Effect of Pyrolysis Temperature on the Characteristics of Wood Vinegar Derived from Chinese Fir Waste: A Comprehensive Study on Its Growth Regulation Performance and Mechanism.

As a high value-added product from biomass pyrolysis, wood vinegar (WV) has been used as a growth regulator for many plant species in agriculture based on the diverse active chemical compounds present. To reveal the relationship between chemical constituents and regulation performance, four kinds of WVs were prepared by slow pyrolysis from Chinese fir waste at different temperature ranges. The chemical constituents of WVs were analyzed by gas chromatography-mass spectrometry, and the regulation performance of WVs was investigated from the aspects of seed germination and root growth of wheat. The results indicated that the chemical constituents of WVs were affected obviously by pyrolysis temperature and the major components were acids and phenols. All types of WVs showed regulation performance but with different effects and levels. The WV collected from 20 to 150 °C exhibited a promoting effect and other three WVs exhibited inhibiting effects. It was considered that the regulation performance of WV was relevant to acids and phenols through a synergy mechanism. Acids caused intercellular acidification and increased root activity, which promoted the seed germination and root growth, while phenols increased the content of malonaldehyde, indicating that phenols caused the oxidative stress to damage cell structure and inhibit growth. All these results could be a reference for further utilization of WVs as a sustainable alternative to chemicals for plant growth regulation in agriculture.

Introduction

In recent years, with the aggravation of environmental pollution and shortage of fossil energy, the utilization of biomass energy has attracted people’s attention. The full estimated potential of annual biomass production from agriculture and forestry is nearly 1.08 × 1011 tons.[1,2] Biomass, considered as the environment friendliness and renewability resource, can be converted to liquid fuels, combustible gases, biochars, and high-quality chemicals by the thermochemical and biochemical approaches.[3] Therefore, the main concerns are the comprehensive utilization of biomass and the conversion technologies that convert biomass into high value-added products.

Chinese fir, also named Cunninghamia lanceolate (Lamb.) Hook, with a huge planting area and distribution, is widely used as a raw material for furniture and wood fiber industries. However, a larger quantity of branches and leftover materials as biomass waste were produced from the furniture and wood industries, which affected the living and production environment. In order to improve the utilization of Chinese fir waste (CFW), appropriate measures should be taken to convert the branches and leftover materials to useful products.

Pyrolysis has received special attention because it offers efficient application of biomass, especially for vastly available biomass byproducts and waste. It is generally defined as thermal decomposition of biomass under anaerobic conditions, leading to the formation of gases, liquid products, and biochars.[4,5] Generally, the pyrolysis process takes place at temperatures of 300–1200 °C, and the resultant yields depend on many factors such as operating conditions, biomass types, and so forth. Thangalazhy-Gopakumar et al. investigated the effect of temperature on bio-oil quality using pine wood and the results showed that the highest yield has been obtained at 450 °C; the contents of phenols and derivatives increased as the temperature increased from 425 to 500 °C while that of guaiacol and derivatives decreased.[6] Ingra et al. investigated the physical and chemical properties of liquid products produced at 450 °C, with mass yields of 48–55% for pine wood, 49–56% for oak wood, 42–44% for pine bark, and 43–50% for oak bark.[7]

Wood vinegar (WV), as one of the main liquid products of biomass pyrolysis, consisted of many complex organic components and compounds.[8] WVs were considered potentially valuable chemicals that could be used as soil amendments, pesticides, food additives, and so forth.[9−11] Agriculture is one of the most important application fields of WVs. Recently, the research studies on the application of WVs in agriculture were quite intensive, mainly focusing on the antimicrobial properties, pesticidal effects, and so forth. Mmojieje et al. prepared WV from mixed wood biomass and investigated its pesticidal effects on the red spider mite and green peach aphid, which showed that WV exhibited more than 90% mortality for both pests.[12] Lashari et al. found that WV has a beneficial effect on leaching soluble salts, decreasing the soil pH and resulting in the improvement of crop productivity in saline soils.[13] Many studies report the regulatory effects of WVs on seed germination and plant growth in sustainable agriculture. Pan et al. found that WV had significant effects on the germination and growth of crop seed, which varied with the concentration of WV, preparation conditions, and seed types.[14] Xu et al. found that the different concentrations of bamboo vinegar inhibited the seed germination and seedling growth of tobacco, but spraying 100–400 times bamboo vinegar solution on leaves promoted growth and improved the quality.[15] WVs exhibited the regulatory effects on seed germination and plant growth, but the effects are dependent on the WV properties, concentration, and plant species.[16] However, no reports have been found to investigate the influence of pyrolysis temperatures on the properties of WVs from CFW. Furthermore, the study of the relationship between chemical constituents and regulation performance of WVs is still lacking.

On the basis of the above discussion, the aim of this paper was to study the physical–chemical properties of WVs prepared from CFW and investigate the relationships between chemical constituents and regulation performance of WVs, providing a better guidance for efficient application of WVs in sustainable agriculture.

Results and Discussion

Properties of WVs

The WVs were collected at different pyrolysis temperature ranges, and four samples were obtained. The properties of WVs are shown in Table . In general, with the increase of pyrolysis temperature, the yields of WVs first increased and then decreased, and the maximum yield reached to 34.09% in WV-2. As we know, biomass materials were mainly composed of cellulose, hemicellulose, and lignin. The increase of temperature promoted the pyrolysis process, which led to an increase in yield. However, the pyrolysis process was gradually completed and part of volatile gas was formed in secondary pyrolysis to form noncondensable gas under higher pyrolysis temperature, which resulted in a significant reduction of yield. The moisture content decreased with the increase of temperature because the fracture of aliphatic hydroxyl in the lignin structure and the dehydration of cellulose were extreme at lower temperature and subdued at higher temperature, leading to the reduction of moisture content under higher temperature.[17] Both pH and density of WVs showed no significant difference, which remained steady at 2.75–3.08 and 1.011–1.044 g/mL, similar to the WVs obtained from giant seed and walnut shell.[18,19]

Table 1

Properties of WVs from Four Temperature Ranges

WVs	pH	density (g/mL)	moisture content (%)	yield (%)
WV-1	3.04	1.011	85.61	15.47
WV-2	2.90	1.036	54.55	34.09
WV-3	3.08	1.039	52.03	3.50
WV-4	2.75	1.044	51.73	0.50

Gas chromatography (GC)–mass spectrometry (MS) was used to study the chemical compositions of WVs, and the relative peak area represented the relative content of chemical components. The main chemical components in WVs at different temperature ranges are shown in Figure . The detailed information of different chemical components presented in the Supporting Information is classified into five groups, including acids, phenols, ketones, aldehydes, and alcohols. As shown in Figure , the same chemical components in WVs were identified, but the distribution and contents were varied. Acids and phenols were the major components for all WVs, which accounted for 15.35–64.84% and 30.09–56.17%, respectively. The relative contents of alcohols were less than 6%. It could be seen that the pyrolysis temperature had a significant effect on the chemical compositions of WVs. For WV-1, acids and phenols were the major components, which accounted for 94.93%. The main components were phenols, acids, aldehydes, and ketones in WV-2 while phenols, ketones, and acids in WV-3. In WV-4, phenols, acids, ketones, and alcohols were the main components.

Figure 1

Relative content profiles of five groups of chemical compounds at different pyrolysis temperatures.

The relative content of acids was highest in WV-1, which decreased first and then increased with the increase of temperature. This was due to the fact that hemicellulose and lignin dehydrated to form small molecular acids at low temperature, while cellulose and hemicellulose degraded to produce macromolecular acids at high temperature. For phenols, defined as the major degraded products of lignin, the relative content increased and then decreased, which exhibited the highest relative content in WV-3. In general, the pyrolysis temperature of lignin was in a wide temperature range of 200–550 °C, but the most intense pyrolysis temperature range was 280–420 °C. Ketones were derived from the depolymerization of polysaccharides and the isomerization of monosaccharides on hemicellulose at high pyrolysis temperature, while alcohols were ascribed to the breakage of the side chain of aliphatic alcohol hydroxyl in lignin. Cellulose, hemicellulose, and lignin all produced aldehydes during pyrolysis.[20,21]

A total of 32 chemical components in WVs were identified and shown in Table . The main components of acids were aliphatic acids and phenolic acid, obtained from the depolymerization of cellulose, pyrolysis of hemicellulose, and breakage of fat hydroxyl in lignin. The major phenolic substances were guaiacol and its derivatives, followed by benzenediol and its derivatives. The alkyl side chains of the phenylpropane structure of lignin contained hydroxyl functional groups, which produced phenolic substances by dehydration during the pyrolysis process.[22] Therefore, it could be concluded that the phenylpropane structure of lignin in CFW was mainly of guaiacol-based type. Ketones contained acetone, methoxyacetophenone, cyclopentanedione, and their derivatives, which were formed from the pyrolysis of cellulose and hemicellulose. Furfural and hydroxymethyl-furfural were the major components of aldehydes. Furfural was the typical degradation product of xylan in hemicellulose and hydroxymethyl-furfural was generated by the degradation of the glucosyl group in cellulose.[23] Methoxy-phenethyl alcohol was the main alcohol in WVs, which may be due to the degradation of the side-chain structure of the aliphatic alcohol hydroxyl group in lignin.[24]

Table 2

Identified Compounds in the WVs Produced from the Slow Pyrolysis of CFW

			contents of organic compounds (area %)
class	compounds	molecular formula	WV-1	WV-2	WV-3	WV-4
acids	acetic acid	C2H4O2	2.75	9.17	1.67	1.66
	propanoic acid	C3H6O2		3.18
	heptylic acid	C7H14O2	13.79			14.00
	pentanoic acid	C6H12O2	44.65	6.75	2.03	13.95
	3-methoxy-4-hydroxybenzoic acid	C9H10O4	3.65	3.76	13.68
phenols	phenol	C6H6O	2.66	1.07		1.20
	2-methoxy phenol	C7H8O2	3.99	6.99	8.21	8.03
	2-methyl phenol	C7H8O		1.78
	3-methyl phenol	C7H8O	1.03	2.20
	maltol	C6H6O2		2.52	1.67
	4-methyl-2-methoxy phenol	C8H10O2	1.27	7.35	1.78	5.00
	1,2-benzenediol	C6H6O2	13.17	4.68		9.79
	4-ethyl-2-methoxy phenol	C9H12O2		4.22	22.91
	eugenol	C10H12O2		4.18
	4-propenyl-2-methoxy phenol	C10H12O2		4.10	8.13
	4-propyl-2-methoxy phenol	C10H14O2		3.22	10.27	9.62
	2,6-dimethoxy phenol	C9H12O2			1.16
	3-methyl-1,2-benzenediol	C7H8O2	3.57			1.43
	4-methyl-1,2-benzenediol	C7H8O2	4.40			11.03
ketones	1-hydroxy-2-butanone	C4H8O2		1.38
	acetone	C3H6O	1.54	2.75	0.94	0.81
	2-furanone	C4H4O2		2.54
	1,2-cyclopentanedione	C5H6O2		2.25	2.75	2.43
	3-methyl-1,2-cyclopentanedione	C6H8O2	1.67	3.58	3.00	1.18
	4-hydroxy-3-methoxy hypnone	C9H10O3		2.08	3.71	11.92
	3-methoxy hypnone	C9H9O2			7.55
aldehydes	furfural	C5H4O2		4.65	0.99	1.78
	5-methyl-2-furfural	C6H6O2		1.61	1.99
	5-hydroxymethyl furfural	C6H7O3		7.10	2.19
	vanilline	C8H8O3		3.79
alcohols	2-furfuryl alcohol	C5H6O2			1.28
	4-hydroxy-3-methoxy phenethyl alcohol	C9H12O3	1.86	3.10	4.09	6.17

Effects of WVs on the Germination of Wheat Seed

The seed germination potential (SGP), defined as the important index for indicating the seed germination speed and viability, was used in this study to reveal the effects of WVs on SGP, and the results are shown in Figure . It could be seen that the effects of different WVs on SGP were obvious. Compared with the sterile water without WV (CK), the SGP increased when treated with WV-1 and decreased when treated with WV-2, WV-3, and WV-4. It also manifested that the SGP was influenced by the culture time. In the first 3 days, the influences of different WV treatments on SGP were not obvious. However, the SGP began to show a significant difference from the fifth day with the extension of culture time. These results indicated that WVs could promote the seed germination but could not break the dormancy of seed and make them germinate earlier. Consistently, the results of this study were consistent with those of Chang et al.[25]

Figure 2

Effects of WVs on SGP of wheat seed treated with CK, WV-1, WV-2, WV-3, and WV-4. Different small letters represent significant difference among the different WV treatments, which were analyzed by SPSS20.0 (P < 0.05).

Table shows the detailed germination characteristics of wheat seed treated with WVs. The seed germination rate (SGR) increased significantly by 43.13% in WV-1 and decreased by 7.84, 29.41, and 17.65% in WV-2, WV-3, and WV-4 treatments, respectively, compared with that in CK. The variation trend of seed germination index (SGI) was similar to SGR and sequenced as WV-1, CK, WV-2, WV-4, and WV-3. Moreover, the relative SGR (RSGR) revealed the effects of WVs on the germination rate of wheat seed, where the positive numbers indicated the promotion effect and the negative numbers indicated the inhibitory effect. It manifested that WV-1 exhibited a promotion effect, while WV-2, WV-3, and WV-4 exhibited an inhibitory effect. WV-1 with the SGR of 60.83% had the highest promotion effect. These results indicated that WVs can be used as a conditioning agent in sustainable agriculture.

Table 3

Germination Characteristics of Wheat Seed

number	3rd day SGP	5th day SGP	SGR	RSGR	SGI
CK	16.67a	33.33b	42.50b		1.00b
WV-1	17.50a	47.50a	60.83a	43.13	1.69a
WV-2	16.67a	32.50b	39.17b	–7.84	0.78c
WV-3	10.83c	22.50d	30.00c	–29.41	0.28e
WV-4	12.50b	26.67c	35.00c	–17.65	0.49d

Effects of WVs on Root Growth of Wheat

The main root length (MRL), lateral root number (LRN), root uniformity (RU), and total fresh root mass (TFRM) are the major morphological characteristics, which could be used to evaluate the growth of root. The main root is the major part of the plant root system and helps plant to absorb water and nutrients from deep soil, the lateral root and RU are the important parts that affect the specific surface area of the root system and root activity, and TFRM indicates the healthy degree of the plant root growth. In this study, we selected the four indicators as the morphological characteristics of root, and the results are shown in Figure . The MRL (Figure a) was affected obviously by different WVs. Compared with CK, the MRL was the longest and reached to 10.5 cm by the treatment of WV-1, which increased nearly 1.2 times, while it was reduced to 3.55 cm with the treatment of WV-3. The MRL increased by the treatment of WV-1 and decreased by the treatments of WV-2, WV-3, and WV-4. Figure b shows the effects of WVs on LRN. As shown, the LRN increased to 105 when treated with WV-1, which was 1.5 times that of CK, and it decreased to 46 when treated with WV-3. The effects of WV-2 and WV-4 on LRN were not significant. Consistently, the RU and TFRM exhibited similar trends to MRL and LRN (Figure c,d). These results indicated that WV-1 promoted the growth and development of the wheat root system, while other WVs (WV-2, WV-3, and WV-4) exhibited inhibitory effects. For the development of the root system and growth of plant, both water and nutrients in soil play important roles. Organic acids, phenols, and other organic compounds are typical allelochemicals, which regulate plant growth via affecting ion absorption, respiratory metabolism, hormone balance, and protein synthesis.[26−28] The WVs, which are obtained from different feedstock and pyrolysis conditions, contained different kinds and contents of chemical substances and exhibited different regulation performances on root development and plant growth.

Figure 3

MRL (a), LRN (b), RU (c), and TFRM (d) of wheat culture treated with CK, WV-1, WV-2, WV-3, and WV-4. Different small letters represent significant difference among the different WV treatments, which were analyzed by SPSS20.0 (P < 0.05).

Effects of WVs on Root Vigor of the Wheat Root System

Root system is an important absorption and metabolic organ of plants. The development of the root system was directly affected by the crop yield and the growth of shoots and leaves above ground. Root vigor (RV), a comprehensive index reflecting the absorption function of the root system, refers to the metabolic activity of the root system. Therefore, RV has become an important index in the study of the root system. Figure shows the impact of RV treated with WVs. It can be seen that the RV of the root was affected obviously by WVs and the order indicator sensitivity to RV with different WV treatments was WV-1 > CK > WV-2 > WV-4 > WV-3. Compared with CK, the RV increased to 92.75 μg/g·h in WV-1 treatment and decreased to 72.48, 62.45, and 48.91 μg/g·h in WV-2, WV-4, and WV-3 treatments, respectively. Coincidentally, Liu et al. stated that the wheat straw extract and decomposition liquid, which contained the similar contents with WV such as phenolic acids, exhibited the regulatory effects on the RV and respiratory action of the cotton root system and affected the growth of root.[29] In addition, organic compounds such as phenolic acids showed different allelopathic effects on the rice root system, which had important effects on RV and plant growth.[30] In general, plant root was affected by allelopathic substances, stress, irrigation, soil, and other environmental conditions, which led to the change of root metabolism and activity and affected their growth. WV contains a large amount of organic compounds, and the type and contents of organic compounds are considered as the key factors that influence the regulatory effects of WV.

Figure 4

RV of wheat culture treated with CK, WV-1, WV-2, WV-3, and WV-4. Different small letters represent significant difference among the different concentrations of WV treatments, which were analyzed by SPSS20.0 (P < 0.05).

Oxidative Stress for Root Growth Caused by WVs

The production and accumulation of malonaldehyde (MDA) in target cells are frequently used as an indicator to assess oxidative stress condition and contaminant toxicity to organisms. Under stress condition, continuous accumulation of MDA will lead to cell deterioration and cell membrane damage. Therefore, the higher MDA content indicates the greater cell damage, which is considered as a disadvantage to the plant growth. The impacts of WVs on the contents of MDA in wheat root are shown in Figure . The MDA content was highly dependent on the kinds of WVs. Treated with WV-1, the MDA content decreased to 0.00925 μmol/g, which was reduced nearly 13% compared with CK groups. The MDA in WV-3 treatment significantly increased by 1.4 times and reached to 0.01485 μmol/g, while it had little changes in WV-2 and WV-4. These results indicated that WV-3 exposure triggered MDA overproduction in root cells and was the stress condition for root growth. The increase of MDA content may be due to the oxidation stress induced by excessive reactive oxygen species (ROS) generation by WV-3 exposure.[31] It was also reported by Guo et al. that the MDA content in Microcystis flos-aquae increased by the gallic acid, which was due to excess ROS accumulation resulting from oxidative stress.[32] Overall, the increased content of MDA indicated that the wheat root cells suffered an intense oxidative stress caused by the treatment of WVs, leading to lipid oxidation and root growth inhibition.[33]

Figure 5

Content of MDA of wheat culture treated with CK, WV-1, WV-2, WV-3, and WV-4. Different small letters represent significant difference among the different concentrations of WV treatments, which were analyzed by SPSS20.0 (P < 0.05).

Correlation Analysis on Chemical Compounds and Regulatory Effects of WVs

In order to investigate the relationship between chemical constituents and regulatory effects and to discuss the mechanism, correlation coefficient analysis was used in this study. Table shows the analysis on correlation coefficient between chemical components in WVs with germination and growth indices. For seed germination, acids were positively correlated with SGR and SGI, while phenols and ketones were negatively correlated with SGR and SGI; aldehydes and alcohols were not significantly correlated with SGR and SGI. As for root growth indices, acids were positively correlated with LRN and RV; ketones were negatively correlated with PRL, LRN, and TFRM; and alcohols were negatively correlated with MRL and TFRM. Phenols exhibited a significant negative correlation with all growth indices. As shown in the component analysis, acids and phenols were the main components in WVs, which accounted for 70–95% of the total components. Especially in WV-1, acids and phenols accounted for about 95%, and WV-1 showed the strongest promotion effect. It could be concluded that acids and phenols played important roles in regulatory effects rather than ketones, aldehydes, and alcohols.

Table 4

Analysis on Correlation Coefficient between Chemical Components in WVs with Germination and Growth Indicesa

	SGI	SGR	MRL	LRN	RU	TFRM	RV	MDA
acids	0.949*	0.968*	0.881	0.950*	0.814	0.848	0.904*	–0.833
phenols	–0.959*	–0.951*	–0.988**	–0.956*	–0.982**	–0.955*	–0.961*	0.972*
ketones	–0.990**	–0.996**	–0.930*	–0.991**	–0.840	–0.929*	–0.819	0.815
aldehydes	–0.266	–0.334	–0.082	–0.272	–0.003	–0.024	0.112	0.122
alcohols	–0.898	–0.869	–0.905*	–0.898	–0.814	–0.958*	–0.866	0.702

*P < 0.05, **P < 0.01.

Treated with WVs, H+ which is provided by acids broke the physical interaction between xyloglucan and other compounds in the cell wall, which helped H+ to enter the cells and increased organic acid to cause intercellular acidification. Intercellular acidification made the cells more prone to division and differentiation, thus promoting the increase of RV and the development of the root system. Furthermore, there were abundant phenolic compounds in WV. Phenolic compounds exhibited the hormesis effect as stimulation at lower concentration and the inhibition at higher concentration. The mechanism involving increased free radicals and activated protease led to the cell proliferation in low concentration while decreased the activities of enzymes and inhibited the growth in high concentration.[34,35] Meanwhile, different phenols exhibited different regulatory effects, which reported that monophenol inhibited plant growth and diphenol or polyphenol promoted plant growth.[36] Phenols in WV-1 were mainly diphenol, such as 1,2-benzenediol, 3-methyl-1,2-benzenediol, and 4-methyl-1,2-benzenediol, while in other WVs, especially in WV-3, phenols were primarily monophenol, such as 2-methoxy phenol, 4-ethyl-2-methoxy phenol, and 4-propyl-2-methoxy phenol. The balance and concentration of different phenols affected the plant growth, which was considered as the high concentration, and breaking the balance of phenols would decrease the RV and increase the MDA content that damaged cell structure and led to the disadvantage of plant growth.

In addition, we further studied the correlation between RV, MDA, and growth indices, and the results are shown in Table . The RV, as an indicator of plant growth activity, was negatively correlated with MDA and was positively correlated with MRL, LRN, RU, and TFRM. This result indicated that the higher content of RV promoted the growth of the root system, especially for principal root growth. MDA was negatively correlated with MRL, RU, and RV. There were many factors that interfered and damaged the structure and function of the membrane in plant growth, and lipid peroxidation was one of the important factors. MDA, the final product of lipid peroxidation, affirmed the indicator of the degree of stress and reflected the accumulation of harmful substances in plant cells under stress condition. The higher the content of MDA, the greater the damage degree of cell membranes.[37,38] Under stress condition, the root adapted to stress by an increase in respiration. However, the long high-intensity respiration would consume a large number of carbohydrates and then reduce the respiratory rate because of the insufficient respiratory substrates. Furthermore, reduction of the respiratory rate may cause the decrease of root metabolism and ATP production, which could not meet the needs of root growth, thus leading to the decrease of RV and inhibition of growth.[39]

Table 5

Analysis on Correlation Coefficient between RV, MDA, and Growth Indicesa

	RV	MDA	MRL	LRN	RU	TFRM
RV	1	–0.950*	0.997**	0.972*	0.976*	0.977*
MDA		1	–0.934*	–0.861	–0.982**	–0.873
MRL			1	0.970*	0.973*	0.989**
LRN				1	0.896	0.968*
RU					1	0.937*
TFRM						1

*P < 0.05 , **P < 0.01.

Conclusions

In this study, the physicochemical properties of WVs from the pyrolysis of CFW were studied, and comprehensive assessment of chemical constituents and regulation performance was also investigated. With the increase of pyrolysis temperature, the yield of WVs increased first and then decreased, and WV-2 reached the maximum yield of 34.09%. GC–MS analysis of WVs showed that the chemical compositions of WVs were significantly affected by the pyrolysis temperature and the acids and phenols were the most dominant components. WVs obtained under four temperature ranges exhibited different regulation performances on seed germination and root growth. Compared with CK, WV-1 promoted the seed germination and root growth, whereas WV-3 inhibited the seed germination and root growth. The correlation analysis results indicated that the regulation performance was relevant to acids and phenol contents, which show that acid substances exhibited a promoting effect and phenolic substances exhibited an inhibiting effect. The regulatory effects were studied through a synergy mechanism which influenced the changes of RV and MDA caused by acids and phenols in WVs.

Overall, this paper underlined the interest for a clear understanding of the relationship between chemical constituents and regulation performance of WVs. This knowledge helps to select suitable WVs for the best application in sustainable agriculture and provides a promising way for biomass waste utilization.

Materials and Methods

Materials

The CFW used in this study was the residues generated in the utilization process (especially, furniture industry), which were collected from Jiangxi (China) and smashed into small pieces (0.5–2.0 mm). All of the chemical reagents used in this study were of analytical grade and purchased from local chemical stores.

Preparation and Characterization of WVs

Slow pyrolysis process was used in this study to obtain WVs, and the schematic diagram is shown in Figure . To maintain an inert atmosphere, N2 with a flow rate of 100 mL/min was fed into the reactor during the pyrolysis process. CFW samples (20 g) were loaded in the middle of the tube in a vacuum tube furnace (VTL 1200, China) and pyrolyzed from the initial temperature to the target temperature with a heating rate of 10 °C/min and maintained for 30 min to ensure complete pyrolysis. A pipe with pyrolytic smoke was connected to a 0 °C ice bath and applied to collect WVs at different temperature ranges by means of a condenser. With the four temperature ranges, the WVs were collected and placed in brown bottles and kept in dark. WV-1 indicates that the WV was obtained at the temperature range from 20 to 150 °C, WV-2 from 150 to 250 °C, WV-3 from 250 to 350 °C, and WV-4 from 350 to 450 °C.[40,41] Three parallel experiments were performed for each pyrolysis condition.

Figure 6

Schematic diagram of pyrolysis (1) N2; (2) gas flow meter; (3) quartz tube; (4) VTL; (5) temperature controller; (6) condenser.

The density of WV was measured using an optical density meter, pH was measured by a pH meter (PHS-3CHS, INESA, China), and the moisture content was measured using a moisture analyzer (ZSD-2J, Anting, China). The chemical composition was characterized using GC–MS (7890A-5975C, Agilent, Palo Alto, USA) equipped with an HP-5 column (30 m × 0.05 μm × 0.32 nm). The following conditions were used for GC analysis: initial temperature 50 °C for 2 min, ramped at 5 °C/min to 280 °C and held for 20 min, a split ratio of 100:1, and injected sample of 0.2 μL. The MS analysis was obtained under 230 °C at 70 eV. The relative contents of compounds were calculated by corresponding peak areas.[42]

Regulatory Effect of WVs

The culture experiments were used to study the regulatory effects of WVs on the seed germination and root growth of wheat. The wheat seeds were sterilized using 75% ethyl alcohol solution and washed with sterile water 2–3 times before the test. Next, 1 mL of different WVs was diluted in 4000 mL of sterile water to obtain the culture solutions; sterile water without WV was denoted as sample CK. The culture experiments were carried out under a light/dark cycle of 12:12 h at 28 ± 1 °C for 7 days in an intelligent incubator (KBWF-720, Binder, Germany). For every culture experiment, 30 wheat seeds were placed in a watch glass (d = 9 cm) with two filter papers, and different WV culture solutions were added under sterile conditions with three conducted repetitions.

Morphological and Physiological Characterization

The morphological characteristics used in this study were SGR, SGP, RSGR, SGI, MRL, LTN, TFRM, and RU. The SGR, SGP, RSGR, and SGI were analyzed following the methods of Yu et al.:[30] SGR (%) = n/N × 100% (where n is the number of germinative seeds at the final period of germination and N is the total number of experimental seeds), SGP represents the 3rd day, 5th day, and 7th day of SGR, SGI = (SGRt × MRLt)/(SGRCK × MRLCK) (where t is represented for treating with WVs, while CK stands for CK groups). The cornier caliper was used to measure the root length; thereby, the MRL was obtained and the RU was calculated as the ratio of >1 cm root number to the total root number. The MDA content was assayed according to the thiobarbituric acid (TBA) method, and RV was analyzed following the triphenyltetrazolium chloride (TTC) method.[43]

Statistical Analysis

The culture experiment data were analyzed using analysis of variance (ANOVA), and the results were expressed as the mean values of three repetitions with standard deviation. Different small letters in tables and figures indicate the significance of the various parameters with different concentrations of WV treatments. The correlation was analyzed using a Pearson test at P < 0.05 by means of SPSS 20.0.