Long-term continuous rice cultivation depletes soil nutrients and disrupts microbial nitrogen cycling, whereas tobacco-rice rotation (TRR) serves as a potential strategy for restoring soil functionality. This study reveals that TRR integrates the plant secondary metabolite nicotine into the nitrogen cycle through synergistic bacterial-archaeal metabolism, forming a “nicotine-molybdenum (Mo)-nitrogen” coupled metabolic network. Nicotine is degraded into ammonia and other nitrogen-containing intermediates via cross-domain collaboration involving bacteria (e.g., Ramlibacter, Streptomyces) and archaea (e.g., Nitrososphaera, Methanopereden). This process is supported by significant upregulation of key nitrogen cycling genes in TRR soils, including nifK (nitrogen fixation), nasA (nitrate reduction), and ndhABC (nicotine degradation. Available Mo content in TRR soils is significantly reduced (0.27 ± 0.02 mg/kg compared to 0.35 ± 0.03 mg/kg in continuous rice cultivation, p < 0.05), indicating that Mo ions are a critical factor in this process. However, long-term TRR may deplete soil Mo reserves, potentially limiting nitrogen availability and highlighting the need for Mo supplementation strategies. This study proposes a “nicotine-Mo-nitrogen” theoretical model, refining the conventional understanding of soil nitrogen cycling and providing a theoretical foundation and practical insights for microbially driven soil health management in sustainable agriculture.

Food flavor is closely related to the type and content of its flavor compounds. The degradation of beta-carotene by microorganisms to produce volatile aroma compounds has shown great potential in enhancing the flavor quality of foods. This study aims to investigate the ability and mechanism of Bacillus thuringiensis 4-1 to degrade beta-carotene and to identify key aroma compounds produced during this process. To our knowledge, this is a relatively fast microbial beta-carotene degradation reported (>90 % degradation within 3 h at 10 mg/L), outperforming published fungal systems that typically require more than 24 h. During this rapid process, Bacillus thuringiensis 4-1 produced key degradation products with floral, fruity, and woody aromas, such as 6-methyl-5-hepten-2-one, beta-cyclocitral, beta-ionone, and dihydroactindiolide. Our study further revealed that sodium nitrate induced the strain to produce small molecules, including benzaldehyde, isovaleric acid, isobutyric acid, and acetic acid, which contributed to beta-carotene degradation by cleaving its double bonds at various positions.

Two strains of Bacillus subtilis designated YY-10 and BY-2, were isolated from the surface of tobacco and found to be capable of significant lignin degradation. The predominant lignin-degrading enzymes produced by these strains were lignin peroxidase (LiP) and manganese peroxidase (MnP), respectively. A notable distinction was observed in the organoleptic evaluation and volatile flavor compounds, as determined by sensory evaluation and GC–MS analysis. The content of volatile flavor compounds, such as geranylacetone, meglumine trienone B, and meglumine trienone C, was found to be significantly increased in roasted tobacco treated with YY-10. This treatment has been shown to reduce the astringent flavor of the roasted tobacco and improve the aroma, which in turn could improve the quality of the roasted tobacco. Conversely, the quality and aroma levels of the roasted tobacco treated with the crude enzyme solution of the BY-2 strain were reduced. Transcriptome analysis revealed that the expression of genes related to amino acid metabolism, genetic material biosynthesis, and protein synthesis was up-regulated in the YY-10 strain compared with the BY-2 strain, which promoted the biosynthesis of LiP. This study provides a preliminary elucidation of the potential mechanism by which YY-10 enhances the quality of tobacco leaves through lignin-degrading enzyme production, thus establishing a research foundation for the subsequent treatment of waste tobacco raw materials and industrial applications.

The growing demand for biocatalysts in biomass processing highlights the necessity of enhancing the thermostability of glycoside hydrolases. However, improving both thermostability and activity is often hindered by trade-offs between backbone rigidity and the flexibility of substrate-binding regions. In this study, Bacillus subtilis cellulase and β-glucanase were engineered using a two-step process incorporating the computational tools Pythia and ESM-2, which were found complementary in improving stability and activity. The engineered cellulase and β-glucanase exhibited increases in their apparent melting temperatures (5.8 °C and 8.4 °C), accompanied by up to a 1.5-fold increase in initial activities. At 50 °C, while the wild-type cellulase lost 60% of its activity after 24 h and wild-type β-glucanase lost activity completely in 2 h, the engineered cellulase-M5 retained its initial activity, and β-glucanase-M7 displayed a 2.2-fold increase in its half-life. Structural analysis indicated that Pythia-identified mutations likely enhanced backbone robustness through refined polar and hydrophobic interactions, while beneficial mutations from ESM-2 appeared to affect polysaccharide-binding regions. This two-step computational redesign offers a promising approach for optimizing both thermostability and activity in glycoside hydrolases and other enzyme families with extensive sequence diversity.

Tobacco leaves need to aged under certain temperatures and humidity for 1 to 3 years. Natural aging, also called fermentation, is a slow process. This article explores the correlation between microorganisms on the surface of flue-cured tobacco and key flavor substances during this process. High-throughput sequencing was used to characterize the composition of bacterial and fungal communities on the surface of flue-cured tobacco from Hunan, Liaoning, Guizhou, Shandong, Yunnan, Fujian, Brazil, and Zimbabwe. Gas chromatography-mass spectrometry (GC-MS) was employed to determine the flavor substances in flue-cured tobacco and correlation analysis between microbial diversity and flavor substances was performed. The function and abundance of key enzymes involved in the degradation of macromolecules on the surface of flue-cured tobacco were predicted using PICRUSt and FUNGuild. Different flavor styles of flue-cured tobacco possessed different flora structures, and the abundance of the genus Bacillus was high and significantly positively correlated with the content of the main flavor substances-carotenoid and cembranoid degradation products (p < 0.05). The content of the Enterobacter genus was higher in light-flavor style flue-cured tobacco and significantly negatively correlated with the above substances (p < 0.05). Among the fungal genera, Wallemia exhibited a significantly positive correlation with phenylacetic acid, palmitic acid, and & alpha;-ionol (p < 0.05). The abundance of the macromolecule-degrading enzymes in the bacterial community was generally higher than in the fungal community, especially the enzymes that hydrolyzed, transferred, and isomerized starch, protein, and cellulose. Classifying the tobacco leaves according to the flavor style revealed that the expression levels of a series of enzymes acting on pectin hydrolysis and cleavage and laccase were higher in strong-flavor flue-cured tobacco. This study explored the microorganisms related to the key substances of flue-cured tobacco and provided a theoretical basis for using microorganisms to improve the flavor.

Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is a serious soil-borne disease, which seriously damages the growth of tobacco crops. Bacillus velezensis A5 was isolated from 3000 m deep-sea sediments of the Pacific Ocean, and was found to be antagonistic to TBW. Here, we report the complete genome sequence of strain A5, which has a 4,000,699-bp single circular chromosome with 3,827 genes and a G + C content of 46.44 %, 87 tRNAs, and 27 rRNAs. A total of 12 gene clusters were identified in the genome of strain A5, which were responsible for the biosynthesis of antibacterial compounds, including surfactin, bacillaene, fengycin, difficidin, bacillibactin, and bacilysin. Additionally, strain A5 was found to contain a series of genes related to the biosynthesis of carbohydrate-active enzymes and secreted proteins. Our results indicate that strain A5 can be considered a promising biocontrol agent against TBW in agricultural fields.

The changes of microbial community on tobacco leaves are affected by several factors during fermentation. However, the relative contribution of different factors in determining microbial community is not clear. This study investigated the effects of fermentation time (fermentation for 0, 3, 6, 9 and 12 months), leaf position (middle and top tobacco leaves) and fermentation site (Longyan and Xiamen warehouses) on bacterial community of tobacco leaves using 16 S rDNA sequencing. The results demonstrated that fermentation time had a much stronger impact on bacterial diversity, composition, co-occurrence network and functional profiles than leaf position and fermentation site. With the fermentation progressed, the difference of bacterial community between middle and top tobacco leaves was gradually reduced or even disappeared. The bacterial community diversity and network complexity at three, six and nine months of fermentation were significantly lower than those at fermentation initiation. Specific bacterial genera with desired functions were recruited at different fermentation stages, such as Terribacillus, Pantoea and Franconibacter at three or six months of fermentation and Pseudomonas at nine months of fermentation. The recruited microorganisms would form biofilms on tobacco leaves and compete for polysaccharide or protein substances to accelerate the degradation of tobacco macromolecular substances. In conclusion, fermentation time was an important factor in determining the composition and function of microbial community on tobacco leaves during the fermentation process.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives. Cellulase solutions obtained from Bacillus subtilis FX-1, a bacterium originating from tobacco, were applied to flue-cured tobacco during the aging process. The cellulase structure of tobacco was analyzed using 13C cross-polarization magic angle spinning NMR spectroscopy. Sensory evaluation was utilized to assess the sensory attributes and quality characteristics. Targeted and untargeted metabolomics were utilized to investigate the chemical components present in the cellulase solutions. The inclusion of cellulase additives altered the cellulose structure in tobacco and introduced various flavor-related precursors and components. The utilization of cellulase additives derived from B. subtilis FX-1 substantially improved the overall quality of the tobacco product.

Starch is an essential factor affecting the quality of flue-cured tobacco, and high starch content can affect the sensory quality and safety. Recently, the degradation of macromolecules in tobacco raw materials by using additional microorganisms to improve their intrinsic quality and safety has become a new research hotspot in the tobacco industry. However, the technical maturity and application scale are limited. Our study analyzed the correlation between microbial community composition and volatile components on the surface of tobacco leaves from 14 different grades in Fujian tobacco-producing areas. The PICRUSt software was utilized to predict the function of the microbial community present in tobacco leaves. Furthermore, dominant strains that produced amylase were screened out, and an enzyme solution was prepared to enhance the flue-cured tobacco flavor. Changes in the content of macromolecules and volatile components were determined, and sensory evaluations were conducted to assess the overall quality of the tobacco leaves. The results showed that the dominant bacterial genera on the surface of Fujian tobacco leaves were Variovorax, Sphingomonas, Bacillus, etc. Bacillus was positively correlated with various volatile components, which contributed to the sweet and aromatic flavors of Fujian flue-cured tobacco. The main genetic functions of Fujian flue-cured tobacco surface bacteria were carbohydrate metabolism and amino acid metabolism. After treating flue-cured tobacco with an enzyme preparation prepared by the fermentation of Paenibacillus amylolyticus A17 #, the content of starch, pectin, and cellulose in flue-cured tobacco decreased significantly compared with the control group. Meanwhile, the content of total soluble sugar and reducing sugar was significantly increased, and the volatile aroma components, such as 3-hydroxy--damascone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-Pyran-4-one, ethyl palmitate, ethyl linolenic acid, etc., were significantly increased. The aroma quality and quantity of flue-cured tobacco were enhanced, while impurities were reduced. The smoke characteristics were improved, with increased fineness, concentration, and moderate strength. The taste characteristics were also improved, with reduced irritation and a better aftertaste. In conclusion, Bacillus, as the dominant genus in the abundance of bacterial communities on tobacco surfaces in Fujian, had an essential impact on the flavor of tobacco leaves by participating in carbohydrate metabolism and finally forming the unique flavor style of flue-cured tobacco in Fujian tobacco-producing areas. Paenibacillus amylolyticus A17 #, a target strain with amylase-producing ability, was screened from the surface of Fujian flue-cured tobacco. The enzyme preparation, produced by the fermentation of Paenibacillus amylolyticus A17 #, was utilized to reduce the content of macromolecules, increase the content of water-soluble total sugar and reducing sugar, and produce a variety of crucial volatile aroma components, which had a significant improvement on the quality of tobacco leaves.

•Ti3C2Tx was used as an efficient initiator to reduce GO upon microwave irradiation.•Microwave sintering to construct rGO/Ti3C2Tx networks in PSU composite.•Sintered composite with just 10 s irradiation showed a high EMI SE of 52.8 dB.•Microwave heating is a clean method for manufacturing rGO-based composite.

Microbial degradation of organic compounds is an environmentally benign and energy efficient part in product processing. Fermentation of plant leaves involves enzymatic actions of many microorganisms. However, microbes and enzymes discovered from natural degradation communities were still limited by cultural methods. In this study, we used a metagenomics sequence-guided strategy to identify the microbes and enzymes involved in compound degradation and explore the potential synergy among community members in fermented tobacco leaves. The results showed that contents of protein, starch, pectin, lignin, and cellulose varied in fermented leaves from different growing sites. The different compound contents were closely related to taxonomic composition and functional profiles of foliar microbial communities. Microbial communities showed significant correlations with protein, lignin, and cellulose. Vital species for degradations of protein (Bacillus cereus and Terribacillus aidingensis), lignin (Klebsiella pneumoniae and Pantoea ananatis) and cellulose (Pseudomonas putida and Sphingomonas sp. Leaf20) were identified and relating hydrolytic enzymes were annotated. Further, twenty-two metagenome-assembled genomes (MAGs) were assembled from metagenomes and six potential cellulolytic genomes were used to reconstruct the cellulose-degrading process, revealing the potential metabolic cooperation related to cellulose degradation. Our work should deepen the understanding of microbial roles in plant fermentation and provide a new viewpoint for applying microbial consortia to convert plant organic components to small molecules.

BACKGROUND:During the biomass-to-bio-oil conversion process, many studies focus on studying the association between biomass and bio-products using near-infrared spectra (NIR) and chemical analysis methods. However, the characterization of biomass pyrolysis behaviors using thermogravimetric analysis (TGA) with support vector machine (SVM) algorithm has not been reported. In this study, tobacco was chosen as the object for biomass, because the cigarette smoke (including water, tar, and gases) released by tobacco pyrolysis reactions decides the sensory quality, which is similar to biomass as a renewable resource through the pyrolysis process.RESULTS:SVM algorithm has been employed to automatically classify the planting area and growing position of tobacco leaves using thermogravimetric analysis data as the information source for the first time. Eighty-eight single-grade tobacco samples belonging to four grades and eight categories were split into the training, validation, and blind testing sets. Our model showed excellent performances in both the training and validation set as well as in the blind test, with accuracy over 91.67%. Throughout the whole dataset of 88 samples, our model not only provides precise results on the planting area of tobacco leave, but also accurately distinguishes the major grades among the upper, lower, and middle positions. The error only occurs in the classification of subgrades of the middle position.CONCLUSIONS:From the case study of tobacco, our results validated the feasibility of using TGA with SVM algorithm as an objective and fast method for auto-classification of tobacco planting area and growing position. In view of the high similarity between tobacco and other biomasses in the compositions and pyrolysis behaviors, this new protocol, which couples the TGA data with SVM algorithm, can potentially be extrapolated to the auto-classification of other biomass types.

To investigate the differences of bacterial community structure among tobacco strips from different growing areas and stalk positions during aging, the bacterial 16S rDNA of strip samples of upper, middle and lower leaves from 4 areas (Youxi in Fujian, Zhanyi in Yunnan, Changshun in Guizhou and Queshan in Henan) during aging were sequenced by Illumina MiSeq. The results showed that 940 operational taxonomic units (OTUs) were obtained from 12 samples. The samples (C3F) from Youxi had the most abundant bacterial biodiversity. The dominant phyla in 12 samples were Proteobacteria, and the dominant genera in most samples were Sphingomonas. The Venn diagram of bacterial OTUs of strips from different areas during aging revealed that 195 of OTUs were common to the strips from 4 areas, the numbers of specific OTUs for Youxi, Zhanyi, Changshun and Queshan were 300, 9, 4 and 0, respectively.

To investigate the diversity of fungal community in tobacco strips during aging, the fungal ITS DNA in 12 samples of upper, middle and lower stalk leaves from four growing areas (Zhanyi in Yunnan, Changshun in Guizhou, Youxi in Fujian and Queshan in Henan) were sequenced by MiSeq. The results showed that: 1) A total of 1 155 operational taxonomic units (OTUs) were obtained from the 12 samples. Samples B2F and C3F from Youxi had the highest fungal community richness and the most abundant biodiversity, respectively. 2) Ascomycota was the dominant phyla in the 12 samples. Phoma, Alternaria and Monographella were the dominant genera in samples C3F and X2F from Zhanyi, samples B2F, C23F and X2F from Queshan and sample X23F from Changshun. Aspergillus was the dominant genera in the other 6 samples. 3) 121, 79 and 168 of OTUs were common to the samples of upper, middle and lower stalk leaves from the 4 areas. The number of reads from the common OTUs accounted for more than 70% of the total number of reads in each sample, except for the sample C3F from Youxi. The differences in fungal community structure from the same stalk position of different areas mainly reflected in the proportion of dominant genera.