Bioconversion of Organosolv Lignin by Rumen Bacterium: Isolation, Characterization and Metabolic Profiling

Abstract

Residual lignin generated by pulp, paper, and biorefining industries is commonly burned for energy, despite its potential as a renewable source of aromatic compounds. Studies focusing on microbial lignin degradation contribute to lignin valorization and represent a sustainable strategy to enhance biomass circularity. Here, we report the isolation of Klebsiella sp. IL2_9 from a ruminal consortium and demonstrate its ability to degrade and metabolize organosolv lignin. After 24 h of cultivation, the strain removed 22% of the initial lignin content. FTIR analysis revealed alterations in functional groups associated with guaiacyl and syringyl units, indicating structural modification of the polymer. GC–MS analyses further showed the consumption of lignin-derived aromatics, including vanillin, 2-aminobenzoic acid, and 4-hydroxybenzoic acid, along with the formation of vanillyl alcohol and phenyllactic acid derivatives. Overall, these findings highlight the potential of Klebsiella sp. IL2_9 as a promising biotechnological candidate for lignin valorization under anaerobic conditions.

1. Introduction

Organosolv lignin is a technical lignin primarily obtained as a by-product of the eucalyptus-based pulp industry and lignocellulosic biorefineries, representing a promising feedstock for the sustainable production of value-added chemicals [1,2,3]. In Brazil, the pulp and paper sector is among the largest worldwide and generates substantial amounts of this lignin-rich byproduct [4]. Compared to other technical lignins, organosolv lignin generally exhibits a higher purity level and lower sulfur content; however, it undergoes structural alterations during pretreatment, including cleavage and condensation reactions, which increase its chemical heterogeneity and recalcitrance relative to native lignin [5,6]. These characteristics represent a major bottleneck for the efficient and economically viable production of value-added aromatic products, as lignin deconstruction constitutes the first and most critical step in its valorization strategies [7].

The biological conversion of technical lignins has therefore attracted increasing interest as a milder and potentially more sustainable alternative to harsh chemical methods [8,9]. This approach is particularly attractive due to the extensive metabolic diversity observed among microorganisms. This metabolic versatility contrasts with conventional chemical approaches, which often converge toward a limited set of products, and further supports the exploration of multiple biological systems as complementary strategies for lignin valorization. In addition, given the high structural and compositional heterogeneity of technical lignins, particularly those derived from cellulose biorefineries, whose chemical constitution varies according to the lignocellulosic feedstock, it is unlikely that universal biological solutions for lignin conversion exist. In this context, the continued prospection and characterization of novel lignin-degrading microorganisms remains essential for the development of efficient lignin valorization strategies.

Aerobic bacteria, such as those from the genera Pseudomonas, Rhodococcus, Sphingomonas, Bacillus, and Aeromonas, have been reported to degrade lignin through oxidative processes [10,11]. In addition, an increasing number of anaerobic and facultative anaerobic lignin-modifying strains have been isolated from different environments, primarily using purified and kraft lignin as carbon sources [12,13,14,15]. Although some anaerobic bacteria have been shown to modify and depolymerize lignins, the mechanisms underlying their cleavage and subsequent metabolization under anaerobic conditions remain largely unresolved. Genomes of facultative anaerobic bacteria, such as those from the genera Klebsiella and Enterobacter, harbor genes encoding reductive and oxidative enzymes, including DyP-type peroxidases, dehydrogenases, and aryl-alcohol oxidases, as well as components of classical aerobic catabolic pathways. However, the functional involvement of these enzymes in lignin depolymerization and metabolization remains poorly supported by experimental evidence. Therefore, studies using isolated strains are essential to validate their metabolic potential and clarify their contribution to lignin bioconversion [16,17].

Our research group previously established and characterized two anaerobic microbial consortia capable of converting kraft lignin using bovine rumen microbiota as a microbial source [18]. These consortia contained Proteobacteria as the dominant phylum, and bacteria classified in orders Enterobacterales and Enterococcaceae. Furthermore, we aimed to obtain and characterize bacteria actively related to lignin degradation and metabolization. In the present study, we report the characterization of a bacterium and evaluate its potential to modify organosolv lignin under anaerobic conditions. The strain, designated IL2_9, was identified and assessed for its ability to grow in the presence of organosolv lignin. Changes in lignin functional groups and the presence of aromatic compounds were evaluated using FTIR and GC–MS analyses. The results provide experimental evidence that a rumen-derived bacterium can promote chemical modifications of organosolv lignin and generate aromatic compounds under anaerobic conditions, thereby adding to the limited body of knowledge on anaerobic lignin bioconversion.

2. Results and Discussion

2.1. Bacterial Strain Isolation and Identification

Isolate IL2_9 was identified as a species of the Klebsiella genus based on its 16S rDNA sequence. Best hits and high coverage were obtained in comparison to sequences of Klebsiella pneumonia, Klebsiella quasipneumonia and Klebsiella variicola (Table S1). The phylogenetic tree showed our isolate clustered with K. pneumoniae and K. variicola (Figure 1). Distinguishing species inside the Klebsiella genus including K. pneumoniae and K. variicola remains a labor-intensive task, due to the high degree of conservation in the 16S rDNA sequences among them [19]. Challenges in differentiating K. pneumoniae from closely related taxa, such as K. variicola, emphasize the need to explore new barcoding regions that can effectively distinguish these species.

2.2. Growth Curve and Lignin Degradation Analysis

Klebsiella sp. IL2_9 grew in liquid medium containing organosolv lignin as the sole carbon source in a time-dependent manner, reaching maximum growth at 48 h. Maximal degradation of organosolv lignin occurred within the first 24 h, achieving a degradation of 22% (Figure 2). This result is consistent with our previous work, which showed kraft lignin degradation and consumption by ruminal anaerobic consortia used as a source of lignin-converting bacteria mainly composed of members of the order Enterobacterales. Taxonomic analysis also revealed enrichment of the genus Dickeya. Therefore, our results support the hypothesis that facultative anaerobic Enterobacterales, such as Klebsiella spp., play a relevant role in lignin conversion within anaerobic microbiomes.

Previous studies have reported comparable degradation levels for technical lignins by Klebsiella species, including degradation of kraft and alkaline lignin by K. pneumoniae NX-1, 23.8% [20], and K. aerogenes TL3, 14.8%, respectively [13]. However, bacterial degradation of organosolv lignin has not been previously documented for Klebsiella spp.

In agreement with our results, Klebsiella species have been described as contributors to the deconstruction and utilization of hydrocarbon-derived compounds as carbon sources in different aerobic or anaerobic microbiomes [21,22,23]. These findings reinforce the broad metabolic diversity within this genus and support its potential involvement in the cycling of lignin-derived and other hydrocarbon-derived compounds across distinct ecosystems, including soil and the rumen. To date, most ligninolytic bacteria described in the literature are aerobic or microaerophilic, such as Pseudomonas, Rhodococcus, Streptomyces and Arthrobacter [8,24].

2.3. Characterization of Degradation Products by FTIR and GC-MS

FTIR spectra showed structural modifications in the residual lignin of cultures inoculated with Klebsiella sp. IL2_9 compared to the negative control, indicating that lignin modification was mediated by a biological agent (Figure 3; Table S2). The most evident alterations were detected at 1595, 1515, 1270, 2900, 853 cm⁻¹ and 618 cm⁻¹, while more moderate changes were detected at 1420 and 1325 cm⁻¹. Comparable FTIR alterations have been reported in studies investigating lignin degradation by bacterial co-cultures isolated from cow dung using alkaline lignin as substrate [25]. In that work, pronounced spectral changes were observed within the 3500–700 cm⁻¹ region following microbial treatment, indicating substantial lignin structural modification and supporting the previous results that Klebsiella sp. IL2_9 is capable of promoting structural rearrangements in technical lignin. Although differences exist between the lignin types and between microbial systems, the overlap in affected spectral regions supports the interpretation that bacterial metabolism in both cases can promote comparable structural rearrangements in technical lignins. Attenuations at 1595, 1513, and 618 cm⁻¹, associated with aromatic skeletal vibrations, suggest partial disruption or modification of aromatic structures. Reductions at bands 1270 and 853 cm⁻¹ indicate modification in guaiacyl and syringyl substructures, decreasing guaiacyl (G) and syringyl (S) units, consistent with alterations in C–O linkages and possible ether bond cleavage. In addition, the decrease at 2900 cm⁻¹ indicates changes in aliphatic side chains and methoxyl groups. Moderate attenuation at 1420 cm⁻¹, together with changes at 1325 cm⁻¹, supports structural rearrangements affecting aromatic ring vibrations and syringyl-associated substructures, indicating partial modification of lignin aromatic domains. Moreover, the absorption band at 1076 cm⁻¹, attributed to secondary alcohols and aliphatic ethers, was absent in lignin treated with strain IL2_9, while the intensity of the 1047 cm⁻¹ band, associated with primary alcohols and ether groups, was notably reduced (Figure 3).

These FTIR spectral changes indicate that Klebsiella sp. IL2_9 promotes structural modifications leading to partial lignin depolymerization and structural simplification, consistent with lignin-modifying activity. The decrease in bands associated with guaiacyl and syringyl units, together with the attenuation of aromatic and aliphatic signals, suggests lignin conversion involving aromatic rearrangements, ether bond cleavage and maybe demethylation. The disappearance and reduction of bands related to alcohols and ethers further indicate the consumption of lignin-derived compounds. Previous reports have shown that under aerobiose, laccases, DyP-type peroxidases, lignin peroxidase (LiP), and manganese peroxidase (MnP) are responsible for the initial modification of lignin through one-electron oxidation, ether bond cleavage, and demethylation [26,27,28,29]. Furthermore, K. pneumoniae NX-1 was described as presenting detectable laccase and lignin peroxidase activities during aerobic cultivation with kraft lignin as the sole carbon source and a soil isolate of Klebsiella sp. uncovered glutathione peroxidases and DyP-type peroxidases [17,20]. Cytochrome P450 monooxygenases have been reported to participate in lignin-derived compound transformation and in the oxidation of structurally complex xenobiotics in other microbial models [30].

Since these classical ligninolytic activities are associated with oxygen-dependent mechanisms, the modifications observed under anaerobic conditions suggest that Klebsiella sp. IL2_9 may rely on alternative enzymatic systems. This may likely involve a cooperative system in which DyP-type peroxidases initiate oxidation, while non-classical oxidoreductases support redox cycling and transformation of lignin-derived intermediates. At this stage, the enzymatic route responsible for lignin degradation by Klebsiella sp. IL2_9 cannot be clearly identified, highlighting the need for further targeted biochemical and genomic investigations.

GC–MS analysis revealed the presence of lignin-derived aromatic compounds, such as 4-hydroxybenzaldehyde, 2-amino benzoic acid, vanillin/isovanillin, and 4-hydroxybenzeneacetic acid in the supernatant of the control (non-inoculated) sample, which were absent in the Klebsiella sp. IL2_9 culture (Figure S1;

Table 1. Compounds identified by GC/MS in the supernatant of the Klebsiella sp. IL2_9 strain culture after 72 h of cultivation. “RT” indicates retention time. The symbols “+” and “-” indicate the presence and absence of the compound, respectively. Control: non-inoculated media subjected to the same growth conditions as the inoculated media and to the same preparation procedures for GC analysis.

No	RT (min)	Identified Compounds	Molecular Form	Control	Klebsiella sp. IL2_9
1	6.367	2,3-Butanediol, 2TMS/Propane-1,2-diol, 2TMS *	C10H26O2	-	+
2	6.691	1,3-Propanediol, 2TMS	C9H24O2	-	+
3	6.837	D-(-)-Lactic acid, 2TMS	C3H6O3	+	+
4	7.167	Glycolic acid, 2TMS	C2H4O3	+	+
5	9.100	Isovaleric acid, 2-hydroxy, 2TMS/2-Hydroxyvaleric acid, 2TMS/2-Methylbutyric acid, 2TMS *	C11H26O3	-	+
6	10.235	Ethyl phosphoric acid, 2TMS	C10H14O2	-	+
7	10.413	Pentanoic acid, 2-hydroxy-4-methyl, 2TMS/2-Hydroxy-3-methylvaleric acid, 2TMS *	C6H12O3	-	+
8	11.089	Phosphoric acid, 3TMS	H3O4P	+	+
9	11.661	Succinic acid, 2TMS	C4H6O4	+	+
10	12.061	Uracil, 2TMS	C4H4N2O2	+	+
11	12.488	Benzaldehyde, 4-hydroxy, TMS	C7H6O2	+	-
12	13.849	4′-Hydroxyacetophenone, TMS/2′-Hydroxyacetophenone, TMS/3′-Hydroxyacetophenone, TMS *	C11H16O2	+	+
13	14.308	4-Hydroxybenzyl alcohol, 2TMS	C13H24O2	-	+
14	14.584	Pyroglutamic acid, 2TMS	C5H7NO3	+	+
15	14.713	Vanillin, TMS/Isovanillin, TMS *	C11H16O3	+	-
16	15.372	2-Phenyl lactic, 2TMS/DL-3-Phenyllactic acid, 2TMS *	C15H26O3	-	+
17	15.691	Benzoic acid, 2-amino, 2TMS	C13H23NO2	+	-
18	15.810	p-Hydroxybenzoic acid, 2TMS /m-Hydroxybenzoic acid, 2TMS *	C13H22O3	+	+
19	15.956	Benzeneacetic acid, 4-hydroxy, 2TMS	C14H24O3	+	-
20	15.983	Isovanillylalcohol, 2TMS/Vanillyl alcohol, 2TMS *	C14H24O3	-	+
21	16.712	Syringaldehyde, TMS	C12H18O4	+	+
22	17.230	cis-Aconitic acid, 3TMS/trans-Aconitic acid, 3TMS *	C15H30O6	+	+
23	17.382	Vanillic acid, 2TMS	C14H24O4	+	+
24	18.100	Citric acid, 4TMS	C6H8O7	+	+
25	18.797	Syringic acid, 2TMS	C15H26O5	+	+
26	18.867	4-Hydroxyphenyllactic acid, 3TMS	C18H34O4	-	+
27	19.175	p-Coumaric acid, 2TMS	C15H24O3	+	+
28	20.639	Isoferulic acid, 2TMS /Ferulic acid, 2TMS derivative *	C16H26O4	+	+

* Compounds detected as isomers.

and Table S3). The absence of these metabolites after cultivation indicates their consumption by strain IL2_9, and their potential use as a source of carbon and/or energy. Although their consumption could theoretically occur via classical aerobic pathways (Figure 4), the absence of protocatechuate or catechol as intermediates, together with the detection of vanillyl/isovanillyl alcohol and 4-hydroxybenzyl alcohol in the culture supernatant, suggests that lignin-derived aromatic compounds are not predominantly transformed through classical aerobic pathways, such as the protocatechuate or catechol branches of the β-ketoadipate pathway [8]. Instead, our results suggest the activation of reducing reactions related to anaerobic branch metabolism, including the conversion of vanillin to vanillyl alcohol and of 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol, catalyzed by NAD(P)H-dependent reductive enzymes previously described in Klebsiella species [22,23] (Figure 4).

Transformation of aromatic compounds under oxygen-limiting conditions is associated with their conversion via the benzoyl-CoA pathway, in which substrates such as 2-amino benzoic acid are initially activated, forming benzoyl-CoA that then undergoes reductive dearomatization, hydration, oxidation, and cleavage of the dearomatized ring, resulting in the formation of aliphatic CoA-linked metabolites, including acetyl-CoA (Figure 4). These compounds are ultimately directed into central cellular metabolic pathways, including branches of the tricarboxylic acid cycle operating incompletely under anaerobic conditions and fermentative pathways [31,32]. Although genes encoding the canonical benzoyl-CoA pathway have not been identified in Klebsiella genomes available in public databases, the metabolic evidence obtained in this study supports the possibility that functionally related, yet-uncharacterized reductive mechanisms may contribute to the transformation of aromatic compounds during lignin degradation.

Phenyllactic acid, 4-hydroxyphenyllactic acid and the products associated with fermentative metabolism and amino acid catabolism, 2,3-butanediol and 2-hydroxyvaleric acid/isovaleric, were detected exclusively in the supernatant of the Klebsiella sp. IL2_9 culture (Table 1). The accumulation of phenyllactic and 4-hydroxyphenyllactic acids has been previously linked, under aerobic conditions, to the transformation of lignin-derived hydroxycinnamic acids, as p-coumaric and ferulic acids [33]. In anaerobic systems, microbial transformation of lignin fragments has frequently been associated with the formation and accumulation of phenyl acids and their derivatives [34], indicating the occurrence of alternative routes, which differ substantially from the classical aerobic pathway. However, the specific metabolic pathways involved in these transformations remain unknown.

2,3-Butanediol is a typical fermentation product derived from pyruvate via the acetoin pathway, which shares a central metabolic step with branched-chain amino acid (BCAA) biosynthesis [35]. Consistently, the formation of 2-hydroxyvaleric acid or 2-hydroxyisovaleric acid, metabolites associated with BCAA pathways, was also observed. Although the acetoin pathway has previously been described in Klebsiella strains cultivated anaerobically in the presence of D-glucose as a carbon source, in the case of strain IL2_9, cultivated under anaerobic conditions in the absence of simple sugars, the pyruvate required for the formation of 2,3-butanediol and BCAA-related metabolites may be generated from intermediates of aromatic metabolism, derived from the degradation of lignin. The accumulation of BCAA biosynthesis products and intermediate molecules is not typically observed during Klebsiella spp. fermentation [35], mainly due to the essential role of these amino acids for bacterial cells.

Although lignin-derived aromatics do not appear to be fully metabolized through canonical central pathways, their reduction may contribute to the maintenance of fermentative metabolism, probably acting as redox sinks, facilitating NAD(P)H reoxidation under anaerobic conditions. In fact, partial metabolization of lignin-derived compounds highlights the biotechnological potential of Klebsiella sp. IL2_9 for organosolv lignin valorization since several of the detected lignin-derived compounds, such as vanillyl alcohol, 4-hydroxybenzyl alcohol, phenyllactic acid and 4-hydroxyphenyllactic acid, are high-value aromatics. Vanillyl alcohol and 4-hydroxybenzyl alcohol have been reported to have applications in the flavor, fragrance, and pharmaceutical industries, whereas aromatic acids such as phenyllactic acid and 4-hydroxyphenyllactic acid have been associated with antimicrobial and antifungal activities and are being investigated as potential natural food preservatives [36,37,38].

Overall, our results indicate that Klebsiella sp. IL2_9 is capable of partially degrading organosolv lignin under oxygen-limited conditions and channeling aromatic derivatives into reductive metabolic pathways. In parallel, the reduction of aromatic aldehydes to alcohols suggests the activation of detoxification mechanisms and redox balancing. Taken together, the consumption of compounds detected in the control sample and the formation of reduced aromatic metabolites and fermentative products support a model in which strain IL2_9 not only modifies the lignin structure but also integrates its degradation products into cellular metabolism, thereby enabling growth in lignin-enriched media. Considering its ruminal origin, these results suggest that this bacterium may contribute to the microbial transformation of lignin in the rumen, acting in a complementary manner under the aerobic and anaerobic conditions present in this complex environment.

3. Materials and Methods

3.1. Lignin Preparation

Organosolv lignin was extracted from sugarcane bagasse using 70% (v/v) ethanol under acidic conditions at an elevated temperature of 190 °C. The resulting lignin was recovered from the liquid fraction and stored at 4 °C until further use.

3.2. Bacterium Isolation

The ruminal lignolytic consortium (KL), obtained as described by Silva et al. (2024) [18], was used as the source of bacteria. To obtain bacterial isolates, initially, 100 µL aliquots of the ruminal lignolytic consortium (KL) were inoculated into serum bottles containing solid reducing medium under anaerobic conditions and containing kraft lignin (0.35% w/v). This concentration was previously established by Silva et al. (2024) [18]. Kraft lignin was initially used because the consortium had been originally enriched using this substrate as a carbon source. The reducing medium consisted of NaH₂PO₄ (5 g L⁻¹), Na₂HPO₄ (2.5 g L⁻¹), NH₄Cl (0.5 g L⁻¹), (NH₄)2SO₄ (0.5 g L⁻¹), NaHCO₃ (0.5 g L⁻¹), MgCl₂ (0.09 g L⁻¹), mineral solution (5.0 mL L⁻¹), vitamin solution (0.5 mL L⁻¹), NaOH (0.04 g L⁻¹), Na₂S·9H₂O (0.125 g L⁻¹), cysteine (0.05 g L⁻¹), phytagel (15 g L⁻¹) and the carbon source [39]. Anaerobic conditions were established by boiling the liquid medium to remove dissolved oxygen, followed by the addition of reducing agents (cysteine and Na₂S·9H₂O). The medium was then dispensed into serum bottles under aseptic conditions, and the headspace was flushed with sterile nitrogen gas. The bottles were sealed with butyl rubber stoppers and aluminum crimps to maintain an oxygen-free environment throughout incubation. After two days at 37 °C, emerging colonies were selected and re-inoculated.

This procedure was repeated until pure isolates were obtained. Purified isolates were then cultured under static and anaerobic conditions for two days in reducing liquid medium, as described above, without phytagel and supplemented with 0.35% (w/v) kraft lignin. The cultures were cryopreserved at −80 °C with 50% (v/v) glycerol.

3.3. Bacterial Growth in the Presence of Organosolv Lingin

To further evaluate the ability of the isolates to utilize different types of lignin, they were also subsequently grown in liquid media containing 5% ethanol-soluble organosolv lignin as a carbon source. The 5% concentration was determined empirically, as it provided lower background color interference, fewer suspended particles, and reduced ethanol-related growth inhibition, thereby improving analytical reliability. Among the tested isolates, Klebsiella sp. IL2_9, which demonstrated growth in the presence of organosolv lignin, was selected for further analysis.

Klebsiella sp. IL2_9 ability to consume organosolv lignin was first evaluated based on its growth under anaerobic conditions at 37 °C in reducing liquid media, as described above, without phytagel and containing 5% (v/v) of organosolv lignin for 96 h. Culture samples were withdrawn every 24 h up to 96 h of growth to evaluate both growth and lignin degradation. For growth estimation, optical density (OD) at 600 nm was measured using a spectrophotometer, with non-inoculated medium containing lignin as a control to account for background interference [40]. To evaluate lignin degradation, culture samples were centrifuged at 10,000× g for 10 min at room temperature. The resulting cell-free supernatants were diluted 10-fold in 50 mM sodium phosphate buffer (pH 7.6), and absorbance at 280 nm was measured to monitor changes in soluble aromatic compounds derived from lignin [41]. Although organosolv lignin presents limited solubility in aqueous media, microbial degradation leads to the release of low-molecular-weight aromatic products that remain soluble and can be detected spectrophotometrically. Absorbance values were corrected using non-inoculated medium containing lignin incubated under the same conditions to account for background solubilization. This experiment was performed in biological triplicate. Absorbance at 280 nm was used as a preliminary indicator of lignin structural modification, which was further confirmed by complementary analytical approaches, including FTIR analysis and GC-MS.

3.4. Fourier Transform Infrared Spectroscopy Analysis

Klebsiella sp. IL2_9 was grown under anaerobic conditions at 37 °C in reducing liquid media, as described above, without phytagel and containing 5% (v/v) of organosolv lignin. After 72 h, the culture supernatant was centrifuged at 10,000× g for 20 min at 4 °C. The residual lignin obtained was used for FTIR analysis, and the supernatant was used as a sample source for GC-MS analysis. The resulting lignin was freeze-dried and stored at 60 °C to prevent reabsorption of moisture. Infrared spectra were recorded using a Bruker ALPHA FT-IR spectrometer (Billerica, MA, USA) operating in attenuated total reflectance (ATR) mode. Data acquisition was performed with 24 scans per sample over the spectral range of 4000 to 400 cm⁻¹.

3.5. Gas Chromatography-Mass Spectrometry

GC-MS analysis was performed as described by Silva et al. (2024) [18]. Initially, the supernatants obtained after 72 h of growth were centrifuged to remove cells and residual lignin. The resulting cell-free supernatant was acidified to pH 1–2 and extracted with ethyl acetate in a 3:1 (v/v) ratio. The organic layer was collected, dried, and then treated with 40 µL of pyridine, followed by incubation at 37 °C for 30 min. Subsequently, 70 µL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was added, and the mixture was incubated for an additional 30 min at 37 °C. A 1 µL aliquot of each derivatized sample was then injected into a GC-MS system (Agilent 7890–5975 MSD, Santa Clara, CA, USA) equipped with an Optima 5MS capillary column (30 m × 250 µm × 0.25 µm). The oven temperature program was as follows: initial temperature of 80 °C for 5 min, followed by a ramp of 10 °C/min up to 300 °C, with a final hold at 300 °C for 5 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The transfer line and ion source temperatures were maintained at 250 °C. Mass spectra were acquired in electron ionization (EI) mode at 70 eV, with a scan range of 30–550 m/z. Compound identification was performed using the NIST spectral library and the MSSearch software (version 2.3), and by comparing the chromatograms with authentic phenolic standards. Compounds that gave a match over 800 are reported in Table 1. Only compounds detected in at least two of the technical triplicates were considered. Where multiple putative matches were obtained, typically for isomers, all are reported.

3.6. Molecular Identification of Isolate IL2_9 (IL2-9)

To obtain the 16S rDNA sequence for IL2_9 (IL2-9) identification, the isolate was cultivated in 50 mL of reducing liquid medium in 100 mL serum bottles and incubated at 37 °C under static conditions and under an anaerobic atmosphere. After two days, the entire culture was centrifuged, and the harvested cells were used for genomic DNA extraction using DNeasy PowerSoil Pro Kit (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. The integrity and quality of the extracted DNA were assessed by electrophoresis on 0.8% (w/v) agarose gels and quantified using a NanoDrop spectrophotometer (ND-2000: NanoDrop™ 2000, Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA was used as template in polymerase chain reactions (PCRs) using the primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGAC-3′). PCR reactions were performed in a final volume of 25 µL, containing 50 ng of genomic DNA, 5 µM of each primer, 200 µM of dNTPs, 1 unit of Taq DNA polymerase (Cellco Biotech, São Carlos, Brazil), and 2.5 µL of Taq DNA polymerase buffer (Cellco Biotech, Brazil). Thermal cycling was carried out as follows: initial denaturation at 95 °C for 8 min; 35 cycles of 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, and a final extension at 72 °C for 5 min.

Amplicons were visualized using electrophoresis in 0.8% (w/v) agarose gel, and the DNA band was excised and purified using the Wizard^® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA), following the manufacturer’s instructions. Purified amplicon was sequenced by the company ACTGene Análises Moleculares (Alvorada, RS, Brazil) by Sanger sequencing. The quality of the obtained sequences was assessed using Phred quality scores through the online platform Electropherogram Quality Analysis (http://lbi.cenargen.embrapa.br/phph/, accessed on 10 August 2025). Trimmed sequences were then aligned using BLAST^® available online at the National Center for Biotechnology Information (NCBI) against the NCBI 16S ribosomal RNA sequence database. A phylogenetic tree was constructed using reference sequences retrieved from the NCBI GenBank database. The analysis was performed in MEGA software (version 11) using the neighbor-joining algorithm with 100 bootstrap replicates.

3.7. Statistical Analysis

All experiments were performed in triplicate. Statistical analyses were conducted using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) was applied to assess statistically significant differences among groups, followed by Tukey’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.

4. Conclusions

In summary, this study demonstrates that Klebsiella sp. IL2_9 is capable of modifying organosolv lignin under oxygen-limited conditions and transforming aromatic compounds derived from this macromolecule. These findings underscore the importance of facultative anaerobic bacteria from the rumen as key contributors to the modification of lignin-derived aromatic compounds, thereby participating in the transformation dynamics of plant biomass in anaerobic ecosystems. Finally, these results expand current knowledge on bacterial lignin modification under anaerobic or oxygen-limited conditions and reinforce the potential of Klebsiella sp. IL2_9 as a promising platform for lignin valorization strategies. Further biochemical, genomic, and transcriptomic analyses will be essential to elucidate the underlying enzymatic pathways and to support the development of sustainable bioprocesses for converting industrial lignin residues into value-added products.