3.1. Biomass and Biochar Properties
Results from proximate and elemental analyses are summarized in
Table 1. As expected, these results were primarily dependent on pyrolysis operating conditions. Results from physicochemical analyses of produced biochars are reported in
Table 2, from which it can be concluded that nutrient contents of produced biochars were mainly affected by the biomass feedstock.
B600 had a higher fixed carbon and ash compared to B400, which conversely had a higher amount of volatile matter. The hydrogen, nitrogen, and oxygen fractions decreased with increasing pyrolysis final temperature. Both specific surface area (Sbet) and pore volume increased with rising pyrolysis final temperature, as a result of the removal of H and O during the enhanced devolatilization process, which resulted in more developed pore structures. The total pore volume increased with pyrolysis peak temperature.
Physicochemical analyses were carried out in the same way as any other organic amendment, in order to quantify the levels of available nutrients. Principal and secondary nutrient levels showed no major differences between B400 and B600 that would justify different application rates for the same crop.
It was interesting to analyze total N content in biochars, also taking into account the ammonia fraction instead of just the organic N. However, the biochar´s resistance against digestion with acids should be considered when analytical methods, such as total N Kjeldhal, are applied [
9]. This explains the results reported in
Table 2 for N total Kjeldhal, which are slightly lower than those reported in
Table 1 for organic N from elemental analyses.
WHC was found to increase with increasing pyrolysis final temperature. Both B400 and B600 showed higher values of WHC than those measured for the initially selected soils (see
Table S1).
Particle size distributions of biochars after the mechanical agitation process are listed in
Table 3.
The obtained mass fraction for particle sizes below 2 mm was very low. In addition, 69–77% of the biochar applied in this work was in the form of particle sizes ranging from 20 mm to more than 40 mm. Smaller particle sizes are usually adopted in most experiments in which the influence of this parameter is evaluated [
50,
52]. However, it is necessary to reach a trade-off concerning the most appropriate particle size, since grinding the biomass feedstock before pyrolysis is an energy-intense pretreatment and grinding biochar could lead to an excessive production of powder-like particles, which are difficult to apply into soil.
Results from proximate, elemental, ash analysis, and biomass components of vine shoots are summarized in
Table S2.
Table S3 reports the biochar mass yields from pyrolysis experiments. As expected, the biochar average mass yield (ƴ
char) notably decreased when the pyrolysis final temperature increased (B400—ƴ
char = 0.38 and B600—ƴ
char = 0.29).
3.2. Phytotoxicity Test
The germination index (GI) is an integrative measure of compounds of low toxicity (affecting root growth) and high toxicity (affecting germination) [
50].
Table 4 shows the main GI values and standard deviation for the selected species. One-way analysis of variance evaluating the effect of biochar temperature on GI for this species did not detect significant differences (
p = 0.182) between treatments. However, in agreement with Zucconi et al. [
49], GI values between 50%–80% represent moderate phytotoxicity. These low values were only observed for high-temperature biochars (600 °C) for both watercress and lettuce. On the other hand, GI values above 100 reveal phytostimulant effects of the tested solution. A slightly higher value was found in basil results under B400 treatment, and an apparent phytostimulant effect for B400 was detected in lettuce. Values between 80%–100% denote a lack of phytotoxicity. Therefore, no adverse effects of biochar were found on sorghum germination through this type of phytotoxicity test. According to Busch et al. [
53], high ash content in biochar can cause negative effects due to saline stress. It could explain some of the effects in the smaller seeds (watercress, lettuce and basil) which showed greater sensitivity to the B600 extract than the larger seeds. Biochar could still be able to affect seeds germination through other ways like direct contact or volatiles emission [
54]; however, the evaluation of these alternative ways is out of the scope of the present paper.
From the results obtained from a phytotoxicity test, it can be concluded that a preliminary washing step of biochar with water is not absolutely required. In other words, for the pyrolysis operating conditions adopted in this study, the organic compounds available on biochar surface did not inhibit germination of selected seeds.
3.4. Growing Substrates Changes and Leaves’ Nutrient Contents
A significant increase in WHC was observed by the addition of biochar (
p < 0.05 between Control treatment and biochar amendment); the increase in WHC was observed for both studied substrates textures. There was also a significant influence of thepyrolysis final temperature on water retention, which increased by 3–6% with biochar temperature (
p ≤ 0.05 between B600 and B400;
p ≤ 0.05 between biochars and control treatment without biochar). Only in clay-loam substrate, WHC increased significantly (
p ≤ 0.001) with application rate. This behavior was also reported by Ali et al. [
57] using biochar application rates of 25 and 50 Mg ha
−1 of biochar.
Figure 3 illustrates the behavior of each type of growing substrate. Numerous studies have demonstrated that biochar amendment improves WHC [
3,
57,
58]; the present study reports a direct relationship between WHC and pyrolysis final temperature, which is directly correlated with WHC values already measured for individual biochar analyses (see
Table 2). In agreement with Marshall et al. [
39], who also reported a high hydrophobicity in vine shoots-derived biochar produced at 400 °C, it could be an important parameter to take into account, and not only the macroporous structure of biochar which could led to attribute higher WHC at lower pyrolysis peak temperatures. Accordingly, the dependence of this parameter on pyrolysis temperature is an aspect that could be of interest for agronomic contributions.
Differences in nutrient concentrations in growing media and leaves are deduced from
Table 7. In the present experiment, biochar application had a significant influence (p < 0.05) on pH in the sandy-loam substrate, decreasing values over 0.1 points. The obtained results are in agreement with those by Liu and Zhang [
59], who reported that biochar application decreased growing media pH in a sandy-loam texture. In their study, pH reduction was enhanced with an increasing biochar application rate and incubation time, related to the production of acidic materials from biochar oxidation.
No differences were observed in total SOM or CEC between treatments (
Tables S5 and S6). It could be explained by the large particle size of biochar adopted in this experiment. In this sense, when substrates’ chemical analyses were carried out, particles of biochar were considered thick elements, and were separated from the analytical samples; the fine fraction able of interacting with clay and organic matter in the soil represented 4–7% of the total amount of biochar applied, which is a small addition to influence soil parameters.
In contrast to other studies [
14,
43,
46], the three-way ANOVA showed that the application of biochar did not significantly modify the cation exchange capacity, soil organic matter, total nitrogen concentration, and available phosphorus concentration. Significant differences (
p < 0.05) were found for both types of textures in K and Ca concentrations. The content of available K was significantly related to the texture of the substrate (
p < 0.0001). In addition, there was a positive interaction between texture growing media and application rate (
p = 0.006), and between biochar and application rate (
p = 0.037). Two-way ANOVA for a given texture showed that significant differences (
p < 0.05) on K concentration in S1 are related to B600 addition but not to application rate. S2 showed significant differences (
p < 0.05) for biochar addition and application rate in this texture. B600 application at 3 wt. % improved soil K content by 67% in comparison with control treatment (see
Figure 4).
K concentration in leaves also showed significant differences (p < 0.005) related to biochar application.
Positive interactions were found between the effect of texture growing media and biochar (p < 0.0001) and between texture and application rate (p = 0.035) on Ca concentrations. Analyzing each textural type individually, it was observed that significant differences (p ≤ 0.05) in Ca concentration in S1 were related to B600 applied at the higher rate (3 wt. %). Different behavior was observed in S2, where significant differences (p ≤ 0.05) in Ca concentration were observed for B400 application.
Biochar addition at higher doses had significant effects (p < 0.0001) on Mg concentration only in the clay-loam growing substrate, regardless of the pyrolysis final temperature (p = 0.950).
In both substrates, the K/Ca ratio used resulted in being extremely low because of the elevated Ca content, indicating the high difficulty of K absorption by plants. In general, this ratio ranges between 2–10, and the values obtained in this study vary from 0.044 to 0.089.
Significant differences were observed for this ratio between growing substrates (p < 0.0001), also between biochar temperatures (p < 0.0001) and between application rate (p = 0.0002). Biochar addition increases K/Ca for both textures of growing media in comparison with control treatment (p < 0.05), and K/Ca increases with application rate only in the clay-loam growing substrate (p < 0.05).
Another cation ratio related to soil and crop fertility is K/Mg. This ratio followed the same trend observed for the previous one. Three-way ANOVA showed significant differences derived from biochar application in both textures (p < 0.0001) except the application of B400 at lower doses in the sandy-loam substrate compared to control treatment, where no differences were detected. Otherwise, analyzing the effects in the clay-loam growing substrate treatments, an evident increment in K/Mg, was observed when both pyrolysis temperature and application rate were increased (p = 0.0008 between B2 and B1; p = 0.025 between S2 and S1).
According to earlier studies [
54,
59,
60,
61], biochar application has an evident influence on essential macronutrients’ availability. Significant differences have been reported in this experiment for K, Ca, Mg, and their ratios. Incorporation of biochar as a soil amendment stimulates plant growth by increasing the availability of essential nutrients [
22]. However, a nutritive solution was necessarily adopted in this experiment to maintain plants development and to supply nutrient deficiencies shown by the crop. Complementary field-scale experiments, where plants growth is not limited by pot dimensions and growing substrate mixture, are needed to confirm the results reported in this study.