Process for the preparation of flavorants from tobacco waste pyrolysis with microbial pretreatment

Abstract

The present application relates to a kind of microbial pretreatment tobacco waste pyrolysis preparation spice process, comprising the following steps: S1, strain selection: bacillus subtilis V16 strain is aimed at tobacco stem, bacillus pumilus V35 strain is aimed at tobacco leaf, trichoderma viride CE1 is aimed at tobacco dust;S2, preparation of microbial preparation;S3, microbial pretreatment: preparation is separately sprayed on tobacco stem, tobacco leaf, tobacco dust, then is loaded into sealed bag, under certain temperature and humidity conditions, after a certain time, high-temperature inactivation and drying;S4, grinding dry distillation extraction bio-oil: after the pretreatment of tobacco waste drying and grinding, it is placed in reactor and carries out dry distillation reaction, after a certain time under the dry distillation temperature set, obtain bio-oil and biochar.The present application uses microbial target pretreatment technology, optimizes pyrolysis reaction path and improves the selectivity of product, provides solid theoretical basis and technical reference for high-value utilization of tobacco waste.

Description

Technical Field

[0001] This invention belongs to the field of biomass energy utilization and tobacco waste resource utilization technology, specifically relating to a process for preparing fragrances by microbial pretreatment of tobacco waste through pyrolysis. Background Technology

[0002] Tobacco waste (such as tobacco stems, broken tobacco leaves, and tobacco dust) is generated in huge quantities during tobacco production, and traditional disposal methods mainly involve landfilling or incineration. These methods not only waste resources but also cause serious environmental pollution; for example, landfilling occupies a large amount of land, and incineration produces harmful gases.

[0003] While resource utilization is an effective way to solve the tobacco waste problem, traditional resource utilization methods, such as direct use as fertilizer or feed, can negatively impact soil and animal health due to harmful substances in tobacco waste. Another example is the pyrolysis extraction of bio-oil, which mainly relies on a single thermochemical conversion process, following a traditional three-step reaction mechanism (dehydration, main pyrolysis stage, and carbonization). The reaction path is limited by the inherent components of the raw materials. However, the chemical composition of tobacco waste is usually quite complex, and the quality of the products from traditional pyrolysis is uneven. Bio-oil often contains high levels of off-flavor substances, limiting its application in high-value-added fields such as fragrances.

[0004] In recent years, microbial pretreatment technology has received widespread attention in the field of biomass energy. Microorganisms can selectively degrade complex organic matter in biomass by secreting enzymes (such as cellulase, hemicellulase, and pectinase), optimizing their chemical structure and thus improving the pathway and product distribution of subsequent pyrolysis reactions. Therefore, this invention proposes a process for preparing flavorings from tobacco waste through microbial pretreatment, aiming to target the degradation of specific components in tobacco waste through microbial pretreatment, optimize the pyrolysis reaction pathway, and thereby achieve a more efficient pyrolysis process and higher-quality products. Summary of the Invention

[0005] This invention provides a process for preparing fragrances by microbial pretreatment of tobacco waste through pyrolysis.

[0006] The specific technical solution is: a process for preparing fragrances by microbial pretreatment of tobacco waste through pyrolysis, comprising the following steps: S1. Strain selection: Bacillus siamensis V16 strain: Targets tobacco stems; Bacillus pumilus Van35: Targets broken tobacco leaves; Trichoderma viride CE1: for tobacco dust; S2. Preparation of microbial preparations: (1) The preparation methods of Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation are the same: the strain is inoculated into LB liquid medium, and after two shaking cultures, it is centrifuged, washed, and water is added and shaken to prepare Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation. (2) Method for preparing crude enzyme solution of Trichoderma viride CE1: After inoculating it into 50 mL of LB medium and activating it twice, it was inoculated into 200 mL of PDA liquid medium for fermentation culture. The supernatant was filtered and the crude enzyme solution was obtained by ammonium sulfate precipitation and dialysis. S3. Microbial pretreatment: The Bacillus simulans V16 preparation, Bacillus pumilus V35 preparation and crude enzyme solution obtained in step 2 were evenly sprayed onto tobacco stems, broken tobacco leaves and tobacco dust, respectively, and then put into sealed bags. After being treated under certain temperature and humidity conditions for a certain period of time, they were inactivated at high temperature and dried. S4. Extraction of bio-oil by pulverization and dry distillation: The tobacco waste pretreated in step S3 is dried until the moisture content is below 7% and pulverized to a particle size of below 60 mesh. The dried and pulverized tobacco waste is then placed in a reactor for dry distillation. After being treated at the set dry distillation temperature for a certain period of time, bio-oil and biochar are obtained.

[0007] Further, in step S2, the preparation method of Bacillus simulans V16 and Bacillus pumilus V35 is as follows: the strain is inoculated into 50 mL of LB liquid medium and cultured at 37℃ and 180 r / min for 14 h to obtain liquid culture; then the liquid culture is inoculated into 200 mL of LB liquid medium at an inoculation amount of 0.1% and cultured at 37℃ and 180 r / min for 14 h until the OD value is 2.0 to obtain culture solution; finally, 20 mL of culture solution is taken, centrifuged at 8000 rpm / min for 10 min, the supernatant is removed, and the precipitate is washed with sterile water. Finally, 200 mL of sterile water is added to the precipitate and shaken evenly to obtain Bacillus simulans V16 and Bacillus pumilus V35 preparations.

[0008] Further, in step S2 (2), the method for preparing crude enzyme solution from Trichoderma viride CE1 is as follows: Trichoderma viride CE1 is inoculated into 50 mL of LB medium and cultured at 37 ℃ and 180 r / min for 14 h for activation twice. Then, it is inoculated into an Erlenmeyer flask containing 200 mL of PDA liquid medium and fermented at 28 ℃ and 180 rpm for 5 days. The supernatant is then filtered and collected. Ammonium sulfate is then slowly added to the supernatant until it reaches 80% saturation. The mixture is allowed to stand overnight at 4 ℃. The precipitate is collected by centrifugation and dissolved in a small amount of buffer solution. The mixture is then dialyzed with the same buffer solution to obtain crude enzyme solution.

[0009] Furthermore, the buffer solution is 0.02 mol / L Na2HPO4 and NaH2PO4, and the pH value is 7.3.

[0010] Furthermore, in step S3, the dosage of Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation is 0.2 mL / g tobacco waste; the dosage of crude enzyme solution is 60 U / g cellulase / tobacco waste.

[0011] Furthermore, in step S3, the Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation were treated at 30°C for 48 hours before being heated to 120°C and held for 20 minutes for high-temperature inactivation when pretreating tobacco stems and broken tobacco leaves; the crude enzyme solution was treated at 30°C and 25% relative humidity for 5 hours before being heated to 80°C and held for 20 minutes for high-temperature inactivation when pretreating tobacco dust.

[0012] Furthermore, in step S4, the dry distillation temperature is 300℃-700℃ and the holding time is 30min.

[0013] Furthermore, the optimal temperature for the dry distillation of tobacco stems is 350℃.

[0014] Furthermore, the optimal temperature for the dry distillation of small pieces of tobacco is 500℃, and the optimal temperature for the dry distillation of medium-sized pieces of tobacco is 450℃. Even further, the optimal temperature for the dry distillation of tobacco dust is 350℃.

[0015] The beneficial effects of this invention are as follows: This invention utilizes microbial targeted pretreatment of tobacco waste. By secreting enzymes such as cellulase and hemicellulase, it effectively degrades polysaccharide components, reduces lignocellulose content, and increases the content of water-soluble sugars and aroma components, thereby optimizing the pyrolysis reaction pathway and improving product selectivity. This process not only significantly improves pyrolysis characteristics and reduces the content of off-flavor substances in bio-oil, but also improves the quality of bio-oil, making the resource utilization of tobacco waste more efficient. Furthermore, by determining the optimal pyrolysis temperature for different types of waste, the degradation of off-flavor substances and energy consumption are balanced, providing a solid theoretical foundation and technical reference for the high-value utilization of tobacco waste. In summary, this scheme demonstrates great potential in improving product quality, reducing energy consumption, enhancing environmental friendliness, and promoting technological development in the field of biomass energy. It has significant economic and environmental implications for achieving the resource utilization, reduction, and harmless treatment of tobacco waste. Attached Figure Description

[0016] Figure 1 A comparative chart showing the changes in the content of the three major components of different tobacco wastes before and after microbial treatment; Figure 2 XPS spectra of TS and TS-V16; (a) XPS spectra of TS and TS-V16; (b), (c), and (d) are the fitting curves of the high-resolution XPS spectra of TS for N1s, C1s, and O1s, respectively; (e), (f), and (g) are the fitting curves of the high-resolution XPS spectra of N1s, C1s, and O1s of TS-V16, respectively; Figure 3 XPS spectra of STP and STP-V35; (a) XPS spectra of STP and STP-V35; (b), (c), and (d) are the fitting curves of the high-resolution XPS spectra of STP for N1s, C1s, and O1s, respectively; (e), (f), and (g) are the fitting curves of the high-resolution XPS spectra of STP-V35 for N1s, C1s, and O1s, respectively; Figure 4 XPS spectra of MTP and MTP-V35; (a) XPS spectra of MTP and STP-V35; (b), (c), and (d) are the fitting curves of the high-resolution XPS spectra of MTP in N1s, C1s, and O1s, respectively; (e), (f), and (g) are the fitting curves of the high-resolution XPS spectra of MTP-V35 for N1s, C1s, and O1s, respectively; Figure 5 XPS spectra of TD and TD-CE1; (a) XPS spectra of TD and TD-CE1; (b), (c), and (d) are the fitting curves of the high-resolution XPS spectra of TD for N1s, C1s, and O1s, respectively; (e), (f), and (g) are the fitting curves of the high-resolution XPS spectra of TD-CE1 for N1s, C1s, and O1s, respectively; Figure 6 TG-DTG images of different tobacco wastes before and after microbial treatment; Figure 7 TG-DTG images of different tobacco wastes before and after microbial treatment Figure 8 The graph shows the changes in aroma components in TS and TS-V16 bio-oils at different pyrolysis temperatures; Figure 9 The graph shows the changes in the content of off-flavor substances in TS and TS-V16 bio-oils at different pyrolysis temperatures. Figure 10 Figure showing the changes in aroma components in STP and STP-V35 bio-oils at different pyrolysis temperatures; Figure 11 The graph shows the changes in the content of off-flavor substances in STP and STP-V35 bio-oil at different pyrolysis temperatures. Figure 12 Figure showing the changes in aroma components in MTP and MTP-V35 bio-oil at different pyrolysis temperatures; Figure 13 The graph shows the changes in the content of off-flavor substances in MTP and MTP-V35 bio-oil at different pyrolysis temperatures. Figure 14 The graph shows the changes in aroma components in TD and TD-CE1 bio-oils at different pyrolysis temperatures; Figure 15 The graph shows the changes in the content of off-flavor substances in TD and TD-CE1 bio-oils at different pyrolysis temperatures; Figure 16 SEM images of pyrolytic biochar from tobacco waste at 350 °C are shown below: (a) TS group of tobacco stems; (b) TS-V16 group of tobacco stems; (c) STP group of small pieces of tobacco leaves; (d) STP-V35 group of small pieces of tobacco leaves; (e) MTP group of medium pieces of tobacco leaves; (f) MTP-V35 group of medium pieces of tobacco leaves; (g) TD group of tobacco dust; (h) TD-CE1 group of tobacco dust. Detailed Implementation

[0017] To make the technical problems and solutions solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0018] In this embodiment, taking tobacco waste (including tobacco stems, broken tobacco leaves, and tobacco dust) generated by a tobacco factory as an example, the process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis as described in this invention is used to extract fragrance (bio-oil) from the tobacco waste. The specific implementation is as follows: S1. Strain selection: Bacillus siamensis V16 strain: Targets tobacco stems; Bacillus pumilus Van35: Targets broken tobacco leaves; Trichoderma viride CE1: for tobacco dust; S2. Preparation of microbial preparations: (1) The preparation methods of Bacillus simulans V16 and Bacillus pumilus V35 are the same: the strain is inoculated into 50 mL of LB liquid medium and cultured at 37℃ and 180 r / min for 14 h to obtain liquid culture; then the liquid culture is inoculated into 200 mL of LB liquid medium at an inoculation amount of 0.1% and cultured at 37℃ and 180 r / min for 14 h until the OD value is 2.0 to obtain culture solution; finally, 20 mL of culture solution is taken, centrifuged at 8000 rpm / min for 10 min, the supernatant is removed, and the precipitate is washed with sterile water. Finally, 200 mL of sterile water is added to the precipitate and shaken evenly to obtain Bacillus simulans V16 and Bacillus pumilus V3 preparations.

[0019] (2) Method for preparing crude enzyme solution from Trichoderma viride CE1: Trichoderma viride CE1 was inoculated into 50 mL of LB medium and cultured at 37 ℃ and 180 r / min for 14 h for activation twice. Then, it was inoculated into an Erlenmeyer flask containing 200 mL of PDA liquid medium and fermented at 28 ℃ and 180 rpm for 5 days. The supernatant was then filtered and collected. Ammonium sulfate was then slowly added to the supernatant until it reached 80% saturation. The mixture was allowed to stand overnight at 4 ℃. The precipitate was collected by centrifugation and dissolved in a small amount of buffer (0.02 mol / L Na2HPO4 and NaH2PO4, with a pH of 7.3). The mixture was dialyzed with the same buffer to obtain the crude enzyme solution.

[0020] S3. Microbial pretreatment: (1) Pretreatment of tobacco stems (TS): Take 20 mL of Bacillus subtilis V16 preparation and spray it evenly onto 100 g of tobacco stems using a small sprayer (referred to as treatment group TS-V16). At the same time, spraying sterile water serves as the control group (referred to as control group TS). Pack the tobacco stems into sealed bags and poke a few small holes in the bags. After treating at 30℃ for 48 h, raise the temperature to 120℃ and maintain it for 20 min to inactivate the bacteria at high temperature. Dry the tobacco stems in a 120℃ oven to reduce the moisture content to 11%-13%.

[0021] (2) Pretreatment of small-leaf tobacco (STP) and medium-leaf tobacco (MTP): Two 20 mL portions of Bacillus pumilus V35 preparation were taken and evenly sprayed onto 100 g of small-leaf tobacco (STP-V35 treatment group) and medium-leaf tobacco (MTP-V35 treatment group) using a small sprayer. Simultaneously, two control groups (STP and MTP control groups) were prepared by spraying with sterile water. The small-leaf and medium-leaf tobacco were placed in sealed bags with a few small holes punched in them. After treatment at 30℃ for 48 h, the temperature was raised to 120℃ and maintained for 20 min to inactivate the bacteria at high temperature. The small-leaf and medium-leaf tobacco were then dried in a 120℃ oven until the moisture content decreased to 11%-13%.

[0022] (3) Pretreatment of tobacco dust (TD): Take the corresponding amount of crude enzyme solution according to the dosage of 60 U / g of cellulase in tobacco waste, dilute it with an appropriate amount of sterile water, and spray it evenly on the tobacco dust using a sprayer (referred to as treatment group TD-CE1). At the same time, a control group was sprayed with sterile water (referred to as control group TD). The tobacco dust was placed in a sealed bag and treated at 30℃ and 25% relative humidity for 5 h. Then, it was transferred to an 80℃ oven and kept for 20 min to inactivate the enzyme. The tobacco dust was then dried in a 120℃ oven to reduce the moisture content to 11%-13%.

[0023] S4. Extraction of bio-oil by pulverization and dry distillation: (1) Sample preparation: The tobacco waste (tobacco stems, tobacco leaves, tobacco dust) after the pretreatment in step S3 is dried to reduce the moisture content to below 7% and crushed to a particle size of below 60 mesh (to ensure that the sample is heated evenly during the dry distillation process). (2) Reactor Preparation: Install the reactor vertically and place steel pipes through the holes in the furnace, ensuring that all parts of the reactor (gas supply section, dry distillation section, catalytic dry distillation section, condensation section, and tail gas treatment section) are tightly connected and well sealed. Simultaneously, install a vertical condenser at the bottom of the reactor and maintain the condenser temperature at approximately 5°C using ice water. Ensure the distance between the furnace bottom and the condenser is approximately 30 cm. Furthermore, insert dry glass fibers into the reactor to support the sample and prevent direct contact between the sample and the reactor wall.

[0024] (3) Sample loading: Accurately weigh 0.5g of dried and pulverized tobacco waste (tobacco stems, tobacco leaves, tobacco dust) and evenly place it onto the glass fiber in the reactor. Each feeding amount is 0.5g to ensure uniform sample distribution.

[0025] (4) Reactor filling and purification: Before the experiment begins, nitrogen (N2) is filled into the reactor for 5 minutes to remove the air in the reactor and prevent the sample from being oxidized during the dry distillation process.

[0026] (5) Dry distillation process (i.e., pyrolysis process): Starting from room temperature, the reactor is heated at a rate of 20℃ / min until the set dry distillation temperature range (300℃-700℃) is reached. This dry distillation temperature is maintained for 30 minutes to allow the sample to undergo a complete dry distillation reaction at this temperature, generating products such as bio-oil and biochar. Throughout the dry distillation process, the nitrogen flow rate is maintained at 150 mL / min to carry the gaseous products generated during the dry distillation process out of the reactor and into the condensation section. At this time, the gaseous products are cooled to about 5℃ by the condenser, and the bio-oil condenses from the gas and flows into the collection device.

[0027] To understand the bio-oil obtained from the dry distillation and pyrolysis of the three microbial pretreatment methods, the following further detection and analysis were conducted: 1. Sample processing Bio-oil processing: The collected bio-oil is diluted with methanol, an excellent solvent that effectively dissolves the various components in the bio-oil. The diluted bio-oil is then filtered through a 0.22 μm filter membrane to remove impurities and ensure its purity.

[0028] 2. Testing and analysis equipment and conditions The diluted and filtered bio-oil sample was analyzed by GC-MS under the following conditions: Chromatographic column: DB-5MS capillary column (60 m × 0.25 mm × 0.25 μm); Carrier gas: Helium (He), flow rate 1 mL / min; Injection volume: 1 μL; Injector temperature: 250℃; Split ratio: 10:1, constant flow mode temperature program: 40℃ (2 min) to 280℃ (20 min), heating rate 4℃ / min; Transfer line temperature: 240℃; Ionization mode: EI; Ion source temperature: 230℃; Ionization energy: 70 eV; Quadrupole temperature: 150℃; Mass spectrometry scan range: 20-350 amu; Scan mode: Full scan mode and selected ion scan mode.

[0029] 3. Analysis of changes in the content of the three major components The three main components refer to hemicellulose, cellulose, and lignin. Figure 1It can be seen that the changes in the content of the three major components of different tobacco wastes before and after microbial treatment are as follows: The contents of TS-V16, STP-V35, and MTP-V35 in the treatment group all showed a decreasing trend compared to the control group, especially cellulose and hemicellulose. This indicates that *Bacillus simulans* V16 and *Bacillus pumilus* V35 preferentially degrade polysaccharide components (cellulose and hemicellulose) by secreting cellulase and hemicellulase, leading to a significant reduction in their content. This helps to alter the chemical structure of tobacco stems and broken tobacco leaves, optimizing the subsequent pyrolysis reaction pathway. The contents of cellulose and lignin in the treatment group TD-CE1 showed a significant decreasing trend compared to the control group TD. This indicates that *Trichoderma viride* CE1 modifies the lignin in tobacco dust (lignin is a complex organic polymer in tobacco waste that is difficult to degrade by traditional methods) by secreting specific enzymes (such as lignin peroxidase and laccase), promoting the oxidative breakage of aromatic ring structures in lignin to generate smaller molecular fragments. These fragments are more easily converted into valuable products during pyrolysis. The exception was the TD-CE1 group, which had a 0.73% higher hemicellulose content than the control group TD. This may be because Trichoderma viride CE1 secretes some extracellular polysaccharides while degrading lignin. These polysaccharides accumulate during the treatment process, leading to an increase in hemicellulose content.

[0030] 4. Surface chemical bond analysis To confirm the impact of microbial treatment on the surface chemical bonds of tobacco waste, XPS (X-ray photoelectron spectroscopy) analysis was performed, such as... Figure 3-5 As shown in the analysis, the chemical bonds on the surface of tobacco waste underwent rearrangement after microbial treatment, and the ratio of pyrrole nitrogen (nitrogen atoms in N-heterocyclic compounds, such as pyrrole) to pyridine nitrogen (nitrogen atoms in N-heterocyclic compounds, such as pyridine) changed. This indicates that microbial treatment altered the types and quantities of nitrogen-containing heterocyclic compounds in tobacco waste. This rearrangement of chemical bonds and the change in nitrogen-containing heterocyclic compounds directly affect the formation pathways of nitrogen-containing heterocyclic compounds (such as pyridine and quinoline) during pyrolysis.

[0031] 5. Analysis of changes in chemical bonds and functional groups like Figure 6 The infrared spectra of different tobacco wastes before and after microbial treatment are shown, displaying the samples at a range of wavenumbers (in cm⁻¹). -1 The transmittance (%) changes at a given wavenumber reflect the presence and concentration of different chemical bonds in the sample. Higher transmittance indicates less infrared light absorbed by the sample at that wavenumber, which usually means that the chemical bonds corresponding to that wavenumber are present in a lower concentration in the sample.

[0032] Depend on Figure 6It can be seen that the microbial treatment groups were all at 3440 cm. -1 1600 cm -1 1500 cm -1 1040 cm -1 and 825cm -1 The absorption peak intensity increases at a nearby location, at 2920 cm⁻¹ -1 and 2850 cm -1 The changes were not significant and were mainly related to changes in chemical composition and functional groups caused by microbial metabolic activities. 3440 cm -1 The vicinity exhibits OH stretching vibrations, and the polysaccharides produced by microorganisms are rich in hydroxyl groups (-OH), significantly increasing the intensity of these vibrations. (1600 cm) -1 The vicinity represents the C=O / CN stretching vibration. Microbial-secreted enzymes epoxidize the aromatic rings in lignin, generating more conjugated C=O or CN rings (such as nitrogen-containing heterocycles synthesized by microorganisms). 1500 cm -1 The area near the C=C vibration of the aromatic ring indicates that after partial degradation of lignin by microorganisms, the C=C vibration of the remaining aromatic ring structure is enhanced. (1040 cm⁻¹) -1 The vicinity exhibits CO stretching vibrations. The CO-C glycosidic bonds and C-OH vibrations are enhanced in extracellular polysaccharides secreted by microorganisms (such as cellulose and hemicellulose degradation products). 825 cm -1 Nearby, there is the out-of-plane bending vibration of the CH group of the aromatic ring, which is the result of microbial degradation of lignin.

[0033] 6. Elemental composition analysis Elemental composition analysis of the treated and control groups (as shown in Table 1) reveals that nitrogen (N) and oxygen (O) content generally decreased after microbial treatment, while carbon (C) content mostly increased. Hydrogen (H) and sulfur (S) content remained relatively stable, although some samples showed an increase in hydrogen content and others a decrease in sulfur content. The decrease in nitrogen content helps reduce nitrogen-containing heterocyclic compounds generated during pyrolysis, such as pyridine and quinoline, which may affect the quality and application of bio-oil. The decrease in oxygen content helps improve the thermal stability and combustion performance of bio-oil, while also reducing acidic substances and improving its quality. The increase in carbon content is generally related to carbon fixation and volatile matter reduction during pyrolysis, which improves the thermal and chemical stability of biochar, making it more suitable as an adsorbent or catalyst carrier. Changes in hydrogen content may affect the hydrogen-to-carbon ratio (H / C) of the pyrolysis products, thus influencing their combustion performance and calorific value. A lower hydrogen-to-carbon ratio helps reduce the thermodynamic losses of smoke and vapors caused by excessive oxidation. Reducing sulfur content helps decrease the emission of sulfur oxides from pyrolysis products, thereby reducing environmental pollution.

[0034] This demonstrates that microbial treatment significantly impacts the pyrolysis characteristics and product distribution of tobacco waste by altering its elemental composition. These changes help optimize the quality of pyrolysis products and enhance their application value.

[0035] Table 1: Comparative Analysis of Elemental Content in Different Tobacco Waste Materials Before and After Microbial Treatment

[0036] 7. Analysis of the Evolution Law of Pyrolysis Characteristics like Figure 7 The thermogravimetric analysis (TG) and differential thermogravimetric analysis (DTG) spectra of different tobacco wastes before and after microbial treatment are shown. The TG curves (Figures a, c, e, g) show the change in the mass percentage (TG%) of the sample during heating as a function of temperature; the decrease in the curve indicates mass loss, i.e., the release and decomposition of volatiles. The DTG curves (Figures b, d, f, h) show the change in the mass loss rate (DTG% / min) of the sample as a function of temperature; the peak value of the curve represents the maximum mass loss rate at a specific temperature, i.e., the maximum weight loss rate.

[0037] Combination Figure 7 As shown in Table 2, the overall trend of the TG curves of all treated groups (TS-V16, STP-V35, MTP-V35, TD-CE1) is similar to that of the untreated groups (TS, STP, MTP, TD). The DTG curves show a decrease in the peak value of the maximum weight loss rate for all treated groups, indicating that microbial treatment reduces the maximum weight loss rate during pyrolysis. The microbially treated samples show a wider peak distribution on the DTG curve, meaning that microbial treatment makes the pyrolysis process more dispersed, allowing the release of volatiles to occur over a wider temperature range. This allows more organic matter to be converted into volatiles, thereby reducing the solid residue rate. Therefore, the effects of microbial pretreatment on the pyrolysis characteristics of tobacco waste (reducing the maximum weight loss rate, reducing the solid residue rate, and altering the volatile release pattern) all contribute to optimizing the pyrolysis process, improving the yield and quality of bio-oil, while simultaneously reducing energy consumption and environmental impact during pyrolysis.

[0038] Table 2: Pyrolysis characteristic parameters of different tobacco wastes before and after microbial treatment

[0039] 8. Synergistic effect analysis of pyrolysis temperature and microbial treatment (1) Tobacco stems (TS and TS-V16) from Figure 8The graphs showing the changes in aroma components in TS and TS-V16 bio-oils at different pyrolysis temperatures reveal that the total aroma component content of TS bio-oil ranges from 31.82% to 50.89%, reaching its highest level at 350 °C. The total aroma component content of TS-V16 bio-oil ranges from 32.45% to 42.22%, slightly lower than TS overall, but exhibits better overall stability, with minimal changes in total bio-oil content across different pyrolysis temperatures, particularly at 350 °C and 500 °C. Furthermore, the ester content of TS-V16 is higher than that of TS, while the contents of other compounds such as alcohols, ketones, phenols, and heterocyclic compounds decrease after treatment. This is primarily because during the pretreatment stage, Bacillus subtilis degrades macromolecules, releasing small-molecule precursors. These precursors, after esterification and deacidification reactions, optimize the composition. Subsequently, during pyrolysis, the modified precursors directionally generate aroma components while inhibiting the formation of other products, resulting in higher quality aroma components in the bio-oil.

[0040] from Figure 9 The graph showing the changes in off-flavor content in TS and TS-V16 bio-oils at different pyrolysis temperatures reveals that the off-flavor substances in tobacco stem pyrolysis bio-oils are mainly heterocyclic and ketone compounds. The treated TS-V16 showed a significant reduction in off-flavor substances, particularly within the pyrolysis temperature range of 350℃-450℃, with the lowest total off-flavor content at 350℃ (only 0.83%). At 500℃, the off-flavor content was 4.98%, presumably due to the high temperature destroying microbial degradation products, leading to the regeneration of some off-flavor substances (such as the surge in ketones, possibly due to the conversion of microbial metabolic intermediates into stable ketone compounds at high temperatures). While the total off-flavor content in the untreated TS group decreased with increasing pyrolysis temperature, it still could not reach the levels seen after microbial treatment at 400℃-500℃, with 5.13% at 500℃, indicating that high temperature alone cannot completely eliminate off-flavor components. It is evident that microbial treatment can significantly reduce the content of heterocyclic compounds and amines in tobacco stem bio-oil, indicating its highly efficient degradation ability for nitrogen- and oxygen-containing heterocyclic compounds.

[0041] Combination Figure 8 and Figure 9 Analysis showed that the TS-V16 treatment group had a higher total amount of aroma components from bio-oil and the lowest content of off-flavor substances at a pyrolysis temperature of 350 ℃, indicating better stability and quality.

[0042] (2) Small pieces of tobacco leaves (STP and STP-V35) from Figure 10The changes in aroma components in STP and STP-V35 bio-oils at different pyrolysis temperatures show that the overall aroma component content in the treated STP-V35 bio-oil is higher than that in the untreated STP group. Furthermore, the contents of alcohols, acids, phenols, and heterocyclic compounds decrease, while the contents of aldehydes, esters, and ketones increase. Esters reach their highest content (35.2%) at 350 °C, indicating that strain V35 promotes esterification. As the temperature increases, the ester content decreases, as the microbially modified ester precursors undergo thermal decomposition at high temperatures, generating small molecule volatiles (CO2, olefins), leading to a decrease in ester yield. Simultaneously, ketones increase while acids decrease, possibly due to further decarboxylation of fatty acids during pyrolysis under microbial pretreatment. Strain V35 degrades lignin derivatives (such as guaiacol) in tobacco leaves, resulting in a decrease in phenol content. Strain V35 uses free amino acids and proteins in tobacco leaves as nitrogen sources, reducing the content of nitrogen-containing heterocyclic precursors during pyrolysis, thus lowering their content.

[0043] from Figure 11 The graphs showing the changes in off-flavor content in STP and STP-V35 bio-oils at different pyrolysis temperatures reveal that the off-flavor substances in small-piece tobacco bio-oils are mainly heterocyclic compounds and amines. The total amount of off-flavor substances in the untreated STP group was lowest at 400℃, dropping sharply from 12.47% at 350℃ to 1.75%. In contrast, the total amount of off-flavor substances in the treated STP-V35 group showed a trend of first increasing and then decreasing with increasing pyrolysis temperature, reaching its highest at 450℃ and its lowest at 500℃ (only 0.6%). This indicates that pretreatment with Bacillus pumilus at 500℃ can degrade nitrogen-containing precursors (such as alkaloids) and prevent the recombination of off-flavor substances.

[0044] Combination Figure 10 and Figure 11 Analysis showed that although the total amount of aroma-producing components of bio-oil was high in the STP-V35 treatment group at a pyrolysis temperature of 350 ℃, its content of off-flavor substances was much higher than that at 500 ℃. In the field of fragrances, off-flavor substances have a fatal impact on product quality. Therefore, the optimal pyrolysis temperature for the STP-V35 treatment group should be determined to be 500 ℃, at which the content of off-flavor substances is the lowest.

[0045] (3) Medium-sized broken tobacco leaves (MTP and MTP-V35) from Figure 12 The changes in aroma components of MTP and MTP-V35 bio-oils at different pyrolysis temperatures show that, compared to the untreated MTP group, the treated MTP-V35 group exhibits increased levels of alcohols, esters, and acids, with little change in ketone content, and decreased levels of phenols, aldehydes, and heterocyclic compounds. These changes not only reflect the impact of microbial catalysis on the bio-oil composition but also contribute to improved aroma quality.

[0046] Specifically, microbial catalysis promotes the esterification of fatty acids and alcohols, increasing ester content. Simultaneously, the degradation of polysaccharides (such as cellulose) generates small-molecule alcohols, leading to alcohol accumulation. Furthermore, incomplete degradation of fatty acids by microorganisms, or the hydrolysis of esters during pyrolysis to generate free fatty acids, increases acid content. Microorganisms preferentially consume nitrogen-containing precursors, reducing the formation of heterocycles such as pyrrole and pyridine, and further oxidize aldehydes to acids or reduce them to alcohols via dehydrogenases.

[0047] These changes in components have significantly optimized the aroma quality of bio-oils. The most significant advantage is the substantial increase in alcohols and esters. Esters typically possess pleasant aromas such as fruitiness, sweetness, and wineiness, and are the main components constituting high-end aromas; while alcohols provide a mild, mellow aroma and a certain degree of aroma enhancement. The esterification reaction catalyzed by microorganisms directly synthesizes more of these pleasant aroma molecules, greatly enhancing the richness, intensity, and appeal of the bio-oil aroma.

[0048] A moderate increase in acidic substances is not a sign of deterioration. Low concentrations of acids (such as acetic acid) can provide a slight sour and vinegary aroma, which harmonizes with flavor compounds such as esters and alcohols, suppresses sweetness, and increases the layers and complexity of the aroma, making the overall flavor closer to the aroma of natural, mature tobacco. This is key to quality improvement.

[0049] Meanwhile, aldehydes (such as furfural) typically have a pungent, grassy, ​​and unpleasant odor, and a low aroma threshold, meaning even small amounts can have a negative impact. Heterocyclic compounds (such as pyridine and pyrrole) are even more typical examples of undesirable off-odors, producing unpleasant roasting, burnt, or even ammonia-like smells. Microbial pretreatment significantly reduces the content of these undesirable components by consuming their precursors and transforming aldehydes, effectively eliminating the pungent off-odors of the aroma and making the overall fragrance purer and smoother.

[0050] Phenolic compounds have a smoky aroma, and excessive amounts can cause a burning sensation in the throat. Reducing their content helps to minimize these negative sensations. At the same time, reducing nitrogen-containing heterocyclic compounds not only improves the aroma but also means a decrease in the formation of potentially harmful nitrogenous compounds, thus enhancing product safety and the consumer experience to some extent.

[0051] In summary, compared with the untreated MTP group, the treated MTP-V35 group showed a significant optimization of the aroma components of its bio-oil. The aroma profile was transformed from potentially "pungent and chaotic" to "mellow, rich, and pleasant," and the overall aroma quality achieved a comprehensive improvement of "enhancing the superior and reducing the inferior."

[0052] from Figure 13The graphs showing the changes in off-flavor content in MTP and MTP-V35 bio-oil at different pyrolysis temperatures reveal that the main off-flavor compounds in the untreated MTP group are heterocyclic compounds, amines, and phenols. The total amount of off-flavor compounds in both the untreated and treated MTP-V35 groups increases with increasing temperature. The untreated MTP group has the highest off-flavor content at 500 ℃ and the lowest at 400 ℃ (3.79%, but with a relatively high amine content). In contrast, the treated MTP-V35 group primarily contains heterocyclic compounds, with lower levels of other compounds. Its off-flavor content is highest at 400 ℃ and lowest at 450 ℃ (only 3.33%, and amines are zero).

[0053] Combination Figure 12 and Figure 13 Analysis shows that, while maintaining a low content of off-flavor substances, MTP-V35 performs best at a pyrolysis temperature of 450℃, with the lowest content of off-flavor substances.

[0054] (4) Tobacco dust (TD and TD-CE1) from Figure 14 The graphs showing the changes in aroma components in TD and TD-CE1 bio-oils at different pyrolysis temperatures reveal that, compared to the untreated TD group, the aroma components in the treated TD-CE1 group changed at different pyrolysis temperatures. In the low-temperature region (350 ℃), esters decreased, indicating that *Trichoderma viride* preferentially utilized fatty acids and alcohols from tobacco dust as carbon sources, resulting in insufficient substrate for the esterification reaction. As the temperature increased, the ester content rose. Aromatic acids produced from lignin degradation reacted with alcohols to form aromatic esters during pyrolysis, and some fatty acids may participate in secondary esterification. Compared to common fatty esters, aromatic esters possess a richer, more persistent, and more elegant fruity, floral, or honeyed aroma, significantly enhancing the quality and complexity of the bio-oil's aroma.

[0055] Cellulose degradation products are converted into aldoses by oxidases, which are further pyrolyzed to generate small molecule aldehydes, thus increasing the aldehyde content. These small molecule aldehydes (such as furfural and 5-methylfurfural) can provide pleasant roasted, sweet, and nutty aromas when in appropriate amounts. Moreover, these substances have a low aroma threshold and strong diffusion, which can effectively enhance the intensity and recognizability of the aroma, making the overall aroma more lively and less dull.

[0056] Lignin-derived phenols oxidize to form aromatic ketones, which typically possess delicate sweet, woody, and creamy aromas. Their introduction enriches the aroma spectrum, forming a complex, harmonious, and distinctive aroma profile together with aromatic esters and small molecule aldehydes, preventing the aroma from becoming monotonous. The reduction in acids is due to the conversion of some acids into esters; heterocycles are stable at high temperatures; and heat-resistant nitrogenous metabolites undergo pyrolysis at high temperatures to form heterocycles.

[0057] The pretreatment with *Trichoderma viride* establishes a specific precursor base (such as enrichment of aromatic acids and aldoses), while the subsequent pyrolysis temperature acts as a "switch," precisely triggering the conversion of these precursors into specific aroma compounds (such as low-temperature inhibition and high-temperature promotion of esterification). This combined "bio-thermochemical" strategy provides a powerful technical means for the targeted design of aroma profiles for bio-oils, which is unmatched by single pyrolysis techniques.

[0058] from Figure 15 The graph showing the changes in off-flavor content in TD and TD-CE1 bio-oils at different pyrolysis temperatures reveals that the main off-flavor substances in tobacco bio-oils are heterocyclic compounds, amines, and phenols. The total off-flavor content in the untreated TD bio-oil is lowest at 450 °C (0.43%). The total off-flavor content in the treated TD-CE1 bio-oil is extremely low at 350 °C and 450 °C (0.36% and 0.56%, respectively), superior to the untreated group. At 400 °C and 500 °C, the off-flavor content is higher, possibly related to the recombination of microbial metabolic intermediates at high temperatures.

[0059] Combination Figure 14 and Figure 15 Analysis shows that TD-CE1 has a lower total amount of off-flavor substances at pyrolysis temperatures of 350 ℃ and 450 ℃, and the content of aroma-producing substances produced by the pyrolysis of tobacco dust is higher at 350 ℃. Therefore, choosing a pyrolysis temperature of 350 ℃ for TD-CE1 can achieve the goal of "highest content of aroma-producing substances, excellent quality, and lowest content of off-flavor substances".

[0060] The off-flavor substances mentioned in (1)-(4) above mainly originate from the complex components of tobacco, such as alkaloids, sugars, proteins, and phenols (as shown in Table 3 below). The decomposition, recombination, and oxidation reactions of these components during pyrolysis bring about unpleasant odors such as irritation, bitterness, and burnt smells, affecting the quality and application of bio-oils. Among them, the most representative components include tar, nicotine, carbon monoxide, hydrogen cyanide (HCN), 4-methylnitrosamine-1-3-pyridyl-1-butanone (NNK), ammonia, benzo[a]pyrene, phenol, and crotonaldehyde.

[0061]

[0062] The main transformation mechanisms of off-flavor substances include heterocyclic control, amine degradation, and ketone inhibition, specifically: Microorganisms control heterocyclic compounds by destroying nitrogen-containing precursors (such as nicotine) through deaminases and oxidoreductases, thereby inhibiting the formation of pyridine compounds. Simultaneously, *Bacillus pumilus* V35 converts amines into NH3 / N2, while *Trichoderma viride* CE1 promotes amine oxidation through hydroxylation, thus achieving amine degradation. Furthermore, microbial pretreatment reduces carboxylic acid intermediates, thereby decreasing the amount of ketones (such as acetone) formed during pyrolysis.

[0063] 9. Analysis of the effect of pyrolysis temperature on biochar After the pyrolysis reaction, the residual biochar content of each sample group is shown in Table 4. The residual content of TS ranged from 49.45% to 62%, TS-V16 from 50.4% to 57.2%; STP from 44.4% to 53.6%, STP-V35 from 51% to 58.4%; MTP from 49.4% to 59.2%, MTP-V35 from 47.6% to 58.8%; and TD from 46.8% to 55.4%, TD-CE1 from 50.6% to 58%. In all sample groups, within the temperature range of 350-500 °C, the residual biochar content decreased with increasing temperature. At 450-500 °C, the residual content of most samples reached its lowest value, indicating that high temperature promotes the decomposition and volatilization of organic matter, resulting in a more complete pyrolysis reaction.

[0064] Table 4: Biochar Residue in Tobacco Waste at Different Pyrolysis Temperatures

[0065] Since the biochar residue content is relatively high at 350℃ compared to other temperatures, SEM image analysis was performed on the untreated and treated groups at this temperature to more intuitively compare the surface changes of tobacco waste.

[0066] from Figure 16The SEM images of pyrolysis biochar from tobacco waste at 350 °C show the following: For tobacco stems (a, b): (a) TS, untreated tobacco stems exhibit a porous network surface morphology, reflecting the decomposition of cellulose / hemicellulose and pore formation caused by high-temperature pyrolysis, a typical characteristic of lignocellulosic biomass pyrolysis; (b) TS-V16, the biochar surface treated with *Bacillus simulans* is covered with bacterial biofilm or metabolites, leading to particle deposition. For small pieces of tobacco leaves (c, d): (c) STP, untreated small pieces show a compact, layered microstructure with minimal surface roughness; (d) STP-V35, *Bacillus simulans* treatment leads to surface degradation, with the strain's biocorrosion of the carbon matrix causing edge blunting. For medium pieces of tobacco leaves (e, f): (e) MTP, compared to small pieces, untreated medium pieces show a more pronounced fibrous structure and larger particle aggregates; (f) MTP-V35, *Bacillus simulans* treatment enlarges existing pores or creates new pores, forming a looser microstructure. Tobacco dust (g, h), (g)TD, untreated tobacco dust with wrinkled surface, (h)TD-CE1, after treatment with Trichoderma viride, bacterial biofilm or metabolites may cover part of the surface, resulting in local smoothness and forming microbial-induced depressions.

[0067] Based on the above analysis, it can be determined that the pyrolysis of tobacco stems treated with Bacillus simulans V16 at 350 ℃ can balance the degradation of off-flavor substances and energy consumption; the pyrolysis of small pieces of tobacco leaves treated with Bacillus pumilus V35 at 500 ℃ and the pyrolysis of medium pieces of tobacco leaves treated with Bacillus pumilus V35 at 450 ℃ can well balance the thoroughness of pyrolysis and the stability of microbial metabolites; and the pyrolysis of tobacco dust treated with Trichoderma viride CE1 at 350 ℃ can balance the degradation of off-flavor substances and energy consumption.

[0068] The present invention has been described in detail above through specific and preferred embodiments. However, those skilled in the art should understand that the present invention is not limited to the embodiments described above. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for preparing fragrances by microbial pretreatment of tobacco waste through pyrolysis, characterized in that, Includes the following steps: S1. Strain selection: Bacillus simulans strain V16: Targets tobacco stems; Bacillus pumilus strain V35: for broken tobacco leaves; Trichoderma viride CE1: for tobacco dust; S2. Preparation of microbial preparations: (1) The preparation methods of Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation are the same: the strain is inoculated into LB liquid medium, and after two shaking cultures, it is centrifuged, washed, and water is added and shaken to prepare Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation. (2) Method for preparing crude enzyme solution of Trichoderma viride CE1: After inoculating it into 50 mL of LB medium and activating it twice, it was inoculated into 200 mL of PDA liquid medium for fermentation culture. The supernatant was filtered and the crude enzyme solution was obtained by ammonium sulfate precipitation and dialysis. S3. Microbial pretreatment: The Bacillus simulans V16 preparation, Bacillus pumilus V35 preparation and crude enzyme solution obtained in step 2 were evenly sprayed onto tobacco stems, broken tobacco leaves and tobacco dust, respectively, and then put into sealed bags. After being treated under certain temperature and humidity conditions for a certain period of time, they were inactivated at high temperature and dried. S4. Extraction of bio-oil by pulverization and dry distillation: The tobacco waste pretreated in step S3 is dried until the moisture content is below 7% and pulverized to a particle size of below 60 mesh. The dried and pulverized tobacco waste is then placed in a reactor for dry distillation. After being treated at the set dry distillation temperature for a certain period of time, bio-oil and biochar are obtained.

2. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 1, characterized in that, In step S2, the preparation method of Bacillus simulans V16 and Bacillus pumilus V35 is as follows: the strain is inoculated into 50 mL of LB liquid medium and cultured at 37℃ and 180 r / min for 14 h to obtain liquid culture; then the liquid culture is inoculated into 200 mL of LB liquid medium at an inoculation amount of 0.1% and cultured at 37℃ and 180 r / min for 14 h until the OD value is 2.0 to obtain culture solution; finally, 20 mL of culture solution is taken, centrifuged at 8000 rpm / min for 10 min, the supernatant is removed, and the precipitate is washed with sterile water. Finally, 200 mL of sterile water is added to the precipitate and shaken evenly to obtain Bacillus simulans V16 and Bacillus pumilus V35 preparations.

3. The process for preparing fragrances by microbial pretreatment of tobacco waste through pyrolysis according to claim 1 or 2, characterized in that, In step S2, (2) the method for preparing crude enzyme solution from Trichoderma viride CE1 is as follows: Trichoderma viride CE1 is inoculated into 50 mL of LB medium and cultured at 37 ℃ and 180 r / min for 14 h for activation twice. Then, it is inoculated into an Erlenmeyer flask containing 200 mL of PDA liquid medium and fermented at 28 ℃ and 180 rpm for 5 days. The supernatant is then filtered and collected. Ammonium sulfate is then slowly added to the supernatant until it reaches 80% saturation. The mixture is allowed to stand overnight at 4 ℃. The precipitate is collected by centrifugation and dissolved in a small amount of buffer solution. The mixture is then dialyzed with the same buffer solution to obtain crude enzyme solution.

4. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 3, characterized in that, The buffer solution is 0.02 mol / L Na2HPO4 and NaH2PO4, and the pH value is 7.

3.

5. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 1, characterized in that, In step S3, the dosage of Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation is 0.2 mL / g tobacco waste; the dosage of crude enzyme solution is 60 U / g cellulase / tobacco waste.

6. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 1, characterized in that, In step S3, the Bacillus simulans V16 preparation and Bacillus pumilus V35 preparation were treated at 30°C for 48 hours before being heated to 120°C and held for 20 minutes for high-temperature inactivation when pretreating tobacco stems and broken tobacco leaves. The crude enzyme solution was treated at 30°C and 25% relative humidity for 5 hours before being heated to 80°C and held for 20 minutes for high-temperature inactivation when pretreating tobacco dust.

7. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 1, characterized in that, In step S4, the dry distillation temperature is 300℃-700℃ and the holding time is 30min.

8. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 7, characterized in that, The optimal temperature for the dry distillation of tobacco stems is 350℃.

9. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 7, characterized in that, The optimal temperature for dry distillation of small pieces of tobacco is 500℃, and the optimal temperature for dry distillation of medium-sized pieces of tobacco is 450℃.

10. The process for preparing fragrance from tobacco waste by microbial pretreatment pyrolysis according to claim 7, characterized in that, The optimal temperature for the dry distillation of tobacco dust is 350℃.

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