Multiple measurements of plant growth were evaluated in order to comprehensively assess the potential of BC as an alternative to peat in soil-free substrates . Germination rates were determined by daily counts for the first 10 days following sowing, after which seedlings were thinned to 1 per pot. Seedlings were transplanted into pots that had zero germination. Replacement seedlings were used from substrates with equivalent %BC but no pH adjustment and were the same age as seedlings in the experimental trial. Weekly measurements over 9 weeks were taken for plant height and for relative chlorophyll content as leaf greenness using a SPAD 502 Plus Chlorophyll Meter . SPAD meters measure the difference between red light and infrared light absorbance, and for a given species and cultivar under the same growing conditions SPAD values can be used as an indicator of relative chlorophyll content . To ensure accurate measurement of new leaf tissue, four separate points were consistently measured on the second fully extended leaf from the top of the plant . SPAD measurements were taken between the tip and apex of the leaf to better reflect chlorophyll content and reduce measurement variability . At early stage flowering in week 9 , above-ground biomass was harvested. Fresh and dry biomass was measured individually for shoots, flowers, and buds. Total N was determined separately for non-flowering and flowering biomass by dry combustion using an elemental analyzer . Total above-ground biomass N was calculated from non-flowering and flowering shoot biomass and N measurements.
To examine fertigation effects on substrate properties over the 9- week growing period,nft growing system root-free substrates were analyzed for pH, electrical conductivity , and plant-available nitrogen and phosphorus . Available N was determined by extraction with 2 mol L−1 KCl with shaking for 60 min. Ammonium and nitrate N in the centrifuged extract were measured colorimetrically using the salicylate-hypochlorite method and vanadium chloride reduction method , respectively. Available P was determined by extraction with 0.5 mol L−1 NaHCO3 at pH 8.5 with shaking for 30 min, and orthophosphate P in the filtered extract was estimated as molybdate-reactive P . Available N and P in post-harvest substrates were corrected for substrate moisture content, which was determined gravimetrically by drying at 105 °C.Analyses of variance was used to analyze differences among the treatments for plant growth and substrate properties. Assumptions of normality and homoscedasticity of residuals were tested with the Shapiro-Wilk and Levene tests, respectively, using SAS Version 9.4 . Data were transformed when possible to meet these assumptions, including log transformation , square root transformation and Poisson transformation for variables with zero values . ANOVA was first performed using an exploratory model to test for potential inter actions of substrate and pH adjustment for each response variable. If there was no interaction, simple mean differences of response variables were evaluated. If there was a significant interaction of BC substitution and pH adjustment, effects were analyzed separately for each factor.If transformations were not successful, non-parametric analysis was performed with JMP Version 11 using a Welch ANOVA, and significant differences in means for BC substitution treatments relative to the non-substituted control were evaluated using the Steel test. Relationships among post-harvest substrate properties were explored using linear correlation analysis with PROC CORR in SAS v9.4.By evaluating an alkaline BC at high volumetric rates in soil-free substrates, this study addresses a potential obstacle to the feasibility of BC-based substrates for plant production .
The present data demonstrate that substituting a softwood BC with strongly alkaline pH for peat at high rates in soil-free substrates does not require pH adjustment under common greenhouse conditions because germination, shoot biomass and N content, and flowering of marigold did not significantly differ between substrates with and without initial adjustment to pH 5.8. BC substitution may even improve plant growth, as marigold plants with intermediate BC substitution exhibited higher relative chlorophyll content relative to 0% BC . These results are in mixed support of the stated hypothesis because BC substitution and pH adjustment effects on marigold depended on the stage of growth. As hypothesized, increasing BC substitution decreased plant height and chlorophyll content in the early stages of marigold growth. Though pH adjustment of BC substrates negatively affected germination and height, this may have been due to phytotoxicity of PLA used to decrease pH of high %BC substrates. By week 9, plant growth was similar regardless of BC substitution and initial pH adjustment, failing to support the hypotheses that high BC substitution rates would impair plant growth and that this would be alleviated by pH adjustment. However, since fertigation provided excess nutrients, pH was likely less important for nutrient availability. Equivalent and slightly positive effects of BC substitution at high rates and without pH adjustment can be partially attributed to the convergence of pH over 9 weeks of fertigation and plant growth to pH 4.4–7.4. As this high-temperature softwood BC has a higher pH than most BCs and was used at high substitution rates , it represents a ‘worst-case scenario’ liming effect. BCs produced from other feed stocks and/or at lower temperatures may not have as pronounced liming effects. De creases in substrate pH over time could reflect a number of processes: a residual liming effect of BC,container size for strawberries which could also account for the slight upward pH drift of substrates initially corrected to pH 5.8; nitrifification; rhizosphere acidification due to cation uptake. Though downward pH drift in peat-based substrates initially limed to a circumneutral pH has been found to be inverse to the base saturation of peat , the 0% BC substrates initially limed to pH 5.8 in this study did not exhibit significant pH changes.
The availability and plant uptake of N may be impacted by substrate pH, as indicated by extractable inorganic N, relative differences in chlorophyll content, and above-ground plant N. This may explain initial decreased plant height and relative chlorophyll content in high BC substrates with high initial pH . Foliar chlorosis in ornamental plants, including marigold, grown in high pH substrates has been induced by liming in peat substrates and could reflect non-N deficiencies such as iron and manganese . Similar above-ground biomass and total N despite greater relative chlorophyll content in high BC substrates by week 9 indicates that initial differences in chlorophyll content by BC substitution did not persist and that initial greater chlorophyll content for marigold in high BC sub strates did not necessarily translate to greater biomass and N uptake. A lack of N defificiency under conditions of fertigation is further evidenced by overall high concentrations of available N in substrates at week 9 and by the absence of correlation between available N with marigold above-ground biomass and N content. Elevated chlorophyll content with BC substitution may therefore reflect enhanced plant access to non-N nutrients. Available N was inverse to SPAD values in week 9 and did not reflect similar above-ground plant N concentrations. The disparity be tween marked differences in substrate N availability under conditions of fertigation yet similar above-ground biomass N content could be explained by pH-dependent gaseous losses of N in pH unadjusted substrates and/or differences in extractability influenced by pH-dependent binding. That extractable inorganic P did not differ as much as inorganic N across pH gradient of pH unadjusted substrates could indicate similar anion exchange capacity of substrates. High available N and P in substrates challenges the hypothesis that BC substitution can influence marigold growth by affecting availability of nutrients added by fertigation. For example, post-harvest available P was positively correlated with marigold biomass but was two orders of magnitude higher than thresholds of deficiency . Though high C:N substrates such as peat can entail sufficient N im mobilization so as to compromise plant growth , N fertilization as in this study would be expected to rapidly alleviate N deficiency. This time-dependent effect may have manifested as lower chlorophyll content in high BC substrates in week 1 but not week 9. Similarly, N fertilization alleviated slightly lower biomass accumulation of marigolds grown in pine wood-based substrates com pared to peat . Though the experimental design of this study removed water and nutrient limitations by daily fertigation, the present findings indicate a potential benefit of BC for water availability in soil-free substrates. The increase in WHC with BC substitution that peaked at 30% BC supports this hypothesized benefit of BC at high rates for soil-free substrates , as well as in inorganic matrices like soils .Marigold germination and growth response to BC substitution in pH adjusted substrates was likely due to the use of pyroligneous acid to decrease pH.
An increasing amount of PLA was applied to reduce increasingly elevated pH at high rates of the alkaline BC used. Since pH adjusted substrates had the same target pH , the difference can be attributed to a non-pH effect of the almond shell PLA used in this study. PLAs are a complex mixture of organic compounds of varying biological and phytological activity, including toxicity. These include organic acids , phenols, ketones phenylethers, and furan and pyran derivatives . The survival and equivalent growth of marigold seedlings transplanted into pH adjusted substrates with no seed germination suggests greater sensitivity of seeds than seedlings to PLA effects and is consistent with previous findings of PLA inhibition of germination . Par allel in vitro experiments , revealed full inhibition of marigold and lettuce germination at PLA ≥ 2.50% and ≥ 1.25% , respectively, though a similiar response occurred for acetic acid, a major PLA component at the same concentration. Studies indicate mixed effects of PLA on biological activity, with both plant-growth promoting and toxic effects, and antimicrobial effects. For example, PLA improved in vitro rooting of pear , and at rates of up to 6% increased fruiting of edible mushrooms in sawdust-based substrates . On the other hand, germination of cress was inhibited by exposure to volatiles from pyrolysis, which are captured via condensation in the production of PLA . Similarly, cress germination was inhibited by BCs with high volatile contents . Like BC, feed stock and production conditions can significantly impact PLA composition and anti-biological activity , and thus the negative impacts of PLA observed in this study may be specific to the almond shell PLA used here.The potential of pyrolyzed biomass in soil-free substrates has been investigated since the mid-20th century. For example, Kono investigated the utility of charcoal to improve substrate physical properties such as water holding capacity and bulk density for orchid production . However, the rapidly expanding body of knowledge on BC, including the ability to design BCs based on feed stock and pyrolysis conditions, means that BCs can be engineered to target additional benefits for to soil-free substrates. Significant enrichment in available N and P over the course of 9 weeks of fertigation reflects high input conditions in greenhouse production systems. Compared to peat, the longer decomposition half-life of high-temperature BCs such as the one in this study, and the potential of nutrient ions to bind to BC and re-solubilize when applied to soils raises the possibility of re-using BC-based substrates as fertilizers. BC substitution may increase the longevity of peat-based substrates under conditions of high nutrient availability common in their use . Decomposition of peat during long grow periods, in particular under high N additions, can compromise physical and chemical properties . Partially replacing peat with less decomposable materials can decrease the overall decomposition rate of the remaining peat component of substrates even under N fertilization , raising the possibility of extending the lifetime of peat-based substrates with partial BC substitution. The availability of BC as a secondary product of bio-energy production and/or waste stream management , as well as lower transportation costs made possible by regional or on-site BC production, could further leverage economic advantages over peat and peat alternatives . Recent studies support the unique ability of BC to mediate biological interactions with benefits for greenhouse production such as enhanced pathogen and pest suppression. For example, 1–5% additions of citrus wood BC to peat-based substrates increased expression of pathogen defense genes in strawberry and as a result suppressed fungal disease ; for tomato and pepper , such additions delayed and reduced disease from fungal pathogens and mites .