Kale Seed Germination and Plant Growth Responses to Two Different Processed Biostimulants from Pyrolysis and Hydrothermal Carbonization

Abstract

The cost of producing organic crops is increasing. Agricultural wastes can be used as biostimulants to increase plant growth and productivity and reduce the dependence on chemical fertilizers. A pouch assay and a potted greenhouse experiment were conducted to investigate the effect of pyroligneous acid (PA) and sea lettuce (SL) on kale (Brassica oleracea subsp. acephala (DC.) Metzg) seed germination and growth. Although previous studies have demonstrated that these two biostimulants could promote plant germination and growth, there is little research to compare their effects on seed germination and plant growth. The pouch assay showed that PA liquid affected the seed germination rate under different concentrations; the seed germination rate decreased as the concentration of PA liquid increased. However, the effect of seed germination was less pronounced in SL liquids. Kale seeds treated with 0.01% PA showed the best elongation and seedling growth performance. Moreover, the greenhouse experiment indicates that SL liquids significantly (p < 0.05) affected kale growth production, while PA liquid had less difference on kale growth under various concentrations. The 0.25% PA and 1% SL increased the aboveground fresh weight by ca. 26% and 29%, respectively. Also, the phytochemical contents of kale leaves, including phenolics, flavonoids, ascorbate, and protein, were significantly increased with 0.25% PA and 1% SL application. These results suggest that low concentrations of PA are more suitable for seedling root growth in kale and 1% SL had the most significant growth-promoting effect on kale. Hydrothermal carbonization sea lettuce liquid can be used as a good biostimulant for agricultural production to improve kale germination and growth.

1. Introduction

The pressure to meet global food and nutrition demand has led to over-exploitation and degradation of the environment and agroecological systems, leading to sustaining global climate change and greenhouse gas emissions [1,2]. For instance, burning or landfilling with organic waste does not only cause air and soil pollution but also leads to wasted resources. Effective agricultural waste management converts organic waste into fertilizers and soil conditioners that can be used to improve soil structure, maintain soil fertility, and increase plant yields [3,4]. Modern intensive agriculture relies heavily on chemical fertilizers, and long-term application of inorganic fertilizers can cause various environmental problems, such as soil nutrient loss, groundwater pollution, water eutrophication, greenhouse gas emissions, and soil acidification [5,6]. The application of organic amendments (e.g., manure, compost, and sludge) can serve as an effective alternative to chemical fertilizers and contribute to sustainable agriculture [6,7]. Organic amendments improve soil nutrient composition and promote the activity of soil microorganisms that help maintain soil health [4].

Pyroligneous acid (PA) is a highly oxidized reddish-brown organic liquid obtained by vapor condensation during biomass pyrolysis [8]. Pyrolysis is the conversion of plant biomass into organic gas and biochar in the presence of limited oxygen [9]. The materials used in pyrolysis are mainly forestry and agricultural wastes such as forest waste, corn cobs, and rice husks. Direct burning of these wastes can form aerosols such as volatile organic carbon (VOC), organic matter, and black carbon, which cause environmental pollution and human health hazards [8]. Therefore, waste treatments by pyrolysis reduce biomass waste and reduce associated environmental pollution.

Hydrothermal carbonization (HTC) is a thermochemical process that carbonizes biomass [10], and its main products are solid (hydrochar), liquid, and a small portion of gas (CO₂) [11]. HTC can directly process wet biomass without pre-drying, which is very suitable for processing feedstock with high moisture content, such as algae, sewage sludge, and animal waste [12]. Algae have very high lipid and biomass reserves and are a sustainable, renewable, and environmentally friendly biofuel [13]. Eutrophication of water bodies allows algae to increase rapidly, causing hypoxia, a reduction in aquatic animals, and overgrowth of sea lettuce (Ulva lactuca) [14,15]. Researchers have used the HTC process to turn sea lettuce into a value-added product for agricultural use. Pyrolytic pyroligneous acid and hydrothermal carbonization sea lettuce (SL) liquids are known to have biostimulatory properties that can improve plant growth and yield [16,17,18,19]. The hydrochar improves soil properties and nutrient utilization, and promotes germination and growth [20]. However, organic compound residues on the surface of the hydrochar are phytotoxic and may be taken up by the plant, thereby inhibiting growth [21]. Although previous studies have demonstrated that these two biostimulants could promote plant germination and growth, there is little research to compare their effects on seed germination and plant growth.

Kale (Brassica oleracea subsp. acephala (DC.) Metzg) is an extensive crop and is consumed as a cruciferous vegetable with a unique flavor and aroma. Kale is also considered one of the healthiest vegetables as it provides functional compounds including antioxidants, carotenoids, glucosinolates, lipid-soluble tocopherols, ascorbic acid, mineral nutrients, and dietary polyphenols [22]. It can be hypothesized that low concentrations of PA and SL can improve the germination and growth properties of kale plants. Therefore, this study aimed to determine the effects of PA and SL on kale seed germination, plant growth, and biochemical and the nutritional composition of the edible portion.

2. Materials and Methods

2.1. Location and Materials

This study was conducted in the Compost and Biostimulant Laboratory and Greenhouse located in the Department of Plant, Food, and Environmental Sciences from September 2021 to April 2022. PA, produced through the pyrolysis of white pine (Pinus strobus), was obtained from Proton Power Inc. (Lenoir City, TN, USA). SL liquid was acquired by a hydrothermal carbonization process at a reactor temperature of 200 °C and 100 psi with an average heating rate of 4.9 °C/min and a residence time of 60 min as described in He et al. [23]. Kale ‘Dwarf Green Curled’ seeds were purchased from Halifax Seeds Inc., Halifax, NS, Canada, and Promix-BX™ (Premier Horticulture Inc., Quakertown, PA, USA) was purchased from Co-op Country Store, Truro, NS, Canada.

2.2. Chemical Analysis

PA and SL were separately added at concentrations (%) of 0 (control), 0.01, 0.1, 0.25, 0.5, 1, and 2. All solutions were prepared in 100 mL volumes and mixed thoroughly by stirring for 1 min. Oakton PC Tester 35 multimeters (Oakton Instruments, Vernon Hills, IL, USA) were used to record the pH, total dissolved solids (TDS), electric conductivity (EC), and salinity for each concentration of the PA and SL. The measurements were replicated three times. Samples (100 mL) of each of the liquids were sent to the Nova Scotia Department of Agriculture Laboratory Services, Truro, for nutrient analysis. Total nitrogen (N) was determined by the AOAC-990.03 combustion method using a LECO-Spec Analyzer (TruSpec^® Micro, LECO, St. Joseph, MI, USA), and calcium (Ca), potassium (K), magnesium (Mg), barium (Ba), boron (B), iron (Fe), cobalt (Co), manganese (Mn), molybdenum (Mo), selenium (Se), and zinc (Zn) were determined using the AOAC-968.08 inductively coupled plasma (ICP) spectrometer method.

2.3. Kale Seed Germination and Seedling Growth

Fifteen (15) kale seeds were carefully placed in each pouch (125 × 157 mm; Mega International, Minneapolis, MN, USA), which contained 25 mL of either PA or SL, and kept in the dark for two days. This study was arranged in a completely randomized design (CRD) with three replications. Both PA and SL were applied at concentrations of 0% (control), 0.01%, 0.1%, 0.5%, 1%, and 2% PA/ddH₂O or SL/ddH₂O (v/v). The seed pouches were transferred to a growth chamber with 25 °C and 71% relative humidity and a 16/8 h day/night light cycle for nine days. Kale seeds were considered germinated once the radicle protrusion exceeded 2 mm. During the growth period, an additional 15 mL of deionized water or various concentrations of biostimulants were added. The germinated seeds were counted every two days for 10 days before calculating the germination rate (number of germinated seeds/total number of seeds) × 100%. A digital camera (Nikon D300s, Nikon Inc., Tokyo, Japan) was used for image acquisition, and ImageJ software (version 1.52a) was used to measure the seedling root length from the tip of the primary root to the hypocotyl. Seven seedlings per pouch per treatment were selected for root analysis using an STD4800 root scanner (Régent Instruments, Quebec, QC, Canada) with a dual-lighting system and an optical resolution of 4800 dpi. Root system parameters including total root length (cm), total root surface area, and root volume were measured using WinRhizo software.

2.4. Greenhouse Pot Experiment and Plant Growth

Kale seeds were sown in a 36-cell plastic tray filled with Pro-mix BXTM and grown for four weeks. The seedlings were transplanted into a 25.4 cm diameter plastic pots containing 800 g of moistened Pro-mix BX™. Treatment application was initiated seven days after transplanting and repeated at weekly intervals. Both PA and SL were applied to the soil of each pot at concentrations of 0% (control), 0.25%, 0.5%, and 1% PA/ddH₂O or SL/ddH₂O (v/v). The study was arranged in a completely randomized design with five replications. Additionally, the plants were watered every 2 or 3 days depending on the weather and soil conditions. The kale plants were grown for five weeks under greenhouse conditions with a 16/8 h day/night photoperiod, 24/16 °C day/night temperature cycle, and a relative humidity of 71%. Supplemental lighting was provided by a metal halide lamp (P.L. Light Systems, Beamsville, ON, Canada) to ensure 12 h of active radiation photoperiod.

2.5. Morpho-Physiological Response

The plant growth rate was measured 7 days after transplanting. The youngest leaf about 1 cm in length was tagged and the changes in length were measured every 3 days for 18 days. Plant height was recorded from the stem collar to the highest leaf. The number of green and healthy leaves per treatment was recorded and the stem diameter was measured using Mastercraft’s 150 mm electronic caliper (Masstores (Pty) Ltd., Johannesburg, South Africa). Chlorophyll content was measured with a 502 SPAD meter (Spectrum Technologies Inc., Aurora, CO, USA) from three fully expanded leaves per plant. Leaf color was determined using a colorimeter (Shenzhen Three NH Technology Co., Ltd., NR20XE, Shenzhen, China) according to the L* a* b* color space, where L* represents lightness, a* and b* are chromaticity coordinates (−a*: green, +b*: yellow), c* is chroma, and h* is hue angle [24]. Photosynthesis parameters were recorded from the same fully expanded three leaves per plant with an LCi portable photosynthesis system (ADC Bioscientific Ltd., Hoddesdon, UK). The photosynthesis parameters included the photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), and intercellular CO₂ concentration (Ci). The measurements were collected between 10:00 AM and 2:00 PM under a photosynthetic photon flux density (PPFD) of 1400 µmol m⁻² s⁻¹, a leaf temperature of 30 °C, and 50% relative humidity. Chlorophyll fluorescence indices were determined using a portable chlorophyll fluorometer (Opti-Sciences, Hudson, NY, USA) on the same three fully expanded leaves per plant. Before the measurement, dark adaptation clips were attached to the leaves for 25 min before reading the maximum fluorescence (Fm), minimum fluorescence (Fo), viable fluorescence (Fv = Fm − Fo), potential photosynthetic capacity (Fv/Fo), and maximum quantum yield or maximal photochemical efficiency (Fv/Fm) of photosystem II.

2.6. Biochemical and Quality Analysis

All healthy leaf samples were collected at harvest, rapidly frozen in liquid nitrogen, and ground to a fine powder. The ground powder was stored at −80 °C until further analysis.

2.6.1. Chlorophyll a, Chlorophyll b, and Carotenoid Content

A 0.1 g ground leaf sample was mixed with 1 mL of 80% acetone, vortexed for 1 min, and centrifuged at 12,000 rpm for 15 min. The absorbance of the supernatant was measured at 646.8, 663.2, and 470 nm using a UV–visible spectrophotometer with 80% acetone as a blank. Chlorophyll (Chl a and Chl b) and carotenoid contents were calculated according to the method described by Lichtenthaler [25]. The total chlorophyll and carotenoid contents were expressed as μg/g fresh weight (FW) of the samples.

2.6.2. Ascorbate and Dehydroascorbate Content

Total ascorbate was measured according to the method of [26] with minor modifications. A 0.2 g sample was mixed with 1.5 mL ice-cold freshly prepared 5% (w/v) trichloroacetic acid (TCA). The mixture was vortexed for 2 min and centrifuged at 8000× g for 15 min at 4 °C. An aliquot of 100 µL of supernatant was transferred into new two separate tubes for AsA and AsAt (total ascorbate) determination, and 400 µL phosphate buffer (150 mM potassium dihydrogen phosphate (KH₂PO₄) and 5 mM ethylenediaminetetraacetic acid (EDTA)) was added to each tube. Then, 100 μL 10 mM dithiothreitol (DTT) was added to the AsAt tube and 100 μL deionized water was added to the AsA tube. All tubes were vortexed for 30 sec and incubated for 15 min at room temperature (25 °C). In the AsAt tube, 100 μL of 0.5% (w/v) N-ethylmaleimide (NEM) was added to remove excess DTT, and 100 μL deionized water was added to the AsA tube. The mixture was vortexed for 30 s and 400 μL of 10% TCA, 400 μL of 44% orthophosphoric acid, 400 μL of 4% (w/v) α- α 1 -dipyridyl in 70% ethanol, and 200 μL of 30 g/L ferric chloride (FeCl3) were added to obtain color. The reaction mixture was incubated at 40 °C for 60 min in a shaking incubator and the absorbance was measured at 525 nm. Total ascorbic acid content was expressed as μmol/g FW using L-ascorbic acid standard curve. DHA levels were estimated as DHA = AsAt − AsA.

2.6.3. Total Sugar Content

The total sugar content of kale leaf was determined with the method of DuBois et al. [27]. A 0.2 g sample of ground leaf was homogenized in 10 mL of 90% ethanol. The mixture was vortexed for 30 s and incubated in a water bath at 60 °C for 60 min. The mixture was vortexed and centrifuged at 4000 rpm for 3 min. Then, 1 mL of the mixture was transferred into a thick-walled glass test tube, and 1 mL of 5% phenol was added and mixed thoroughly. After, 5 mL of concentrated sulfuric acid was added and mixed thoroughly. The mixture was incubated in the dark for 15 min, cooled at room temperature, and the absorbance was measured at 490 nm. The total sugar content was determined using a glucose standard curve and expressed as μg of glucose/g FW.

2.6.4. Total Protein Content

The total protein content of the fresh leaf sample was determined using the method of Bradford [28]. First, 0.5 g of ground leaf sample was mixed with 5 mL ice-cold extraction buffer (50 mM potassium phosphate buffer (pH 7.0), and 0.1 mM EDTA). The mixture was vortexed for 30 s and centrifuged at 15,000× g for 20 min at 4 °C. Then, 100 μL of the supernatant was added to 1 mL Bradford reagent and the absorbance was measured at 595 nm after 5 min. The total protein content was estimated using the Bovine serum albumin (200–900 μg/mL) standard curve and expressed as μg/g FW.

2.6.5. Total Phenolic Content

The total phenolic content of the leaf sample was determined using the method of Ainsworth and Gillespie [29]. First, 0.25 g of ground leaf sample was mixed with 2 mL of ice-cold 95% methanol. The mixture was vortexed for 30 s, incubated for 48 h at room temperature in the dark, and centrifuged at 13,000× g for 5 min. Then, 100 μL of the supernatant was added to 200 μL of 10% Folin–Ciocalteau reagent, vortexed for 5 min, and 800 μL 700 mM Na₂CO₃ was added. The mixture was vortexed for 5 min and incubated in the dark at room temperature for 2 h. The absorbance of the sample was measured at 765 nm. Total phenolic content was determined using a gallic acid standard curve and expressed as mg of GAE/g FW.

2.6.6. Total Flavonoid Content

The total flavonoid content was estimated using the colorimetric method described by Chang et al. [30]. First, 0.2 g of ground leaf sample was homogenized in 2.5 mL of 95% methanol, vortexed for 20 sec, and centrifuged at 7000 rpm for 20 min. Then, 500 μL of supernatant was transferred to a mixture of 1.5 mL of 95% methanol, 0.1 mL of 10% AlCl₃, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water, with a blank control lacking AlCl₃. The mixture was incubated for 30 min at room temperature (25 °C) and the absorbance was measured at 415 nm. Total flavonoid content was determined by using quercetin equivalents and expressed as mg/g FW.

2.7. Statistical Analysis

The data collected were analyzed using Minitab version 19.2 statistical software (Minitab Inc., State College, PA, USA), and graphs were plotted using Microsoft Excel and GraphPad Prism version 9.3.1 software (San Diego, CA, USA). Analysis of variance (ANOVA) was carried out to determine treatment differences at p ≤ 0.05. Tukey’s test at α = 0.05 was used to separate treatment means when the ANOVA indicated a significant difference between treatments. Data were presented as mean ± standard deviation (SD). Data for PA and SL were analyzed separately using one-way ANOVA. Tables with results provide numerical data, with different letters indicating the significant difference (p ≤ 0.05) within each concentration.

3. Results and Discussion

3.1. Chemical Properties of Liquids

All the determined chemical parameters of the PA and SL increased as their concentrations, increased except for pH, which showed a slight decline in PA and a slight increase in SL (

Table 1. Chemical properties of each concentration of pyroligneous (PA) liquid.

Treatment	pH	TDS (mg/L)	EC (μS)	Salinity (mg/L)
Control	5.94a	22.87g	39.77g	47.40g
0.01%PA	4.27b	39.50f	56.23f	54.30f
0.1% PA	3.59c	84.00e	101.93e	96.47e
0.25% PA	3.31d	108.33d	137.93d	137.10d
0.5% PA	3.14e	133.67c	196.43c	178.37c
1% PA	2.97f	171.67b	263.67b	235.50b
2% PA	2.82g	217.00a	345.67a	320.00a
p-value	<0.001	<0.001	<0.001	<0.001

EC, electrical conductivity; TDS, total dissolved solid. Means with different letters are significant differences (p ≤ 0.05) within each concentration. Means were derived from three replications and Tukey’s test was used to separate the means.

and

Table 2. Chemical properties of each concentration of sea lettuce (SL) liquid.

Treatment	pH	TDS (mg/L)	EC (μS)	Salinity (mg/L)
Control	6.46e	17.70g	39.77g	35.53g
0.01% SL	6.72d	23.43f	51.03f	46.10f
0.1% SL	6.78d	30.10e	65.33e	58.40e
0.25% SL	7.01c	42.37d	93.07d	83.27d
0.5% SL	7.13bc	63.43c	136.43c	122.37c
1% SL	7.19b	99.33b	218.00b	195.00b
2% SL	7.36a	172.00a	369.33a	332.67a
p-value	<0.001	<0.001	<0.001	<0.001

EC, electrical conductivity; TDS, total dissolved solid. Means with different letters are significant differences (p ≤ 0.05) within each concentration. Means were derived from three replications and Tukey’s test was used to separate the means.

). The pH levels had a significant (p < 0.001) decrease with higher concentrations of PA liquid. Salinity increased significantly (p < 0.001) with increasing concentrations of PA liquid and SL liquid. The electrical conductivity (EC) of 2% liquid showed significant (p < 0.001) differences from other concentrations, particularly for SL liquid.

The nitrogen content (N, HNO₃, NO₂⁻) of PA liquid was markedly higher than that of SL liquid (

Table 3. Chemical element of pyroligneous (PA) liquid and sea lettuce (SL) liquid.

Elements	PA (µg/L)	SL (µg/L)
Kjeldahl Nitrogen	460,000	53,000
Nitrate + Nitrite	100,000	<50
Potassium (K)	180	8600
Magnesium (Mg)	<10	10,500
Calcium (Ca)	100	1300
Iron (Fe)	<20	100
Manganese (Mn)	1	213
Zinc (Zn)	10	7
Boron (B)	<1	31
Selenium (Se)	<1	<5
Barium (Ba)	<1	<5
Cobalt (Co)	<0.1	<0.5
Molybdenum (Mo)	<0.1	<0.5

). However, SL liquid contained higher levels of Mg, Ca, K, Mn, Fe, and B than PA liquid. The content of cobalt (Co), molybdenum (Mo), selenium (Se), and Barium (Ba) were present at levels below the reported limits in both liquids. This composition of PA is consistent with the findings of previous studies [31]. These differences in chemical properties can be attributed to the feedstock and pyrolysis temperature [9].

3.2. Germination Test of Kale

Germination occurred in all treatments after the pouches were incubated in a dark environment for two days, except for the 1% and 2% PA-treated seeds, which did not germinate. PA had a significant (p ≤ 0.05) inhibitory effect on the germination of kale seeds, especially at 1% and 2% PA. The germination rate of kale seeds showed a distinct (p < 0.05) decrease with increasing PA concentration, and 0.5% PA reduced germination rate by ca. 43% compared to the control. (Figure 1A). This may be due to the high PA concentrations, which have high electrical conductivity and low pH. Changes in the pH and EC of acidic solutions affect biological activity, and plants are more sensitive to salinity during germination and seedling growth [32]. Most plants grow and perform well at an optimal pH between 5 and 8. The 1% and 2% PA had a complete inhibition on the germination of kale seeds, and all other concentrations of PA delayed the germination of seeds (Figure 1A). However, the 0.01% and 0.1% PA-treated kale seeds had similar germination rates, but the germination rate of 0.1% PA was slightly higher than that of 0.01% PA at a later stage (Figure 1A). Several studies have demonstrated that low PA concentration can facilitate metabolic activities and enhance plant germination [19,33]. The biostimulatory ability of PA has been attributed to the presence of butanolide, a biologically active compound belonging to the Karrikin family of phytohormones and other phenolic compounds which are known to play a critical role in regulating seed photomorphogenesis [33,34].

There was no obvious difference (p > 0.05) in the germination rate of kale seeds between different concentrations of SL on day 9 (Figure 1B). The germination rate of kale seeds treated with 0.01% SL was less than the germination rate of seeds treated with all other concentrations (Figure 1B). The germination rate of the control was greater than all other SL concentrations at a later stage (after seven days) (Figure 1B). On day 7, the germination rate of the control was 4.8% higher than that of 0.01% SL (Figure 1B). High salinity inhibits seeds due to the high EC in the SL liquid, which could negatively affect seed germination. Similar results were found in previous studies, where the germination rate of lettuce (Lactuca sativa) and corn (Zea mays) seeds decreased with an increasing concentration of the process water generated in the HTC of vinasse and sugarcane bagasse [35]. This study used the same HTC process water as the SL liquid. The diluted hydrothermal charring filtrate had a significant (p < 0.05) inhibitory effect on the germination and growth of maize [36].

It is obvious from Figure 2 that the accumulative rate of root elongation of kale treated with the PA and SL liquids was faster than that of the control. The kale seeds maintained a rapid growth rate in the early stage (first four days), which began to slow down and level off in the later stage (after day 4). The root elongation of 0.1% PA- and 0.01% PA-treated plants was significantly (p < 0.05) greater than that of the control, while that of 0.5% PA was inhibited significantly (p < 0.05) and lowered by ca. 82.8% compared to the control (Figure 2A). The inhibition effect of PA on kale was reduced after seed germination, and low concentrations of PA promoted the growth of kale roots. Infiltration of seeds with 600-fold-diluted PA promotes wheat seed germination and growth and significantly increases yield. This is attributed to the high bioactivity of acids and phenolics in PA, which can promote seedling growth and nitrogen uptake at low concentrations [37]. The 1% and 2% PA had no growth data because they completely inhibited the germination of treated seeds (Figure 2A).

The kale seedling root growth of SL-treated kale seeds was significantly (p < 0.05) promoted, especially at low concentrations of SL. The length of the kale roots at 0.5% SL was 1.8 times longer than the length of the roots in the control. This may be related to the dilution concentration and that the SL liquid was rich in nutrients that promote plant root growth. Similarly, SL contains phenolic substances that promote plant growth and improve plant stress tolerance [15]. The seedling growth of SL-treated plants was longer than the roots of PA treatments, and all concentrations of SL were much better than the control. Also, 0.5% SL had the highest root elongation and the fastest growth rate among all concentrations of SL, and a trend of rapid growth on the eighth day (Figure 2B).

All SL treatments seemed to have similar effects on the germination and seedling growth of kale seeds and had a pronounced growth promotion effect compared to the control. PA liquid had a significant (p < 0.05) reduction effect on all the seedlings in the present study. As clearly shown in Figure 3, PA-treated seeds had a significant (p < 0.05) inhibitory effect on the germination and growth of kale seeds, which increased with an increasing concentration of PA.

There were significant differences (p < 0.05) in the total length, total surface area, and root volume of kale seedlings grown at different concentrations of PA (

Table 4. Seedling growth of kale treated with different concentrations of pyroligneous acid (PA) liquid and sea lettuce (SL) liquid.

Treatment	PA			SL
	TL (cm)	TSA (cm2)	RV (cm3)	TL (cm)	TSA (cm2)	RV (cm3)
Control	12.4a	5.96a	0.045a	12.4a	5.96a	0.045a
0.01%	14.7a	6.95a	0.027ab	13.7a	6.68a	0.050a
0.10%	14.2a	6.72a	0.040ab	13.8a	7.17a	0.050a
0.50%	4.0b	2.46b	0.021b	14.9a	7.16a	0.041a
1%	1.1b	1.25b	0.019b	14.1a	6.59a	0.041a
2%	/	/	/	13.6a	6.07a	0.034a
p-value	<0.001	<0.001	0.001	0.910	0.533	0.620

TL, total length; TSA, total surface area; RV, root volume. Values are means of three replicates and different letters indicate significant differences (p ≤ 0.05) according to Tukey’s test.

). Low concentrations of PA promoted the growth of kale seeds compared to the control. However, high concentrations of PA (≥0.5% PA) had a significant (p < 0.05) negative effect on seedling growth (Table 4). In addition, the 0.01% PA-treated kale seeds had the highest total length, which was higher than that of the control by ca. 18.5% (Table 4). It is suggested that the effectiveness of PA is highly dependent on the concentration used [9]. This indeed confirms that lower levels of PA may contain a tight proportion of bioactive compounds to stimulate kale seedling growth.

There were no significant (p > 0.05) differences in the total length, total surface area, and root volume of kale seedling growth with the different concentrations of SL. However, it was observed that the growth of kale seeds treated with 0.5% SL was relatively higher compared with that of control, followed by 0.1% SL (Table 4). The total length of kale seeds treated with all concentrations of SL liquid was non-significantly (p > 0.05) higher than the control (Table 4).

3.3. Growth Test of Kale

All treatments were photographed to compare the growth and color of the kale (Figure 4). The application of biostimulants had a significant effect (p < 0.05) on the color of the kale. Different concentrations of PA and SL had little effect on the color of the kale, but all treatments were significantly (p < 0.05) higher than the control. Comparing the yellow-blue value b* and red-green value a*, it was obvious that the control leaves were yellowish in color and the treatment leaves were greenish (

Table 5. Color analysis of kale treated with pyroligneous acid (PA) liquid and sea lettuce (SL) liquid.

Treatment	SPAD	L*	a*	b*	c*	h*
Control	40.1b	41.87b	−11.80a	15.46a	19.44a	127.45b
PA 0.25%	45.3a	44.37a	−11.10a	12.75a	16.99a	132.23a
PA 0.5%	44.9a	44.20a	−10.93a	12.19a	16.41a	132.23a
PA 1%	45.1a	42.81ab	−10.91a	14.24a	17.98a	128.78ab
p-value	<0.001	0.011	0.193	0.056	0.101	0.013
Control	40.2b	41.87b	−11.80b	15.46a	19.44a	127.45b
SL 0.25%	44.4a	43.14ab	−10.94ab	12.79ab	16.88ab	131.87b
SL 0.5%	44.7a	44.22a	−10.54a	11.41b	15.31b	134.50a
SL 1%	44.2a	41.77b	−10.08a	11.38b	15.25b	132.23a
p-value	0.001	0.008	0.001	0.001	<0.001	0.002

SPAD, chlorophyll content; L*, lightness; a* and b*, chromaticity coordinates (−a*: green, +b*: yellow); c*, chroma; h*, hue angle. Means with different letters are significantly different (p ≤ 0.05) within each concentration. Values are means of three replications and means were separated using Tukey’s test.

). The 0.25% PA and 0.5% SL-treated plants had the greenest leaf color, while the 0.5% PA- and 0.5% SL-treated plants had the highest chlorophyll content (Table 5). The brightness L* of both PA- and SL-treated plants was significantly (p < 0.05) higher than that of the control. Specifically, the 0.25% PA- and 0.5% SL-treated plants recorded the highest brightness compared to the control (Table 5). Taken together, 0.25% PA and 0.5% SL were greener and brighter in terms of kale leaves.

The physiological response of kale to different concentrations of PA treatment showed a positive trend. The stomatal conductance and transpiration rate of 1% PA-treated plants were significantly higher (p < 0.05) than those of the control group, by ca. 204% and 100%, respectively (

Table 6. Physiological response to kale treated with pyroligneous acid (PA) liquid and sea lettuce (SL) liquid.

Treatment	Fo	Fm	Fv/Fm	Fv/Fo	Ci	E	gs	A
Control	178.3a	954.9a	0.81b	4.37b	437.13a	1.13b	0.08b	0.22b
PA 0.25%	157.5b	916.4a	0.83a	4.91a	335.90b	2.60a	0.13ab	4.05a
PA 0.5%	156.3b	937.8a	0.83a	5.01a	347.07b	2.88a	0.12ab	3.98a
PA 1%	162.7b	936.8a	0.83ab	4.77ab	366.80b	3.44a	0.16a	2.47a
p-value	0.001	0.140	0.002	0.001	<0.001	<0.001	0.056	<0.001
Control	178.3a	954.9a	0.81b	4.37b	437.13a	1.13b	0.08a	0.22b
SL 0.25%	157.1b	961.4a	0.84a	5.14a	363.00b	1.73ab	0.11a	3.33a
SL 0.5%	158.1b	942.3a	0.83a	5.00a	384.20b	1.63ab	0.10a	1.94ab
SL 1%	157.1b	944.4a	0.83a	5.03a	382.60b	2.29a	0.14a	3.02a
p-value	<0.001	0.659	<0.001	<0.001	<0.001	0.017	0.131	<0.001

Fo, minimum fluorescence; Fm, maximum fluorescence; Fv = Fm − Fo, viable fluorescence; Fv/Fm, maximum quantum yield or maximal photochemical efficiency; Fv/Fo, potential photosynthetic capacity; Ci (μmol mol⁻¹), Sub-stomatal CO₂; E (mol H₂O m⁻² s⁻¹), transpiration rate; gs (mol H₂O m⁻² s⁻¹), stomatal conductance; A (μmol CO₂ m⁻² s⁻¹), photosynthetic rate. Means with different letters are significant differences (p ≤ 0.05) within each concentration. Values are means of three replications and means were separated using Tukey’s test.

). The intercellular CO₂ concentration of the control plants was significantly (p < 0.05) higher than that of all PA-treated group plants, while the transpiration rate of the control was lower compared to other concentrations of PA (Table 6). In the study of Ofoe et al. [38], the application of low concentrations of PA was less toxic to the root system and could improve plant nutrient uptake and promote root growth. The results showed that 0.25% PA had the best growth promotion effect on kale with the highest photosynthetic rate and biomass yield. However, the 1% PA exhibited some biological toxicity and a relatively low growth-promoting effect. The application of PA increased the leaf area index and dry matter accumulation of rapeseed and improved plant resistance to low temperatures and diseases. Pyroligneous acid can also be mixed with other plant hormones or liquid fertilizers with significantly greater effect than PA alone [37].

SL liquid had a beneficial effect on the growth of kale leaves. There was no obvious difference in the potential photosynthetic capacity and the maximum quantum yield of kale leaves with the different SL concentrations. However, the intercellular CO₂ concentration of the control plants was significantly (p < 0.05) higher than that of all SL-treated group plants (Table 6). Transpiration rate and stomatal conductance were highest in kale plants treated with 1% SL, and were 103% and 75% higher than the control, respectively. (Table 6). The 0.25% SL significantly (p < 0.05) increased the photosynthetic rate of kale, which was 14 times higher than that of the control (Table 6). SL liquid had a beneficial effect on the growth of kale, and 1% SL exhibited the best performance compared to the other SL treatments. SL liquid contains N, Mg, and K, which are essential nutrients for plant growth and can enhance the water uptake of plants [39]. The rich K content increases the disease resistance of the plant, helps to regulate the water status of the plant, and controls the opening and closing of stomata [32], which explains the significantly (p < 0.05) enhanced photosynthetic rate of SL-treated kale. The SL liquid obtained by the HTC process has a higher carbon content compared to raw sea lettuce, and it contains phenolic substances that contribute to plant growth [15].

Two liquids had a great growth-promoting effect on kale. The effects of the different concentrations of the liquids on leaf growth did not differ much; all treatments were relatively similar in the first period (first nine days), and the promotion effect was more pronounced in the later period (Figure 5A). In the 0.25% PA-treated plants compared to other PA treatments, there was a significant (p < 0.05) difference in the elongation and growth of the leaves (Figure 5A). Moreover, the leaf elongation rate of SL-treated plants changed from slow to fast with time; in the first stage (first 9 days), both 0.25% and 0.5% SL-treated plants had shorter leaves than the control. However, in the later stage (after 9 days), all SL-treated plants were clearly longer than the control, especially 0.25% SL-treated plants (Figure 5B). Overall, 0.25% PA and 0.5% SL performed the best in leaf elongation (Figure 5).

The number of kale leaves was not significantly (p < 0.05) different in the PA and SL treatments. The control treatment had the highest number of kale leaves compared to PA and SL treatments (Figure 6A). The number of leaves of PA- and SL-treated kale decreased with 0.25% and 0.5% applications of the liquids (Figure 6A). Aside from the control, 1% PA had the next highest number of leaves compared to the other treatments (Figure 6A). However, the addition of the solution to the soil did not have an obvious promotional effect on leaf numbers.

There was no difference between the two liquids at different concentrations for the total height of the whole kale plant. Different concentrations of PA and SL liquids have similar promotion effects on height with kale. The average height of kale in the control was the lowest compared to the other treatments, and the data proved that PA liquid and SL liquid still had some promotional effect on the total plant height of kale (Figure 6B). The 0.5% PA and 0.25% SL had the highest effect on the height of kale plants (Figure 6B).

The stem diameter of kale had significant (p < 0.05) effects with PA and SL treatment. The stem diameter of the control was the lowest among all treatments, proving that the two liquids treatments had a significant (p < 0.05) increase in the growth of kale. The results of SL and PA treatments for stem diameter were very similar; the stem diameters of 0.25% PA and SL fluids were ca. 17.1% and 14.6% wider than the control, respectively, and the diameter of treated kale stems decreased with increasing PA or SL concentrations (Figure 6C). The 0.25% PA and SL liquids showed higher performances among the treatments (Figure 6C). There was a significant (p < 0.05) difference between different concentrations of PA on the growth of kale stem diameter (Figure 6C). The growth of kale main stems in 0.25% PA and SL liquids was higher than that of controls.

The fresh weight of kale performed best under the treatment of the liquid, with significant differences (p < 0.05) between the different concentrations. Although there was no significant (p < 0.05) difference in the fresh weight of kale treated with different concentrations of PA, an increase of 25.7% in total fresh weight was observed with 0.5% PA compared to the control (Figure 6D). There was a significant (p < 0.05) difference between different concentrations of SL liquid in the growth of kale. The 1% SL treatment recorded the highest fresh weight of kale compared to the control (Figure 6D). Among all treatments, the control group had the lowest fresh weight, and the data indicated that these two liquids could effectively promote fresh weight and increase the yield of kale (Figure 6D).

3.4. Biochemical Analysis

The content of chlorophyll and carotenoid pigments is closely related to photosynthesis. Biostimulant application significantly (p < 0.05) increased chlorophyll b content, while the effect on chlorophyll a content and carotenoid content were not obvious. Chlorophyll a content was not significantly (p > 0.05) influenced by different concentrations of PA but was significantly (p < 0.05) affected by concentrations of SL treatment. Chlorophyll a content and SL liquid concentrations were positively correlated. The 0.5% SL treatment showed significantly (p < 0.05) decreased chlorophyll a than the control (Figure 7A). The low concentration of biostimulants was not favorable for chlorophyll synthesis. The 0.5% PA and 1% SL treatments had a significant effect (p < 0.05) on chlorophyll b, increasing it by 5.6% and 6.1%, respectively. PA and SL liquids had an inhibitory effect on carotenoids. The 0.5% PA and 1% SL significantly (p < 0.05) decreased carotenoid contents by 17.2% and 18.0%, respectively, compared to the control (Figure 7C).

PA and SL significantly (p < 0.05) elevated the ascorbic acid content of kale, while the dehydroascorbic acid content was not affected. The 1% PA and 0.25% SL treatments significantly (p < 0.05) elevated the ascorbic acid content by ca. 117% and 175% compared to the control (Figure 8A). The 0.25% and 1% PA treatment effectively increased the ascorbic acid and dehydroascorbic acid contents in kale leaves (Figure 8). The concentrations of SL liquid and ascorbic acid and dehydroascorbic acid content were negatively correlated, and the low concentration of SL facilitated the accumulation of ascorbic acid and dehydroascorbic acid content. The contents of ascorbic acid and dehydroascorbic acid contents were increased by 2.75 and 1.39 times, respectively, following the application of 0.25% SL, compared with the control (Figure 8).

PA and SL liquids increased the phytochemical composition of kale leaves, while SL liquid had a more pronounced phytochemical promotion effect. As shown in Figure 9A, the sugar content of both PA and SL treatments showed remarkable changes. The sugar content of 0.25% SL and 1% SL increased by ca. 162% and 148% compared to the control (Figure 9A). There was no great difference in the protein content of PA at different concentrations in the kale leaves. However, SL treatment had a significant (p < 0.05) effect on protein content. The protein content of 0.5% SL significantly (p < 0.05) reduced the protein content compared to the control (Figure 9B). However, 1% SL significantly (p < 0.05) increased the protein content, indicating that a high concentration of SL can effectively increase the protein content of kale leaves (Figure 9B). This suggests that PA and SL could be used to enhance crop quality for human health and nutritional purposes.

PA and SL liquids were effective in increasing the phenolic content in kale leaves, and the phenolic content was more pronounced with lower concentrations of PA and SL. The 0.25% PA and 0.5% SL treatments significantly (p < 0.05) increased phenolic content by ca. 119% and 127%, respectively, compared with the control (Figure 9C). Numerically, the phenolic content of SL treatments was slightly higher than that of PA treatments. PA and SL liquids significantly (p < 0.05) increased flavonoid content, but the flavonoid contents were affected differently by PA and SL treatments. Different concentrations of PA liquid and flavonoid content were inversely proportional, while the flavonoid content of SL treatments increased with increasing concentration. Flavonoid was significantly (p < 0.05) increased with 0.25% PA and 1% SL by ca. 134% and 152%, respectively, compared with the control (Figure 9D). A high concentration of SL liquid or a low concentration of PA liquid can effectively increase the flavonoid content in kale leaves. Kale is considered to be an excellent source of phytochemicals including vitamins, flavonoids, and phenolics, which are crucial for scavenging reactive oxygen species (ROS) radicals [22]. Studies have reported that PA application enhances the antioxidant content of edible parts of leaves and fruits [37,40]. Therefore, the increase in these antioxidant compounds with PA application is expected and can be attributed to the high organic acids and phenolic compounds in PA since previous studies have demonstrated that phenolic compounds in PA exhibited high ROS-scavenging activities, reducing power, and anti-lipid peroxidation capacity [41]. Although the exact mechanism of the biostimulant-mediated increase in antioxidant compound in the leaves has not been examined, it is plausible that the increase in these compounds could be due to the activation of defense responses, improved nutrient absorption and assimilation, and the upregulation of genes and enzymes involved in the phenylpropanoid pathway [42,43].

4. Conclusions

This study demonstrated that PA and SL liquids affect kale germination and growth. The high pH and conductivity of PA inhibited the germination of kale seeds, and the inhibitory effect increased with increasing concentration. After seed germination, this inhibitory effect was reduced, and the low concentration of PA also elongated the root system of kale. The 1% SL sample had a significant effect on the fresh weight, photosynthetic rate, and chlorophyll content of kale and was the most effective for the growth of kale. The 1% SL treatment significantly increased the content of sugars, proteins, and flavonoids, followed by SL at 0.25% and 0.5%. This study showed that SL processed by HTC was more effective at stimulating kale growth, and, therefore, SL is a promising and effective biostimulant for agricultural production. It is worth mentioning that PA displayed better results than more concentrated extracts when applied at lower concentrations during the kale seed germination stage and that PA was rich in nitrogen. This suggests that the application of lower PA concentrations can effectively promote seedling root growth in kale. This study provides some reference for the reuse of organic waste as biostimulants to help better utilize organic waste resources and energy in agriculture. These findings not only build upon previous research but also provide insights into the morpho-physiological and biochemical responses and optimal application rates of these biostimulants on kale growth in a greenhouse. Therefore, it is recommended that producers use low concentrations of PA for kale seedling root growth and use SL to help the germination and growth of kale. The concentration range of biostimulants will be expanded in future studies to further investigate the most suitable biostimulants for kale germination and growth. Additionally, while both biostimulants have significant effects on plant growth, their mechanisms of action and potential synergies require further investigation to maximize their use in sustainable crop production.