In this study, four different biochar application rates and a control were set up using indoor potted tobacco, to study the effects of biochar on the microbial diversity and metabolism of tobacco-growing soil. The five treatments were as follows: control—0% biochar (w/w) + 26 g fertilizer/pot; biochar treatments—1% biochar (w/w) + 26 g fertilizer/pot, 2% biochar (w/w) + 26 g fertilizer/pot, 3% biochar (w/w) + 26 g fertilizer/pot, and 4% biochar (w/w) + 26 g fertilizer/pot. We found that biochar increases the microbial diversity of soils and simultaneously changes the microbial community structure. Under the influence of biochar, soil urease activity increased by 18%, invertase activity increased by 23.40%, polyphenol oxidase activity increased by 59.50%, and catalase activity increased by 30.92%. Biochar also significantly increased the microbial biomass carbon and nitrogen content of the soil. Soil microbial biomass nitrogen had a positive correlation on bacterial diversity, with the highest coefficient, while soil microbial biomass carbon had a positive correlation on fungal diversity, with the highest coefficient. The microbial diversity and metabolic capacity of soil are improved under the influence of biochar, and soil enzyme activity and microbial biomass carbon and nitrogen have positive impacts on soil microbial diversity.

The valorization of tobacco stems, a major agricultural by-product with high lignin content, is hindered by the recalcitrance of plant cell walls. Conventional approaches using single microbes or enzymes suffer from inefficiency, instability, and poor performance on lignin-rich substrates. To address this, we proposed a “microbe-enzyme synergistic” strategy by combining Bacillus megaterium (a multifunctional degrader) with its own extracellular enzymes. Three treatments were designed: microbe-only (T _b ), enzyme-only (T _e ), and microbe-enzyme combination (T _b&e ), with sterile water as the control (CK). Cell wall component contents, enzyme activities, and microbial community structure were analyzed after 7 days of fermentation. The T _b&e treatment achieved the most substantial degradation: 64.93% for cellulose, 57.89% for lignin, and 37.20% for pectin, significantly outperforming T _b and T _e . It also exhibited the highest activities of cellulase, laccase, and pectinase. High-throughput sequencing revealed that T _b&e specifically enriched Bacillus megaterium (94.97% of the community) while suppressing non-functional competitors. Our findings demonstrate that the synergy creates a positive feedback loop: initial enzymatic hydrolysis facilitates microbial colonization, which in turn boosts sustained enzyme production. This study elucidates a mechanistic model for efficient biodegradation and provides an innovative strategy for the high-value utilization of high-lignin agricultural wastes.

The heavy metal ATPase (HMA) family, classified as P_1B-ATPases, plays a pivotal role in maintaining plant metal homeostasis by coupling ATP hydrolysis to the active transport of metal ions across biological membranes. However, comprehensive characterization of the HMA family in tobacco (Nicotiana tabacum) remains insufficient. In this study, we conducted a systematic genome-wide bioinformatic analysis and identified 27 HMA-encoding genes in tobacco, designated NtHMA1 through NtHMA27. Phylogenetic and structural analyses revealed that these genes fall into two major clades, further resolved into six evolutionarily conserved subfamilies. Chromosomal localization showed that NtHMA genes are predominantly distributed on chromosomes 1, 17, and 25, and all retain hallmark domains characteristic of the HMA family. Comparative genomic analysis identified ten segmentally duplicated NtHMA gene pairs, all exhibiting signatures of purifying selection (Ka/Ks < 1), suggesting functional constraint and evolutionary conservation. Cis-regulatory element analysis uncovered 14 types of stress-responsive cis-acting elements associated with responses to biotic and abiotic stresses. Integrated transcriptomic profiling across multiple abiotic stress conditions and heavy metal treatments indicated that NtHMA6, NtHMA14, and NtHMA23 are preferentially upregulated under heavy metal exposure, implicating their roles in metal transport and detoxification, while NtHMA2, NtHMA7, NtHMA10, NtHMA11, NtHMA13, NtHMA18, NtHMA22, and NtHMA25 show significant transcriptional responsiveness to abiotic stresses or hormone stimuli, suggesting involvement in stress adaptation. In summary, this study provides the first comprehensive genomic, structural, evolutionary, and expression-based characterization of the tobacco NtHMA family, establishing a foundation resource for functional genomics studies and offering valuable insights to guide molecular breeding strategies aimed at enhancing tobacco resilience and metal tolerance.