foundation for engineering specialized lignin-degrading strains, including those capable of differentially utilizing the breakdown products of this biopolymer.