Effects of Different N, P, and K Rates on the Growth and Cannabinoid Content of Industrial Hemp

ABSTRACT As a key metabolite with high medical values in industrial hemp, cannabidiol (CBD) has been paid increasing attention. However, how nutrient supply affects the CBD content and yield of hemp remains unclear. Therefore, an aeroponic experiment with nine treatments (A1~A9) was conducted to evaluate the effects of different concentrations of nitrogen (N, at 4.0, 6.0, and 8.0 mmol L−1), phosphorus (P, at 1.0, 1.5, and 2.0 mmol L−1), and potassium (K, at 3.0, 4.0, and 5.0 mmol L−1) on the growth, CBD content, and biomass of hemp. Results showed that at A7 (N8P1K5) (N, P, K: 8, 1, 5 mmol L−1) markedly enhanced the dry weight of shoot and inflorescence with an increment of 208.7% and 435.7% respectively; the equivalent number of total CBD content in inflorescence was increased by 41.8%, and the CBD yield was increased by 7.0 times compared with A3 (N4P2K5). Correlation analysis revealed that N was positively related to the CBD yield, while P or K had a negligible effect on CBD yield. In conclusion, nutrient supply would dramatically influence the growth and CBD yield of hemp. Under the experimental conditions, A7 (N8P1K5) is the most suitable choice to cultivate hemp and achieve a higher CBD yield.


Introduction
Hemp (Cannabis sativa L.), an annual herbaceous plant, belongs to the family of Cannabaceae, and is defined as a kind of Cannabis sativa with Δ-9-tetrahydrocannabinol (THC) lower than 0.3% (Andre, Hausman, and Guerriero 2016). As a multi-purpose crop, it is cultivated and utilize for more than 6000 years in the world, it is extensively utilized in textile, paper, food, medicine, edible oil, and even the construction and biocomposite sectors (Cheng et al. 2016;Zhao et al. 2021). Cannabidiol (CBD), a Cannabis sativa constituent, is a vital non-psychotropic component and performs various functions for the treatment of some diseases such as hypnotic, epilepsy, Alzheimer's, anxiety, pain, and inflammation (Blessing et al. 2015;Burstein 2015;Devinsky et al. 2017;Poleg et al. 2019).
It is well established that nitrogen (N), phosphorus (P), and potassium (K) are the three essential macronutrients that determine many aspects of plant growth and metabolism. Recent studies have demonstrated the significant effects of P Bernstein 2021a, 2021b), N Bernstein 2021, 2022a) and K (Saloner and Bernstein 2022b;Saloner, Sacks, and Bernstein 2019) on medical cannabis function, yield, and secondary metabolite production. It was found that inflorescence yield could be increased under elevated N (Saloner and Bernstein 2021) and P (Shiponi and Bernstein 2021a) supply, but yield response to K supply was reduced under K deficiency (Saloner and Bernstein 2022b). As N input is one of the key factors for plant development and function, extensive studies have been focused on the impact of N supply on plant yield (Bouchet et al. 2016;Leghari et al. 2016). Studies have shown that higher N levels could elevate the biomass of leaves for the fiber varieties, whereas decreasing the content of THC in leaves (Coffman and Gentner 1977). Shiponi and Bernstein found that when the N concentration is at 160 mg L −1 , drug-type (medical cannabis) could contain relatively high secondary metabolism content and even better yield quantity (Shiponi and Bernstein 2021b).
By contrast, evidence that appears to be instrumental for CBD accumulation is surprisingly limited. Moreover, Bernstein, Gorelick, and Koch (2019) found that the biomass of shoot increased with the increment of NPK fertilizer (Bernstein, Gorelick, and Koch 2019); Grabowska, Rbarz, and Chudy (2009) indicated that the ratio of NPK 1:0.7:1.5 could radically boost the yield of fiber hemp, while the ratio of NPK 1:0.8:1 was more suitable for seed hemp (Grabowska, Rbarz, and Chudy 2009). However, how nutrient supply affects the CBD content and yield of drug-type (medical cannabis) remains largely unknown yet.
Therefore, an indoor aeroponics experiment was conducted to investigate the effects of different dosages of N, P, and K as well as their compositions on the growth and CBD accumulation of hemp. By this means, it was expected to obtain one or two suitable NPK rates for higher CBD yield and dip a specialized hydroponic nutrient solution formula for hemp cultivation.

Plant materials and growth conditions
The hemp seeds (variety, Xinma No.1) were grown in a culture room with nutritional soil (peat soil: perlite: vermiculite = 1:1:1) (14 h light/10 h dark at 25°C, RH 60%), irrigating with distilled water every three days. After the emergenced of the second true leaf, the seedlings were transplanted into plastic pots, the male plants were removed and the female plants remained for later use. Light-emitting diode (LED) SZ100 lamps were used as the light source (single LED band size: 2.8 mm × 3.5 mm; the ratio of red to blue light: 1.6:1; photosynthetic quantum flux density 252) were supplied by the Guangdong Weizhaoye Optoelectronic Energy Saving Co., LTD, China.

Experimental reagents and procedures
The concentration of nutrients in the nutrient solution were Ca( The well-grown shoots were cut down from one female plant cultured in the hydroponic equipment. When roots appeared to be 2 ~ 5 cm, they were transferred to the aeration cultivation device. The aeration cultivation device (brought from the Henan Caiju Dongli Agricultural Technology Co., LTD, China, Model number: QWP-DK-1-6) with a monoculture hole was used for planting hemp in the experiment. The size of the cultivation barrel was 27 cm × 27 cm × 38 cm with a volume of 20 L. Each treatment contained 6 seedlings, the test was repeated 3 times.
The experiment was designed using the L9 (3 4 ) orthogonal test (Table 1). The nutrient solution formula was modified from the Hoagland nutrient solution, of which N was provided by Ca(NO 3 ) 2 ·4 H 2 O and KNO 3 , P was provided by KH 2 PO 4 , K was provided by KNO 3 and KH 2 PO 4 . The nutrient solution PH was adjusted to 5.8 and replaced every two weeks. After 20 weeks of treatment, the whole plant was harvested and all indexes were determined.

Morphological indexes and biomass
The hemp plants were harvested when the pistils' color changed from white to yellow or orange (20 weeks). Before harvest, the plant height was measured from the base of the stem to the apex of the plant by using a ruler, the stem diameter was measured with a vernier caliper. After that, the plants were harvested and dried at 65°C in the oven, and the biomass of the shoot (stem and leaves), roots, and inflorescence were recorded.

Cannabinoid content
The inflorescence samples of hemp were firstly separated into leaves and flowers, dried at 70°C in the oven (50 min), and then ground to pass a sieve (≤0.425 mm). The contents of cannabidiol acid (CBDA), CBD, and THC were determined with an approach as reported previously , the chromatographic conditions include Chromatographic column: Water Xbrige (250 mm × 4.6 mm i. d., 5 μm); Mobile phase: 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B; Flow rate: 0.8 mL.min −1 ; Column temperature: 30°C; Sample quantity: 10 μL; Detection wavelength: 220 nm. The conversion formula of CBDA and CBD is as follows:

Statistical analysis
The data were analyzed via one-way analysis of variance (ANOVA) with SPSS 26.0 (IBM, Armonk, NY, USA) and GraphPad Prism 7.0 software. The Pearson correlation analysis and graph were carried out with the R Project (Package of "corrplot"), where the blue circle and red circle refer to negative correlation and positive correlation, respectively; the larger circles mean stronger correlations; *, ** The number after N, P, and K in the name of the treatment refers to the concentration of N, P, and K; the number in the parentheses refers to the concentration of each nutrient; the number of 1, 2 and 3 beside the parentheses were the order of Orthogonal test sequence.
and *** refer to significance at 0.05, 0.01, and 0.001 level, respectively. The figures were made and modified using Originpro 2019b (OriginLab, Massachusetts, USA) and Adobe Illustrator CS6. A 95% confidence level was used in this study (P < .05).

The effects of NPK on the growth of industrial hemp
The NPK rates significantly affected the morphological and dry weight of hemp ( Figure 1). A7 (N8P1K5) could considerably enhance the plant height and stem diameter growth of industrial hemp among all treatments (Figure 1a ~b). Compared with A3 (N4P2K5), the biomass of shoot (stem and leaves), root, and inflorescence were notably increased by A7 (N8P1K5), with increments of 208.7%, 113.2%, and 435.7%, respectively (Figure 1c ~e). The results showed that the NPK ratio of 8:1:5 could significantly increase the biomass of industrial hemp and promote CBD production indirectly.

The effects of NPK on cannabinoid of industrial hemp
The supplements of NPK markedly influenced the cannabinoid content including THC, and total CBD content in leaves and inflorescence (Figure 2). The total CBD content of leaves for A1 (N4P1K3) was the highest among all the treatments (Figure 2a), followed by the A7 (N8P1K5), which was increased by 65.63% and 56.77% compared to A3 (N4P2K5). Simultaneously, in inflorescence, the total CBD content displayed a similar response to both of them compared to A3 (N4P2K5), which increased by 41.81% and 35.84%, respectively (Figure 2c). Interestingly, the content of THC in all treatments was lower than 0.3% (Figure 2b, d). The results exhibited that the ratio of NPK at 8:1:5 or 4:1:3 was more suitable for promoting the CBD content of industrial hemp.

The effects of NPK on CBD yield
NPK rates markedly affected the CBD yield of industrial hemp (Figure 3). Relative to A3 (N4P2K5), the treatments of A7 (N8P1K5), A1 (N4P1K3), and A8 (N8P1.5K3) could radically enhance CBD yield by 7.0 times, 3.7 times, and 3.6 times, respectively. Hence, promoting the inflorescence biomass was one of the most useful access to improve CBD yield.

Correlations between different parameters
Pearson correlation between parameters obtained from both NPK and hemp were calculated (Figure 4). The results that the dry weight of inflorescence, shoot, and stem diameter had a strong positive correlation with N level, the CBDA and total CBD in the inflorescence were significantly and  Red circle and blue circle refer to positive correlation and negative correlation, respectively; the larger circles mean stronger correlations; and *,**,*** refer to significance at 0.05, 0.01, and 0.001 levels, respectively. positively related with the N level; while the P level had a negative correlation with all parameters of the hemp growth, especially for the cannabinoids including CBD or THC in the different tissue; for the K level, it had a negligible effect on CBD yield.

A suitable NPK enhances the growth of hemp
Previous studies have shown that N can dramatically determine hemp growth and flowering, the elevation of N nutrition is conducive to increasing the plant height, biomass, or seed yield of hemp (Curci et al. 2017;Saloner and Bernstein 2020). As we described above, the treatment of A7 (N8P1K5) could significantly enhance the growth and biomass of hemp (variety, Xinma No.1) (Figure 1), as well as the CBD yield (Figure 3 ~4), while this was not in line with the observation for Campiglia et al., who argued the higher N levels can be more inclined to promote the stem development instead of the growth of inflorescence (Campiglia, Radicetti, and Mancinelli 2017). Differing from N, increasing P nutrition could well improve the plant height, but not significantly enhance the biomass (Aubin et al. 2015), excessive P may reduce the biomass of the crop and cause a potential source of environmental pollution (Bevan, Jones, and Zheng 2021;Chen et al. 2017), a possible explanation in our result is that the P absorption could be selected since the P nutrition was significantly negatively correlated with almost all parameters ( Figure 4).
K is mainly concentrated in the stem of hemp, Saloner et al. showed that when K was supplied up to 175 mg L −1 , the biomass of leaves, roots, and the stem diameter of the plants could be increased, but when it was up to 240 mg L −1 , the negative effects would occur (Saloner and Bernstein 2020). Thus, considering the effects of specific nutrients on biochemical, physiological, and molecular processes, more works need to be done to adjust the detailed fertilization scheme at the different stages of plant growth and development.

A suitable NPK enhances the CBD content and yield
The effects of nutrient supplements on cannabinoid content depend on the organ of the plant, the THC content is higher for the upper parts of the hemp (Bernstein, Gorelick, and Koch 2019), and the CBD presented a similar trend. Our data presented that A7 (N8P1K5) had positive effects on the synthesis and production of cannabinoids THC and CBD in the inflorescence and leaves of hemp ( Figure 2), thus, we speculate that high N promotes the accumulation of CBD, which stimulates CBD yield formation under various nutrition conditions. Studies revealed that higher P levels could significantly increase the biomass and THC content of medical hemp (Bernstein, Gorelick, and Koch 2019;Gentner 1975, 1977;Rioba et al. 2015), which may be related to the varieties and cultivation conditions of hemp (Abot et al. 2013).
The synthesis of CBD and THC are controlled by the same gene locus, which is mainly synthesized and accumulated in the secretory sacs of the capitate-stalked glandular trichomes. The different NPK ratios caused varied cannabinoid content in specific tissues, this may be related to key enzymes in the fatty acid pathway (fatty acid biosynthesis) and the terpenoid pathway (DOXP/MEP pathway), the two biological pathways for cannabinoid synthesis (Morimoto et al. 1998). This study provides ideas for solving the shortage of CBD-targeted products in the source of biomedicine. Thereby, multi-omics analysis and synthetic biology approaches may be utilized to illustrate how NPK affects the synthesis of intermediate substances or key enzymes, as well as the cannabinoid concentrations in the cannabinoid biosynthesis pathway.

Conclusion
Through aeroponic experiment, the following conclusions can be drawn: (i) The rate of NPK:8:1:5 could increase dramatically the hemp (variety, Xinma No.1) growth, biomass, and accumulation of cannabinoids in hemp; (ii) the rates of NPK 8:1:5 or NPK 4:1:3 can significantly improve CBD yield by increasing CBD content in hemp as well as by increasing the biomass of inflorescence or leaves.