Evaluation of the Potentials of Tobacco Waste Extract as Wood Preservatives against Wood Decay Fungi

: Utilizing conventional wood preservatives poses potential risks to ecosystems and human health. Therefore, the wood protection industry must develop alternatives that are both efficient and environmentally friendly. In this paper, industrial tobacco waste extracts were used as eco-friendly wood preservatives against wood decay fungi. Three major constituents in the extracts were identified via gas chromatography-mass spectrometry (GC-MS) and included nicotine, neophytadiene, and 2,7,11-cembratriene-4,6-diol. The antifungal activities of waste tobacco extracts and these three major constituents against wood decay fungi were tested. At a concentration of 40 mg/mL, the tobacco waste extract treated with 50% ethanol significantly inhibited the activity of wood decay fungi. This was because the extract contained nicotine as the primary active component and neophytadiene as a synergistic active component. Wood decay resistance tests were conducted on Pinus yunnanensis and Hevea brasiliensis treated with a 50% ethanol extract of tobacco waste at a concentration of 40 mg/mL. The mass losses of Pinus yunnanensis exposed to G. trabeum and T. versicolor were 4.11% and 5.03%, respectively, while the mass losses in Hevea brasiliensis exposed to G. trabeum and T. versicolor were 7.85% and 9.85%, respectively, which were classified as highly resistant. The acute ecotoxicity of the tobacco waste extract was assessed using a kinetic luminescent bacteria test with Aliivibrio fischeri , which revealed significantly lower acute toxicity than a commercial copper-based wood preservative. This study offers insights into high-value utilization of tobacco waste and advancement of natural wood preservatives.


Introduction
Wood has been widely used in the construction and furniture industries for thousands of years due to its renewability, versatility, durability, robust structure, cost-effectiveness, non-toxicity, and abundant availability [1][2][3].However, it is susceptible to degradation due to biotic and abiotic factors.Wood preservatives are employed to enhance their performance and longevity in various applications.Traditional wood preservatives such as copper, arsenic, and chromium-based materials have been used to protect wood from decay, but these metals are highly toxic [4,5], and their release into the environment may adversely affect ecological systems and human health [6].Therefore, increasing attention has been devoted to researching wood preservative alternatives that demonstrate low toxicity and environmental friendliness.
Natural plant extracts have been used to improve wood decay resistance as alternative wood preservatives, including Tung oil [7], tannin [8], wood extractives [9][10][11], propolis extracts [12,13], chitosan, and essential oils [14].Several industrial sidestreams, such as pyrolytic distillate from tree bark [15], coffee silverskin extractives [16], konjac fly powder [17], and coconut shell pyrolytic oil distillate [18], have also been used.The efficacy of natural wood preservatives has been the main focus of previous studies.However, only a limited number of studies have characterized these active compounds in preservatives and evaluated their effectiveness.Furthermore, ecotoxicity has rarely been considered, even though many natural wood preservatives can also be toxic or harmful to the environment.To advance sustainable development in the wood protection industry, a deeper understanding of the impact of extraction concentration, wood treatment method, wood species, wood decay resistance, active compounds, and ecotoxicity in natural preservatives is crucial.
Tobacco is an important economic crop [19], whose cultivation and industrial production generate a significant quantity of biomass waste [20].According to a 2019 report, the global annual cigarette consumption amounts to approximately 6 trillion cigarettes, with tobacco production totaling around 6.68 million tons.China leads in both tobacco cultivation area and production, with an annual production exceeding 2 million tons.However, during cultivation and processing, about 25% of the total tobacco output becomes waste [21].Currently, most of this waste is either discarded or incinerated.Improper treatment of tobacco waste not only wastes resources but also contributes to pollution in the atmosphere, soil, and groundwater, posing environmental risks.Therefore, the high-value utilization of tobacco waste could enhance the ecological, economic, and social benefits associated with the tobacco industry.Tobacco waste biomass contains alkaloids and polyphenols substances.Tobacco waste may be converted into value-added products.Previous studies have reported the antibacterial properties of tobacco extract against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa [22][23][24].The antifungal property of tobacco stalks was confirmed by the previous literature, which described its activity against brown rot fungi Coniophora puteana [25]; however, further validation is necessary to confirm the effectiveness of tobacco waste extract as a wood preservative against wood decay fungi in the field of wood protection.
Pinus yunnanensis and Hevea brasiliensis are two widely planted fast-growing trees in southwest China, whose woods are being gradually applied in construction, furniture, and as raw materials for wood fiber.However, the low natural durability of these woods restricts their potential applications and usage scenarios.Therefore, the development of treatments targeting the enhancement of wood decay resistance in these two tree species is necessary.The objective of this study was to assess the potential of using tobacco waste extract as a natural and environmentally friendly wood preservative.Initially, the tobacco extract was characterized, and then, the antifungal activities of waste tobacco extracts and three major constituents were tested against wood decay fungi.Wood decay resistance tests were performed on Pinus yunnanensis and Hevea brasiliensis species, and the acute ecotoxicity of the tobacco waste extract was assessed using a kinetic luminescent bacteria test with Aliivibrio fischeri and compared with that of commercially available copper-based wood preservatives.This study offers insights into the high-value utilization of tobacco waste and the advancement of natural wood preservatives.
Softwood-Pinus yunnanensis Franch (Pinales: Pinoideae), aged over 25 years old, with a diameter of 28 cm at breast height and an annual growth width of 1.35 mm, and hardwood-Hevea brasiliensis (Willd.ex Juss.)Müll.Arg., aged over 25 years old, with a diameter of 30 cm at breast height and an annual growth width of 4.27 mm were obtained from Yunnan State Farms Group Co., Ltd., Jinghong, China.Mature logs measuring 180 cm in length were selected.According to the Chinese Standard GB/T 13942.1-2009 [26], sap-wood sections were cut into clear, defect-free blocks with dimensions of 20 × 20 × 10 mm (R × T × L).Subsequently, the blocks were dried at 60 • C until they reached a consistent weight (~8% moisture content).The oven-dried density of Pinus yunnanensis and Hevea brasiliensis was approximately 0.48 g/cm 3 and 0.54 g/cm 3 , respectively.
The brown-rot fungus Gloeophyllum trabeum (Pers.ex.Fr.) Murr.(G.trabeum) and the white-rot fungus Trametes versicolor (L.ex Fr.) Qu'el.(T.versicolor) were obtained from the China Forestry Culture Collection Center (Beijing, China).The fungi were cultivated on a culture medium consisting of 3.7% potato dextrose agar (PDA) at 28 • C and a relative humidity of 80% for seven days before use.
The tobacco waste leaves were obtained from China Tobacco Yunnan Industrial Co., Ltd., Kunming, China (N25.75739).They were air-dried, crushed, and sieved through an 80-mesh screen for further use.

Extraction of Tobacco Wastes
Tobacco waste powder (10 g) was macerated in 250 mL of one of three solvents: water, 50% ethanol, or anhydrous ethanol.The resulting mixture was subsequently sonicated for 40 min at 60 • C using an ultrasonicator (CNC Ultrasonic Cleaner, KQ-200KDE, Kunshan Ultrasonic Instrument Co., Ltd., Suzhou, China).The ultrasonicator was operated at a fixed frequency of 40 kHz and a power of 200 W.The extracted substances were then filtered using filter paper and concentrated using a rotary evaporator.The obtained extracts were dried and stored in opaque bottles before use.

Chemical Composition Analysis of Tobacco Waste Extracts via GC-MS
Analyses were performed on a GC-MS (Agilent 6890N, USA) equipped with a 5973N mass spectrometer (Agilent, Santa Clara, CA, USA).An HP-5MS capillary fused silica column (30 m × 0.25 mm × 0.25 µm) was used for separation, and helium (99.999%) was used as carrier gas with a flow rate of 1 mL/min.The temperature program was initiated at 50 • C (1 min), then 10

Antifungal Activity Test against Wood Decay Fungi
The antifungal activities of tobacco waste extracts and three major constituents were assessed.Solutions of tobacco extracts with concentrations of 5, 10, 20, 40, and 80 mg/mL were prepared along with three major constituents at concentrations of 1, 2, 4, 8, and 16 mg/mL.Before incorporating the tobacco waste extracts and three major constituents, molten PDA was cooled to 60 • C. The mixtures were subsequently transferred onto cooled Petri dishes with a diameter of 90 mm.A disc measuring 5.00 mm was obtained from the test fungi and placed in the center of each Petri dish.The incubation process lasted for 14 days at 28 • C and 80% RH in a dark environment.For the brown-rot fungus G. trabeum and white-rot fungus T. versicolor, experiments were conducted on three Petri dishes for each concentration of tobacco waste extracts and three major constituents.After inoculation, the diameters of fungal colonies were measured, and the digital photographs were captured.

Antifungal Activity Test against Wood Decay Fungi
Solutions of tobacco extracts with concentrations of 5, 10, 20, 40, and 80 mg/mL were prepared.The softwood and hardwood blocks were treated by submerging them in treatment solutions under a vacuum (−0.08 MPa) for 30 min, followed by the application of 0.7 MPa pressure for 15 min.The treated blocks were then dried in an oven at 60 • C until they achieved a stable weight.The preservative retention was calculated using Equation (1), with six replicates utilized for each experimental group.
where R represents the preservative retention (kg/m 3 ); V denotes the specimen volume (cm 3 ), and c represents the concentration of a preservative solution (%); m 1 and m 2 refer to the weight of the specimen before and after impregnation with the preservative, respectively (g).

Wood Decay Resistance Tests
Wood decay resistance tests were conducted according to the Chinese Standard GB/T 13942.1-2009.The classification of wood decay resistance is presented in Table 1.The decay fungi were incubated at 28 • C and 80% RH until the mycelium completely colonized the surface of PDA.Subsequently, the sterilized wood block samples from an autoclave were transferred to the fungal cultures.All treated block samples were incubated at 28 • C and 80% RH.After incubation, the mycelium was removed from the wood block, dried, and then weighed.The mass loss was calculated using Equation (2).To determine the average mass loss (ML), six replicates were employed for each experimental group.
where m 3 represents the initial weight before the wood decay resistance tests, and m 4 denotes the weight after 12 weeks of fungal incubation.

Acute Ecotoxicity Assessment
The acute ecotoxicity assessment of tobacco waste extracts was evaluated according to the ISO 11348-3:2007 standard procedure.The inhibitory effect (inhibitory concentration: IC 50 , mg L −1 ) of tobacco waste extracts on the bioluminescence emission of Aliivibrio fischeri was determined using a BioFix ® Lumi-10 luminometer (Macherey-Nagel GmbH & Co. KG, Duren, Germany).The control group employed a commercially available copperbased preservative with a concentration of 40 mg/mL.Meanwhile, the experimental group utilized tobacco waste extracts at an equivalent concentration.The IC 50 values were calculated through regression analysis using the logarithmic-probit analysis method.

Statistical Analysis
All experiment values were recorded as means ± standard deviation (n = 3).SPSS 24 (IBM, Armonk, NY, USA) was used for statistical analysis, and p-values < 0.05 were considered statistically significant.

Chemical Composition of Tobacco Waste Extracts
The GC-MS ion chromatograms of the three tobacco waste extracts are presented in Figure 1 and Table 2.A total of 33 major compounds were identified in the water extract of waste tobacco, including alkaloids (3), acids (2), ketones (9), alcohols (7), esters (7), olefins (2), and aromatic compounds (3).In both the 50% ethanol and absolute ethanol extracts, a total of 24 major compounds were identified, including alkaloids (3), acids (1), ketones (7), alcohols (9), esters (1), olefins (1), and aromatic compounds (2).The alkaloid compounds mainly included nicotine; the ketone compounds included β-damascone, solanone, and four isomers of tabanone; the alcohol compounds included linalool, phytol, 2,7,11cembratriene-4,6-diol, and stigmasterol.The ester compounds included ethyl phenylacetate and methyl palmitate.The olefin compounds mainly included neophytadiene.The concentration of different types of compounds varied significantly.Nicotine, neophytadiene, and 2,7,11-cembratriene-4,6-diol were the predominant volatile components in all three tobacco waste extracts.Their concentrations in the water extract were 7.02%, 10.07%, and 5.15%, respectively, and 6.29%, 8.82%, and 8.78% in the 100% ethanol extract.Significantly higher concentrations of 9.32%, 8.78%, and 13.61% were observed in the 50% ethanol extract.The GC-MS analysis results were consistent with previous studies, indicating the presence of nicotine, 4,8,13-cembratriene-1,3-diol, and neophytadiene [27].Factors such as seasonality, geographical location, time of collection, and extraction parameters (e.g., temperature, duration, and solvent-to-solid ratio) may account for variations between the findings reported in this study and the previous research [28].In previous reports, 2,7,11-cembratriene-4,6-diol acted as an antitumor inhibitor and inhibited the growth of wheat germ worms [29].Nicotine and neophytadiene have exhibited inhibitory effects on Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa [30,31].These findings indicate that these three major constituents may have potential antibacterial and antitumor properties.However, research on the antifungal activity of these compounds remains limited.Further research is, therefore, necessary to investigate their antifungal activity against wood decay fungi and evaluate their role in the antifungal effects of tobacco waste extracts.

Antifungal Activity of Tobacco Waste Extracts against Wood Decay Fungi
The activities against wood decay fungi of three different types of tobacco waste extracts were investigated, as illustrated in Figures 2 and 3.The control PDA showed robust growth of white rot and brown rot fungi and colonized the entire medium's surface after a 14-day incubation period.In contrast, all tobacco waste extracts inhibited the growth of both types of fungi, and their activities against wood decay fungi were enhanced upon increasing their concentration.Extracts with concentrations of 20 mg/mL for each type of extract (50% ethanol, water, and ethanol) exhibited inhibition rates greater than 50% against wood decay fungi.The inhibitory effect of the 50% ethanol extract on wood decay fungi was superior to that of the water and absolute ethanol extractives at equivalent concentrations.A concentration of 40 mg/mL WE extract completely inhibited the growth of brown rot fungi, and the WE extract exhibited a stronger inhibitory effect on brown-rot fungi compared with white rot fungi at all tested concentrations.The previous literature suggests that teak water extract [9], konjac flying powder extract [15], withania somnifera fruit extract [32], and propolis extract [33] inhibited the growth of wood decay fungi at concentrations of 40, 40, 30, and 120 mg/mL, respectively.The antifungal efficacy of plant extracts relies on the characteristics of their active ingredients.Therefore, by evaluating the antifungal activity of the active ingredients, the crucial components that contribute to the antifungal properties of plant extracts can be identified.

Antifungal Activity of Major Constituents against Wood Decay Fungi
As shown in Figures 4 and 5, the inhibitory effect on G. trabeum and T. versicolor exhibited a gradual enhancement as the concentration of the three major constituents increased.The maximum inhibitory effects of nicotine were observed at concentrations above 8 mg/mL during the 14-day incubation period.Nicotine exhibited greater inhibitory effects on G. trabeum than on T. versicolor above 8 mg/mL, which is consistent with previous studies [34].In contrast, neophytadiene exhibited lower inhibitory effects on G. trabeum at a concentration of 16 mg/mL, while its inhibitory effect on T. versicolor was similar to that on G. trabeum.At various concentrations, 2,7,11-cembratriene-4,6-diol did not inhibit any wood decay fungi.Therefore, the results of this study reveal that nicotine was the primary active component and neophytadiene was a synergistic component for inhibiting wood decay fungi.The antifungal mechanisms of natural ingredients include cell wall destruction [35], cell membrane integrity disruption [36], respiration inhibition [37], and enzymatic activity inhibition [38].Previous studies have reported that nicotine inhibited the activity of catalase and glutathione enzymes [39].Thus, nicotine's strong inhibitory effect on wood decay fungi could be attributed to its action on enzymes during wood decay.This information is valuable for guiding research and development efforts for antifungal agents against wood decay fungi.

Retention Analysis
As indicated in Table 3, the retention of wood samples showed an upward trend as the concentration increased.At low concentrations (5 mg/mL and 10 mg/mL), there was minimal difference in retention rates between the two species.However, as the concentration increased, Hevea brasiliensis had a higher retention rate than Pinus yunnanensis.The previous literature has reported that wood density is related to retention; higher wood density results in lower retention rates.In this experiment, Pinus yunnanensis and Hevea brasiliensis had densities of 0.48 g/cm 3 and 0.54 g/cm 3 , respectively, with little variation between them.Therefore, the difference in retention rate may be attributed to Pinus yunnanensis containing more extracts compared to Hevea brasiliensis, which resulted in a decrease in the permeability of the tobacco waste extract in Pinus yunnanensis.

Wood Decay Resistance of Tobacco Waste Extracts
Wood decay resistance was assessed using the mass loss after fungal degradation after 12 weeks of fungal exposure, as illustrated in Figure 6.The control samples of softwood and hardwood exposed to G. trabeum exhibited mass losses of 18.49% and 37.73%, respectively, and those of softwood and hardwood control samples inoculated with T. versicolor were 20.72% and 35.68%, respectively.These findings indicated that hardwood exposed to the two decay fungi demonstrated higher mass loss than the corresponding softwood owing to the differences in inherent structural and chemical composition [40].Treatment with all three waste tobacco extracts reduced the mass loss of softwood and hardwood samples.Both softwood and hardwood samples treated with the 50% ethanol extract achieved the highest resistant classification, while those treated with WT and AE tobacco waste extract did not.In addition, the wood samples treated with the 50% ethanol extract exhibited higher wood decay resistance when exposed to brown rot fungi compared to white rot fungi.The different antifungal components extracted by different solvents may account for this phenomenon.The 50% ethanol extract was more suitable for extracting alkaloids, whereas anhydrous ethanol preferentially extracted lipophilic active substances, and water extracted highly polar compounds [41,42].The mass losses of the softwood exposed to G. trabeum and T. versicolor were only 4.11% and 5.03%, and only 7.85% and 9.85% in hardwood exposed to G. trabeum and T. versicolor, respectively, which were classified as highly resistant.Therefore, considering the cost-effectiveness and by-products of tobacco waste, the 40 mg/mL tobacco waste extract treated with 50% ethanol demonstrated excellent wood decay resistance and showed potential for utilization as an industrial waste product for wood preservation.

Acute Ecotoxicity Test
The EC 50 value of the algal test was used to rank algae to determine the toxicity ranking.According to the acute ecotoxicity test, the tobacco waste extracts showed low ecotoxicity and were ranked as 'not harmful' (EC 50 > 100 mg/L), with an EC 50 of 1065 mg/L.In contrast, the copper-based wood preservative showed much higher ecotoxicity, ranked as 'very toxic' (EC 50 > 0.1-1 mg/L), with an EC 50 of 0.134 mg/L.The ecotoxicity of tobacco waste extract was significantly lower than that of the commercial copper-based wood preservatives.The results of the acute ecotoxicity test did not comprehensively demonstrate all environmental effects, but they have theoretical significance for comparing the ecotoxicity of waste tobacco extract with traditional commercial wood preservatives.The partial or complete substitution of highly toxic components in wood preservatives with waste tobacco extract has the potential to mitigate adverse impacts on both the environment and human health.

Conclusions
The decay resistance of wood treated with tobacco waste extracts against wood decay fungi was evaluated.Three major constituents in the extracts were identified via GC-MS and included nicotine, neophytadiene, and 2,7,11-cembratriene-4,6-diol.The antifungal activities against wood decay fungi of waste tobacco extracts and these three major constituents were tested.At a concentration of 40 mg/mL, 50% ethanol tobacco waste extract significantly inhibited the activity of wood decay fungi because nicotine was the primary active component and neophytadiene was a synergistic component in this extract.Wood decay resistance tests were conducted on Pinus yunnanensis and Hevea brasiliensis, and the 50% ethanol tobacco waste extract at a concentration of 40 mg/mL demonstrated excellent wood decay resistance.The ecotoxicity of tobacco waste extract was significantly lower than that of commercial copper-based wood preservatives.These findings indicate that the tobacco waste extract shows promising potential as a natural alternative to traditional wood preservatives for indoor applications.However, further research is necessary to comprehensively evaluate its performance in terms of anti-ageing, anti-leaching, and mechanical resistance when applied at outdoor conditions.
• C/min to 260 • C (5 min), and 4 • C/min to 280 • C (10 min).The injector temperature was 280 • C. A 0.5 µL sample was injected in split injection mode.Mass spectra were acquired within the 35-455 amu range using a bombardment voltage of 70 eV and an ionization source temperature of 230 • C. Compound identification was performed by comparing mass spectra with those in the Wiley 275 and NIST98 libraries.

Figure 4 .
Figure 4. Colony diameters of (a) G. trabeum and (b) T. versicolor when treated with various concentrations of major extractive constituents.

Figure 5 .
Figure 5. Digital photographs of (a) G. trabeum and (b) T. versicolor when treated with various concentrations of major extractive constituents.

Figure 6 .
Figure 6.Mass loss of softwood samples treated with various concentrations of tobacco waste extracts when exposed to (A) G. trabeum and (B) T. versicolor.Mass loss of hardwood samples treated with various concentrations of tobacco waste extracts when exposed to (C) G. trabeum and (D) T. versicolor.(WE:50% ethanol extractive; WT: water extractive; AE: absolute ethanol extractive).Means within a column followed by the same letter are not significantly different (Tukey, p < 0.05).

Table 1 .
Classification of wood decay resistance.

Table 3 .
Retention of treated wood.