Optimizing hydroponic culture media and NO3−/NH4+ ratio for improving essential oil compositions of purple coneflower (Echinacea purpurea L.)

Medicinal plants represent a valuable commodity due to beneficial effects of their natural products on human health, prompting a need for finding a way to optimize/increase their production. In this study, a novel growing media with various perlite particle size and its mixture with peat moss was tested for hydroponic-based production of Echinacea purpurea medicinal plant under greenhouse conditions. The plant growth parameters such as plant height, total fresh leave weight, fresh root weight, total biomass, total chlorophyll, leaf area, and essential oil compositions were assessed. Perlite particle size in the growing media was varied from very coarse (more than 2 mm) to very fine (less than 0.5 mm), and the ratio between perlite and peat moss varied from 50:50 v/v to 30:70 v/v. In addition, two nitrate (NO3−) to ammonium (NH4+) ratios (90:10 and 70:30) were tested for each growing media. The medium containing very fine-grade perlite and 50:50 v/v perlite to peat moss ratio was found to be most optimal and beneficial for E. purpurea performance, resulting in maximal plant height, fresh and dry weight, leaf surface area, and chlorophyll content. It was also found that an increase in NO3−/NH4+ ratio caused a significant increase in plant growth parameters and increase the plant essential oil content. The major terpene hydrocarbons found in extract of E. purpurea with the best growth parameters were germacrene D (51%), myrcene (15%), α-pinene (12%), β-caryophyllene (11%), and 1-Pentadecene (4.4%), respectively. The percentages of these terpene hydrocarbons were increased by increasing of NO3−/NH4+ ratio. It can be concluded that decreasing the perlite particle size and increasing the NO3−/NH4+ ratio increased the plant growth parameters and essential oil compositions in E. purpurea.

www.nature.com/scientificreports/ While the industrial application of E. purpurea essential oils is well established, several factors such as weather changes, plant growth stage 14 , and method of cultivation may influence both the composition and production of E. purpurea essential oil 15 . The open field cultivation of E. purpurea has some significant limitations such as crop inconsistency, seed dormancy 16 , water stress regims 17 , microbes, heavy metal ions and other pollutants 2 and loss of wild germplasm, that affect the different chemical composition of the plant extract. The above limitations have prompted a shift towards plant production under greenhouse conditions, especially in hydroponic (or soilless) culture systems 18 . Growing in a greenhouse also offer an additional advantages of more effective control of plant nutrition 19,20 .
Different hydroponic cultivation methods, such as artificial substrate media, water culture, and nutrient film techniques have been reported for E. purpurea cultivation 18,21 . However, using artificial substrates in the hydroponic cultivation system reduces the cost of establishing advanced hydroponic cultivation systems and also enables the farmer to make a practical use of it by using commonly raw materials such as cocopeat, sand, and vermiculite as an initial plant growing media 22 . Nevertheless, different inorganic products such as peat moss, perlite, mixed materials, etc. are fully or partially used instead of initial substrates due to their useful physical properties. The particle size of substrates is a critical factor in air and water-holding capacity, root distribution, and plant growth, which are different based on their origin and preparation conditions. A high volume of roots can concentrate at the top portion of the container includes low aeration and high water-holding capacity 22 .
In addition to the importance of substrates properties in the hydroponic culture system, attention to the chemical composition of nutrient solution is important 22 . In terms of chemical composition of nutrient solutions, two major inorganic forms of nitrogen (N), the NH 4 + and the NO 3 − , can differentially impact the various plant properties, based on the plant species 18 . Although the assimilation and metabolism of NH 4 + form require less energy than that of NO 3 − in plants, the majority of plant species grow better on NO 3 − since NH 4 + is toxic for plants and a few species grow well if NH 4 + is the only source of N 4 . The plant species and environmental conditions are two critical factors that affect the optimum NO 3 − /NH 4 + ratio 23 . It has been reported that different N application rates could affect the essential oil compositions of peppermints (Mentha piperita L.) 24 . Previous researches also demonstrated that the inorganic N application rate and sources could affect the essential oil content of sweet basil (Ocimum basilicum L.) and forage maize (Zea mays L.) 25 .
Although many researches have been performed on hydroponic culture of E. purpurea, but the use of culture media with different perlite particle sizes, different NO 3 − /NH 4 + ratios, and their effects on essential oil compositions of E. purpurea has been assessed for the first time in this study. So the main goal of this study was to investigate the growth parameters and essential oil compositions of E. purpurea growing in new hydroponic culture media with various perlite particle sizes and different NO 3 − /NH 4 + ratios.

Materials and methods
Growth conditions. The experiment was performed in a commercial greenhouse at Urmia University, West Azerbaijan, Iran. The air temperature was 22/18 °C (day/night) and the humidity ranged from 70 to 80%. The maximum photosynthetic photon flux density (PPFD) fluctuated from 550 to 750 μmol m −2 s −1 inside the greenhouse. The E. purpurea seeds were purchased from Iranian private joint-stock company, Pakan Bazr Esfahan (www. Pakanbazr.com). The seeds were sowed in plastic cups filled with a mixture of perlite and peat moss substrates as a medium to initiate germination. Irrigation was performed based on greenhouse conditions regularly. Seedlings (with four real leaves) were translocated to experimental plastic pots (2.5 L) containing a different ratios of perlite and peat moss as artificial substrates (100% perlite, 100% peat moss, 50% (v) perlite + 50% (v) peat moss, 70% (v) perlite + 30% (v) peat moss) with various perlite particle size containing less than 0.5 mm, 0.5-1 mm, 1-1.5 mm, 1.5-2 mm, and more than 2 mm. Chemical concentrations of nutrient solution are shown in Table 1. The pH and electrical conductivity (EC) of the nutrient solution were maintained between 5.7 to 6.2 and 1.0 to 1.5 dS m −1 , respectively. According to the stage of the plant growth, 0.5 to 3.5 L day −1 was used in fertigation system 18 . www.nature.com/scientificreports/ Sample preparation. Plants were harvested at the end of the flowering stage (eight months). The plants were divided into roots, stems, flower heads, and lower and upper leaves after washing with tap water. Root, flower heads, and leaves samples were dried at 25 ± 1 °C, ground into a fine powder and collected for further analyses 6 .
Plant growth parameters. The main growth parameters such as plant height (cm), fresh root weight (g plant −1 ), total fresh leave weight (g plant −1 ), total biomass (g plant −1 ), and leaf area (cm 2 ) were determined for each plant at the matured stage. The leaf area was measured by using leaf area meter. Chlorophylls a and b were determined using 0.5 g of dry sample, which was homogenized with 10 mL acetone. Homogenized samples were centrifuged at 10,000×g for 15 min at 4 °C 2 . The supernatant was separated, and the absorbance spectra were measured at 400-700 nm. The total chlorophyll was calculated at 645 nm and 663 nm respectively. So that 26 : where C is the total chlorophyll contents in mg/L of acetone extract, A 645 , and A 663 are the absorption of the extract at 645 and 663 nm.
Extraction of essential oils. The E. purpurea plants which shown the best morphological properties (maximum height, dry and wet weight of leaves and roots, and leaf area) were selected for analysis of essential oil. Distilled water was added to 120 g powder samples (root, leaves, and flower head) at a 1:10 (g mL −1 ) ratio. The essential oil was extract based on the distillation procedure using a commercial Clevenger apparatus 27 .
Analysis of essential oil. The essential oil analysis was performed using gas chromatography (GC) with  Table 2 and Figs. 1 and 2. Overall, plants grown in the 50% perlite + 50% peat moss medium with perlite particle size less than 0.5 mm and 90:10 NO 3 − /NH 4 + ratio had the highest height (mean 105 cm) (Fig. 1), fresh leave weight (mean 30 g plant −1 ), fresh root weight (mean 65 g plant −1 ) (Fig. 2), total biomass (mean 96 g plant −1 ), and leaf area (mean 60 cm 2 ). Decreasing perlite percentage of culture media and perlite particle size improved all the morphological properties (Table 2). There were significant differences in the plant morphological properties at different NO 3 − /NH 4 + ratios. Increasing NO 3 − proportion in the N nutrition of E. purpurea caused to increase in plant height and root weight considerably (Table 2).
Essential oil analysis. The flower head, leaves, and root essential oil compositions of E. purpurea grown at the 50% perlite + 50% peat moss medium with perlite particle size less than 0. 5  www.nature.com/scientificreports/  www.nature.com/scientificreports/ The essential oils were separated into 51 components, 38 of them were identified, comprising 92.8% of the total essential oil yield (Tables 3 and 4). The content and composition of the essential oil exhibited a variable pattern at different plant organs at different NO 3 − /NH 4 + ratios (Tables 3 and 4). The most abundant terpenes including, germacrene D, myrcene, α-Pinene, β-caryophyllene, and 1-pentadecene were found in chemical composition of E. purpurea extract by previous researchers. Comparing of the results in present study with other researches shows the noticeable increase in essential oil composition by using novel growing media and nutrition pattern (Table 5), which is related to improve physical properties of growing media (50% perlite + 50% peat moss medium with perlite particle size less than 0.5 mm and 90:10 NO 3 − /NH 4 + ratio).

Discussion
Based on open hydroponic cultivation system in the present experiment, decreasing perlite particle size, increased the retention time of nutrient solution in the culture media. Increasing nutrient accessibility for plant roots by increasing retention time improves nutrient uptake and plant growth. However, the pure perlite culture system (100% perlite, < 0.5 mm) has a very low air-filled porosity (AFP) of 33% and water holding capacity (WHC) of 56% in comparison with other fine-perlite culture media (Table 6). Accordingly, the lowest growth parameters were obtained in pure perlite medium (Table 2), which can be attributed to the rapid withdrawal of nutrient solution from the culture medium and the inability of the medium to maintain the nutrient solution. Due to the high porosity of peat mass and nutrient solution retention capability, an increase of the plant morphological parameters is expected in the presence of peat moss in various cultural media ( Table 2). The noticeable increase in chlorophyll content by reducing perlite particle size implies the significant effect of culture media on photosynthesizing pigments ( Table 2). It has been reported that the application of N fertilizers in the fine perlite culture media increased N content of the plants, thereby increasing their chlorophyll content, subsequently, and their ability to absorb sunlight and produce photosynthates, which resulted in their higher leaf area, and growth and yield 18,28 . The essential oil was characterized by a higher percentage of terpene hydrocarbons, especially the monoterpenoids, which constituted 60 to 70% of the essential oil composition. The major terpene hydrocarbons found are α-pinene, myrcene, β-caryophyllene, 1-Pentadecene, and germacrene D. The percentages of these terpene hydrocarbons were higher in flower head than leave and root at both NO 3 − /NH 4 + ratios. The most abundant terpene found in the essential oil was germacrene D, which showed a remarkable rise from 1.5% in root to 51% in flower head and 0.95% in root to 47% in flower head at 90:10 and 70:30 NO 3 − /NH 4 + ratios, respectively. Variability was also obtained in the concentration of other compositions. The results (Tables 3 and 4) indicate that the various components of the essential oil of E. purpurea are specific to the plant organs, which influence their concentration.
The variations in the concentrations of various essential oil compositions at different NO 3 − /NH 4 + ratios (Tables 3 and 4) may be due to supply different amounts of NO 3 − to the plant. The presence of N as a key factor can affect the production of essential oils in aromatic plants 29 . Nitrogen is critical factor in biosynthesis pathway of essential oil in medicinal and aromatic plants 30 . Nitrogen increases photosynthetic efficiency and plays an important role in increasing the amount of essential oil by increasing the number and area of leave and providing a suitable condition for receiving sunlight energy and also participating in the structure of chlorophyll and www.nature.com/scientificreports/ enzymes involved in photosynthetic carbon metabolism 31 . Nitrogen is an essential nutrient in plants used to synthesize many organic compounds in plants such as nucleic acids, enzymes, proteins, and amino acids, which are necessary for essential oil biosynthesis pathway 32 . Besides, essential oils are terpenoids compounds whose constituent units (isonoids) such as isopentenyl pyrophosphate and dimethyl ally pyrophosphate are strongly formed into adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH), and due to the effect of N in the production of these compounds, the amount of essential oil increased 33 . Nitrogen increases the essential oil content of plants by increasing the dry weight (Nyalambisa et al. 2016). Comparing of the results in Tables 3 and 4 indicated that increase of NO 3 − concentration could increase the percentage of essential oil composition due to its effect on essential oil biosynthesis as demonstrated in previous researches 34 .
Germacrene D, myrcene, α-Pinene, β-Caryophyllene, and 1-Pentadecene were the major compositions of essential oil of E.purpurea grown in very fine-grade (< 0.5 mm) perlite with 50:50 v/v perlite to peat moss ratio (Tables 3 and 4). The compositions have a valuable beneficial effects in medicine and agriculture industries 7 .
Germacrene D is a natural hydrocarbon, belongs to sesquiterpenes, which is found in aromatic plants 27 . The hydrocarbon is a useful bioactive phytochemical compound in human health Maintains healthy blood pressure www.nature.com/scientificreports/ is one of the important roles of germacrene compounds in humans 8 . The antimicrobial properties of germacrene D were reported in previous researches 10 . Anti-inflammatory, antimicrobial, and antioxidant effects of germacrene D are also well known 8 . The anti-insect influence of germacrene D has been reported in previous studies 10 .
Myrcene is a terpene with anti-inflammatory and anti-depressant effects 14 . Regulating the efficiency of other terpenes and cannabinoids by increasing of myrcene is recognized previously 7 . Pinene has a several of potential benefits, including anti-inflammatory, antimicrobial, antitumor, antioxidant, and neuroprotective effects. It may also help counteract the short-term memory issues that many people experience. Beta-caryophyllene is also known for antioxidant and anti-inflammatory medicinal effects. It is especially useful to decrease pain and anxiety 35 . It was found that the mixture of peat moss into very fine-grade perlite (< 0.5 mm) at 50:50 v/v perlite to peat moss ratio had a significant increase in plant growth parameters, which increased by increasing of NO 3 − /NH 4 + ratio. The essential oil content was significantly highest in the 50:50 v/v perlite to peat moss ratio (perlite particle size less than 0.5 mm) than others. The major terpene hydrocarbons found in extract of E. purpurea with the best growth parameters were germacrene D, myrcene, α-pinene, β-caryophyllene, and 1-Pentadecene, respectively.   www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.