Introduction

During tobacco cultivation, large quantities of tobacco straw are generated, and improper disposal of this biomass can cause significant environmental impacts1. Conventional management approaches such as incineration, landfilling, or utilization as fertilizer or livestock feed demonstrate low efficiency and fail to fully exploit the intrinsic value of tobacco straw2. Notably, tobacco straw contains abundant cellulose (35–45%), hemicellulose (15–25%), and lignin (10–20%)3, presenting substantial potential for valorization. Consequently, developing innovative strategies for efficient tobacco straw utilization—particularly microbial degradation technologies—could address both environmental concerns and resource recovery challenges4.

While Acinetobacter indicus has been demonstrated to degrade phthalate esters5, solubilize phosphorus6, remediate gaseous methyl tert-butyl ether (MTBE) contamination7, and remove heavy metals from soil8, its capacity for lignocellulose degradation remains largely unexplored. In this study, we report the first isolation of a lignocellulose-degrading Acinetobacter indicus strain, designated A. indicus LXX12, from tobacco-planting soil. Through comprehensive analyses including enzymatic activity assays, straw decomposition rate quantification, Fourier-transform infrared spectroscopy (FTIR), and whole-genome sequencing, we systematically elucidated the strain’s functional roles in cellulose and lignin degradation. Our findings provide novel insights into the bioconversion of tobacco straw, offering a sustainable approach for agricultural waste management and environmental protection.

Materials and methods

Experimental materials

Material source

Sampling location: Yangwu, Xixiu District, Anshun City, Guizhou Province, China. Source of material: Tobacco-growing soil.

Main culture media

(1) Enrichment Medium9: NaNO2 0.5 g, KCl 0.5 g, KH2PO4 1.0 g, Fe2(SO4)2·7 H2O (1% stock solution, 1 mL added), MgSO4·7 H2O 0.5 g, alkali lignin 10 g, distilled water up to 1000 mL. (2) Isolation Medium10: Bacteria: Nutrient agar (NA) medium. Fungi: Martin’s agar medium. Actinomycetes: Modified Gause’s No. I medium. (3) LB Medium: Yeast extract 5.0 g, peptone 10.0 g, NaCl 10.0 g, agar (solid medium) 18.0 g, distilled water up to 1000 mL, pH 7.0. (4) Differential Medium11: CMC-Na medium containing CMC-Na 20.0 g, Na2HPO4 5.0 g, peptone 2.5 g, yeast extract 0.5 g, distilled water up to 1000 mL, agar 20 g, pH 7.0–7.2. (5) Fermentation Medium12: CMC-Na 10.0 g, KH2PO4 2.0 g, (NH₄)2SO₄ 4.0 g, MgSO₄·7 H2O 0.5 g, peptone 10.0 g, beef extract 5.0 g, distilled water up to 1000 mL. (6) Tobacco Stalk Solid Medium13: Tobacco stalk powder (40-mesh) 10.0 g, peptone 10.0 g, NaCl 5.0 g, agar 18.0 g, distilled water up to 1000 mL, pH natural. (7) Tobacco Stalk Liquid Medium: Tobacco stalk powder (40-mesh) 10.0 g, (NH4)2SO4 2 g, KH2PO4 1 g, K2HPO4 1 g, MgSO4 0.2 g, CaCl2 0.1 g, MnSO4·H2O 0.02 g, FeSO4·7 H2O 0.05 g, distilled water up to 1 L, pH 7.0–7.2.

Methods

Isolation, purification, and preliminary screening of cellulose-degrading bacteria

A 10.0 g soil sample was suspended in 90 mL of sterile distilled water containing glass beads, shaken at 150 rpm for 1 h, and allowed to settle for 5 min. Subsequently, 2 mL of the supernatant was transferred to sterilized enrichment medium and incubated at 30 °C with 180 rpm shaking for 3 days. Following aseptic protocols, the enriched culture was serially diluted. Aliquots (0.1 mL) of 10− 6, 10−7, and 10−8 dilutions were spread on nutrient agar (NA) for bacterial isolation, while 10−⁴, 10−⁵, and 10−⁶ dilutions were plated on Martin’s medium for fungi and Gauze’s No. 1 medium for actinomycetes14. Purified isolates were inoculated onto CMC-Na agar plates, and cellulose-degrading strains were preliminarily screened using Congo red staining to detect hydrolysis zones. Carboxymethyl cellulase (CMCase) activity was quantified via the DNS colorimetric method15.

Strain identification

Morphological characterization was performed by observing colony color, size, and shape on LB agar after 24 h incubation at 28 °C. Gram staining was conducted using the BASO staining kit, with colony morphology examined under an optical microscope. Growth conditions were assessed at varying temperatures (4 °C, 10 °C, 28 °C, 37 °C, 42 °C, 44 °C, 55 °C), pH levels (4–12), and NaCl concentrations (1–10%, w/v). Growth on tryptic soy agar (TSA) and nutrient agar (NA) was evaluated. Motility was tested using migration agar, and hemolytic activity was determined on Columbia agar supplemented with 5% sheep blood after 48 h incubation. Acid production, hydrolysis of suberin, casein, gelatin, DNA, Tween 20, and Tween 80, as well as catalase activity (3% H2O2 test) and nitrate reduction (Smibert & Krieg, 1994), were systematically analyzed to establish physiological and biochemical profiles.

For molecular identification, genomic DNA was extracted using the Tiangen Biotech (Beijing) bacterial DNA kit. The 16 S rDNA gene was amplified with universal primers 27 F/1492R in a 25 µL PCR mixture containing 1.1× T3 Super PCR Mix, primers, and template DNA. Thermal cycling conditions included initial denaturation at 98 °C for 2 min, followed by 32 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min. PCR products were electrophoresed on 1% agarose gels and sequenced by Tsingke Biotechnology (Beijing). Sequences were aligned using NCBI BLAST, and phylogenetic trees were constructed in MEGA 11 via the neighbor-joining method (Kimura 2-parameter model, 1000 bootstrap replicates), with multiple sequence alignment performed by ClustalW.

Tobacco stalk degradation experiment

The experimental group received a 5% (v/v) inoculum of LXX12, while the control (CK) remained uninoculated. Fresh tobacco straw was cut into 10–12 cm segments (40 g per sample), placed in 1.0 mm mesh nylon bags, and embedded in 150 g soil within plastic containers to ensure soil-straw contact. Degradation was monitored in a 28 °C incubator. Straw weight loss was measured at 7, 21, and 35 days. Post-degradation samples (day 35) were analyzed by scanning electron microscopy (Hitachi SU8010) at 3.0 kV and 1000× magnification to visualize structural changes.

Mechanism of tobacco stalk degradation

Tobacco straw was incubated as the sole carbon source in liquid medium inoculated with 5% (v/v) LXX12 and shaken at 180 rpm (28 °C). Samples collected at 7, 14, and 28 days were freeze-dried, mixed with KBr (1:99 ratio), ground in an agate mortar, and pressed into pellets. Fourier-transform infrared spectroscopy (FTIR; 400–4000 cm−1) was performed to assess functional group changes16.

Whole-genome sequencing and analysis

PacBio SMRT sequencing was conducted using SMRT cells, where DNA polymerase-template binding enabled real-time detection of fluorescently labeled nucleotides. Library preparation followed PacBio protocols: DNA fragmentation, damage repair, end polishing, adapter ligation, exonuclease digestion, and size selection. Predicted amino acid sequences were annotated against CO, CAZy, and KEGG databases using BLASTp. Comparative genomic analysis with NCBI-deposited genomes focused on CAZyme profiles and core metabolic pathways.

Results and analysis

Identification of strain LXX12

Morphological observation

When cultured on LB agar at 28 °C for 24 h, colonies of strain LXX12 appeared circular with entire margins, flat and smooth surfaces, and a white coloration (Fig. 1).

Fig. 1
figure 1

Growth of strain LXX12 on LB medium.

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Molecular characteristics

The 16 S rRNA gene sequence of strain LXX12 was aligned with NCBI database entries, and a phylogenetic tree was constructed using the most closely related strains. As shown in Fig. 2, the strain exhibited the highest sequence similarity (99.78%) to Acinetobacter indicus CIP 110,367 strain A648, confirming its taxonomic designation as Acinetobacter indicus LXX12.

Fig. 2
figure 2

Phylogenetic tree of strain LXX12 based on the 16 S rRNA gene sequence.

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Physiological and biochemical characteristics

Strain LXX12 is a Gram-negative, non-motile, aerobic bacterium, catalase-positive and H₂S-negative. It demonstrated non-hemolytic activity and hydrolyzed Tween 80 but not Tween 20, gelatin, DNA, or casein. The strain utilized benzoic acid, ethanol, acetic acid, DL-lactic acid, D-fructose, β-alanine, and DL-malic acid as sole carbon sources, but not raffinose, cellobiose, xylose, mannitol, glycerol, sucrose, sorbitol, citric acid, adipic acid, L-aspartic acid, L-histidine, L-arginine, L-ornithine, or L-lysine. Notably, it exhibited robust environmental adaptability, growing at pH 6–11, NaCl concentrations of 0-10% (w/v), and temperatures ranging from 15 °C to 44 °C. Comparative analysis revealed phenotypic discrepancies between LXX12 and A. indicus CIP 110,367 strain A648 (Table 1)17.

Table 1 Comparison of physiological and biochemical characteristics between strain LXX12 and A648.
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Table 1 Comparison of physiological and biochemical characteristics between strain LXX-2022-12 and A648.
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CMCase activity and nicotine tolerance of strain LXX12

Strain LXX12 displayed a hydrolysis zone diameter (D) of 1.14 cm and colony diameter (d) of 0.27 cm on Congo red-stained CMC-Na agar, yielding a D/d ratio of 4.28 (Fig. 3). The CMCase activity reached 65.25 U/mL after 24 h incubation. When cultured on tobacco straw as the sole carbon source, the strain formed colonies with a diameter of 1.4 cm within 24 h, confirming its nicotine tolerance (Fig. 4).

Fig. 3
figure 3

Hydrolysis zone formed by strain LXX12 on CMC-Na.

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Fig. 4
figure 4

Growth of strain LXX12 after 24 h on tobacco stalk medium.

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Tobacco stalk degradation by strain LXX12

Strain LXX12 demonstrated exceptional degradation efficiency, achieving a 65.70% weight loss of tobacco straw after 35 days of treatment (Fig. 5), significantly higher than the control group.

Fig. 5
figure 5

Weight loss rate of tobacco stalks treated with strain LXX12.

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Scanning electron microscopy results

SEM revealed distinct morphological changes in tobacco straw after degradation (Fig. 6). Untreated control straw (CK) retained a smooth, tightly organized surface, whereas LXX12-treated straw exhibited structural loosening, surface collapse, and multiple cavities, indicating substantial degradation of structural integrity.

Fig. 6
figure 6

SEM images of tobacco stalks before and after treatment with strain LXX12. Left: The untreated tobacco stalk shows a smooth surface with a tightly structured and organized morphology. Right: After treatment with LXX12, the tobacco stalk surface displays significant structural changes, including loosened fibers, surface damage, collapse, and the formation of multiple pores and cracks, resulting in a more disordered and porous structure.

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Degradation mechanism of tobacco stalks by strain LXX12

FTIR analysis elucidated the degradation mechanism of strain LXX12 on tobacco straw(Fig. 7). The intensified absorption peak at 1109 cm−1 corresponds to Si–O stretching vibrations in silica and C–O stretching vibrations in polysaccharides, indicating disruption of the silicified layer and subsequent exposure/degradation of cellulose and hemicellulose, which releases small-molecule sugars and oligosaccharides. At 1035 cm⁻¹, the attenuation of C–O and C–C stretching vibrations (cellulose/hemicellulose) suggests cleavage of carbon-oxygen bonds, liberating organic fragments18. The diminished peak at 1235 cm−1, associated with O = C– stretching vibrations in hemicellulose-lignin complexes, reflects partial disintegration of this composite structure and initiation of hemicellulose-lignin separation19. Notably, the enhanced peak at 616 cm−1 (aromatic ring out-of-plane bending vibrations) signifies partial degradation of lignin’s aromatic framework, generating phenolic compounds. Concurrently, the intensified peak at 1423 cm−1 (–CH2 deformation vibrations in lignin and aliphatics) implies lignin breakdown and release of aliphatic compounds. The strengthened absorption at 1652 cm−1 (C= C/C= O stretching vibrations) further confirms lignin depolymerization into aromatic aldehydes, ketones, and unsaturated aliphatics. Additionally, the broadened -OH stretching vibration band (3100–3254 cm−1) indicates formation of hydroxylated derivatives (e.g., alcohols or phenols) during lignin decomposition. These spectral shifts collectively demonstrate that strain LXX12 disrupts the intertwined cellulose-hemicellulose-lignin matrix in tobacco straw, accelerating cellulose/hemicellulose degradation and facilitating lignin exposure for subsequent breakdown.

Fig. 7
figure 7

Fourier-transform infrared (FTIR) spectra of tobacco stalks treated with strain LXX12 at 0, 7, 14, and 28 days. The peak at 3100–3254 cm−1 associated with the -OH stretching vibration is enhanced, indicating the formation of alcohol/phenolic products from lignin degradation. The peaks at 1652 cm−1 (C=C/C=O), 1423 cm−1 (–CH2), and 616 cm−1 (aromatic ring) point to the gradual breakdown of lignin, with the accumulation of aromatic and aldehyde/ketone compounds, and the release of aliphatic compounds. The weakening of the O = C–C peak at 1235 cm−1 suggests the dissociation of the hemicellulose-lignin complex, while the enhancement of the Si–O and polysaccharide C–O peaks at 1109 cm−1, and the flattening of the C-O/C-C peaks at 1035 cm−1 reflect the breakdown of the silicate layer, exposing and degrading cellulose/hemicellulose to produce small organic molecules.

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Whole-genome sequencing and analysis

Genome sequencing results of strain LXX12

The complete genome of strain LXX12 comprises 3,372,068 bp with a GC content of 45.35%. A total of 3,235 protein-coding genes were predicted, alongside 83 tRNA, 21 rRNA (including seven 16 S rRNA genes), and an N50 value of 3,362,218 bp(Fig. 8).

Fig. 8
figure 8

Genome circular diagram. The outermost circle indicates the genome size, with markers every 5 kb. The second and third circles represent genes on the forward and reverse strands of the genome, respectively, with different colors indicating different COG functional classifications. The fourth circle shows repetitive sequences. The fifth circle represents tRNA and rRNA, with blue indicating tRNA and purple indicating rRNA. The sixth circle represents GC content: light yellow areas indicate regions with GC content higher than the average genome GC content, with higher peaks indicating greater differences, while blue areas represent regions with GC content lower than the average. The innermost circle represents GC-skew, with dark gray regions indicating areas where G content is higher than C content and red regions indicating areas where C content is higher than G content.

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GO functional classification

GO annotation of the genome identified 2,365 genes assigned to three major categories: cellular components, molecular functions, and biological processes (Fig. 9). Cellular component analysis revealed predominant enrichment in cell membrane, membrane-associated structures, and cytoplasm. Molecular functions were dominated by catalytic activity (e.g., hydrolases, oxidoreductases) and binding activity (e.g., ion binding), while biological processes highlighted active roles in metabolic and cellular processes. Notably, genes encoding enzymes and cofactors critical for lignocellulose degradation were identified, including O-glycoside hydrolases (GO:0004553) for cellulose/hemicellulose hydrolysis, esterases (GO:0016788) targeting polysaccharide ester bonds in hemicellulose, and peroxidases (GO:0004601) coupled with redox processes (GO:0055114) for lignin oxidation. Additionally, carbohydrate metabolic enzymes (GO:0005975) and metal ion-binding genes (Mg2+: GO:0000287; Fe2+: GO:0005506) suggest cofactor-dependent activation of cellulases and laccases, collectively supporting the strain’s lignocellulolytic potential.

Fig. 9
figure 9

GO functional annotation classification chart. The horizontal axis represents the GO functional categories, while the left vertical axis indicates the percentage of genes, and the right vertical axis shows the gene counts. This chart displays the gene enrichment across secondary functions within the entire gene background, reflecting the relative importance of these functions in the given context.

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CAZy database annotation

AZy analysis demonstrated the strain’s robust carbohydrate-active enzyme repertoire, comprising glycosyltransferases (GT, 35.38%), carbohydrate esterases (CE, 27.69%), and glycoside hydrolases (GH, 16.92%) (Fig. 10), which mediate glycosyl transfer, ester bond cleavage, and cellulose depolymerization, respectively. Auxiliary activities (AA, 9.23%) and carbohydrate-binding modules (CBM, 9.23%) further facilitate oxidative degradation and enzyme-substrate interactions. Among 65 annotated CAZyme genes, GH3 family β-glucosidases and glucan 1,4-β-glucosidases hydrolyze β-1,4-glycosidic bonds for stepwise cellulose breakdown, while GH5 endoglucanases target backbone cleavage. AA4 (vanillyl alcohol oxidase), AA6 (1,4-benzoquinone reductase), and AA3 (aryl alcohol oxidase) genes drive lignin derivative oxidation, quinone detoxification, and aromatic alcohol metabolism. The AA3 family cellobiose dehydrogenase generates H₂O₂ to fuel AA peroxidase activity, enabling synergistic lignin-cellulose degradation. CE3 acetylxylan esterases deacetylate hemicellulose side chains, while CBM50 modules enhance substrate binding. This GT-GH-AA-CE-CBM enzymatic cascade orchestrates “backbone depolymerization-side chain modification-redox-driven” degradation of tobacco straw lignocellulose (Fig. 11).

Fig. 10
figure 10

Distribution of carbohydrate-active enzyme proportions.

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Fig. 11
figure 11

KEGG metabolic pathway diagram. The vertical axis represents KEGG secondary classifications, while the horizontal axis represents the percentage.

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KEGG metabolic pathway analysis

Through KEGG annotation, 1,626 genes were identified. The results revealed predominant gene assignments to metabolic pathways including amino acid metabolism (98 genes), carbohydrate metabolism (80 genes), and lipid metabolism (Fig. 12). Notably, strain LXX12 exhibited specialized gene distribution across critical pathways: pyruvate metabolism (43 genes), propionate metabolism (27 genes), butanoate metabolism (27 genes), glycolysis/gluconeogenesis (26 genes), and aromatic compound degradation (22 genes). These genetic features demonstrate the strain’s robust capacity for degrading complex organic matter (e.g., lignin, cellulose) and coordinating energy metabolism. Enzymes such as α-D-glucose and β-D-glucose isomerases in starch/sucrose metabolism pathways were found to mediate carbon source utilization (Fig. 11)20,21,22. Genes associated with aromatic compound degradation likely drive the breakdown of complex substrates into key intermediates that feed into the tricarboxylic acid (TCA) cycle, thereby enhancing energy production. Additionally, oxidoreductases and carbohydrate metabolism-related genes were shown to critically support both energy transduction and lignocellulose deconstruction.

Fig. 12
figure 12

KEGG annotation classification chart. Numbers within boxes indicate EC numbers (Enzyme Commission codes) of key enzymes. Blue-highlighted boxes denote genes detected in the genome encoding corresponding enzymes.

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Discussion and conclusion

In this study, a high-efficiency tobacco straw-degrading strain, Acinetobacter indicus LXX12, was isolated from tobacco-planting soil in Anshun City, Guizhou Province, China. The strain, identified as a Gram-negative, non-motile aerobic bacterium, exhibited remarkable environmental adaptability, thriving under pH 6–11, salinity up to 10%, and temperatures ranging from 15 °C to 44 °C. Despite sharing 99.78% 16 S rRNA sequence similarity with A. indicus CIP 110,367, phenotypic discrepancies in physiological and biochemical traits (Table 1) suggest its potential classification as a novel subspecies or variant, warranting further polyphasic taxonomic validation (e.g., whole-genome ANI analysis, DDH). This finding expands the known diversity of the Acinetobacter genus and provides a candidate microbial resource for developing novel straw-degrading agents.

The degradation performance of strain LXX12 surpassed previously reported strains: its CMCase activity (65.25 U/mL at 24 h) exceeded that of Bacillus subtilis YC-2 (38.65 U/mL)23, while tobacco straw weight loss reached 40.37% at 7 d—nearly fourfold higher than B. subtilis YC-2 (10.14%)24. After 35 d of treatment, the weight loss rate peaked at 65.7%, with SEM confirming structural disintegration (surface collapse, cavity formation). Genomic insights corroborated these capabilities: GO-annotated catalytic activities (O-glycoside hydrolase GO:0004553, peroxidase GO:0004601) and CAZy enzyme systems (GH3, AA4/AA6, CE3) synergized via a “backbone depolymerization–side chain modification–redox-driven” network to degrade cellulose, hemicellulose, and lignin. The AA3 family cellobiose dehydrogenase-generated H₂O₂ facilitated lignin oxidation (evidenced by FTIR C = O peak intensification at 1652 cm⁻¹), while KEGG-annotated benzoate metabolism pathways supported quinone detoxification and carbon assimilation, underscoring metabolic robustness.

Notably, this study reports the first Acinetobacter strain with dual cellulose-lignin degradation efficacy. Compared to traditional Bacillus spp., LXX12’s salt-alkali tolerance (10% NaCl, pH 11) and nicotine resistance enhance its applicability in agricultural waste composting, particularly in high-salinity environments. However, cross-crop adaptability (e.g., corn, rice straw) requires validation. Future studies should focus on enhancing AA/GH enzyme thermostability or engineering synthetic consortia with nitrogen-fixing bacteria to optimize industrial-scale efficiency.

In conclusion, the isolation and functional characterization of A. indicus LXX12 advance lignocellulose bioconversion technology. Its annotated genome and multi-enzyme synergy provide a molecular blueprint for targeted enzyme engineering, positioning this strain as a promising candidate for sustainable agricultural waste valorization.