Difference between revisions of "Journal:A spectroscopic study to assess heavy metals absorption by a combined hemp-spirulina system from contaminated soil"
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==Materials and methods== | ==Materials and methods== | ||
===Materials=== | ===Materials=== | ||
3-(Trimethylsilyl)-2,2,3,3-tetradeutero-propionic acid sodium salt (TSP, CAS N. 24493-21-8, 99 %D, Armar Chemicals, Döttingen, Switzerland), hydrochloric acid (HCl, 37%, CAS N. 7647-01-0; ≥ 99.5%, Sigma-Aldrich, Milan, Italy), sodium oxalate (Na<sub>2</sub>C<sub>2</sub>O<sub>4</sub>, CAS N. 62-76-0; ≥99.5%, Sigma-Aldrich, Milan, Italy), sodium azide (NaN<sub>3</sub>, CAS N. 26628-22-8; ≥99.5%, Sigma-Aldrich, Milan, Italy), and deuterium oxide (D<sub>2</sub>O, CAS. N. 7789-20-0, 99.86 %D, Eurisotop, Saclay, France) were used for sample preparation. NMR tubes (Norell 509-UP 7) were provided by Norell, Landisville NJ, United States. The NMR samples were prepared using an automated system for liquid handling (SamplePro Tube, Bruker BioSpin). | |||
The soil used during cultivation was Plagron Lightmix (Plagron, Ospel, The Netherlands), which had pH 6-7, electric conductivity (E.C. 1:5) 310-470 µS•cm<sup>−1</sup> and (E.C. 1:1.5) 0.7-1.1 µS•cm<sup>−1</sup>, NPK (12-14-24) 1.5 kg•m−3, total N 180 g•m<sup>−3</sup> (105:75, NO<sub>3</sub>:NH<sub>4</sub>), P<sub>2</sub>O<sub>5</sub> 210 g•m<sup>−3</sup>, K<sub>2</sub>O 360 g•m<sup>−3</sup>, dry matter 37% (of which 82% organic matter), and water retention of 6 mL•g<sup>−1</sup> dry matter. | |||
The soil was contaminated by Cd(NO<sub>3</sub>)<sub>2</sub>‧4H<sub>2</sub>O (CAS N. 10022-68-1, Carlo Erba Reagents, Milan, Italy), K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub> (CAS N. 7778-50-9, Carlo Erba Reagents, Milan, Italy), Cu(SO<sub>4</sub>)‧5H<sub>2</sub>O (CAS N. 7758-99-8, Prolabo, Paris, France), Pb(CH<sub>3</sub>COO)<sub>2</sub> (CAS N. 301-04-2, Carlo Erba Reagents, Milan, Italy), Ni(NO<sub>3</sub>)<sub>2</sub>‧6H<sub>2</sub>O (CAS N. 13478-00-7, Carlo Erba Reagents, Milan, Italy), Zn(CH<sub>3</sub>COO)<sub>2</sub>‧2H<sub>2</sub>O (CAS N. 5970-45-6, Carlo Erba Reagents, Milan, Italy). | |||
Hydrochloric acid (HCl 37%, Merck, Darmstadt, Germany), nitric acid (HNO<sub>3</sub> 65%, Merck, Darmstadt, Germany), double-distilled water (DDW ≥ 99.5%), standard multi-elemental reference solution (Ag, Al, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sr, Tl, V, Zn [10 mg/l]) were used for heavy metals analysis by ICP-AES. | |||
===Algal strain and growth conditions=== | |||
An ''Arthrospira platensis'' strain was cultivated by ApuliaKundi S.r.l. in open ponds (3,000 L per pond) under controlled greenhouse conditions with natural light at a temperature ranging from 22 to 28 °C. | |||
The production cycle was monitored by turbidity measurements through a Secchi disk (200 mm diameter, Scubla, Remanzacco (UD), Italy). Once the disk was lowered into the algae suspension at 5÷6 cm and could not be seen anymore, the algae were collected. Overall, the production cycle lasted approximately three days. The collected algal biomass was filtered (40 μm filter), extruded, and cold dried at a temperature value lower than 38 °C. The study was conducted using the dried biomass in powder form, commonly named "spirulina." | |||
===Cultivation of ''Cannabis sativa'' L. (''cv.'' Kompolti)=== | |||
The cultivation of hemp by Enjoy Farm Soc. Coop. (Bitetto, Bari, Italy) took place indoors with controlled light and temperature conditions using a vegetative photoperiod of 18/6 h of light/dark for the first nine weeks, and a flowering photoperiod of 12/12 h of light/dark until harvest. (De Backer et al., 2012) Germination occurred 10 days after the seed sowing. After 50 days from germination, six plants were transplanted into 15 L pots filled with four kilograms of 75-days-old contaminated soil, in addition to two control plants transplanted into pots filled with four kilograms of uncontaminated soil. The pre-contaminated soil was prepared upon controlled addition into the soil of six different heavy metals, namely cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), nickel (Ni), and zinc (Zn). The choice of heavy metals and quantities used for contamination was based on the typical content of metals detected in different sites of the Apulia region of southern Italy, as provided by Agenzia Regionale per la Prevenzione e la Protezione dell'Ambiente (ARPA Puglia) and Acquedotto Pugliese S.p.A. Specifically, the pre-contaminated soil contained 16, 527, 200, 434, 357, and 448 mg•kg<sup>−1</sup> of Cd(NO<sub>3</sub>)<sub>2</sub>4H<sub>2</sub>O, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, Cu(SO<sub>4</sub>)<sub>2</sub>‧5H<sub>2</sub>O, Pb(CH<sub>3</sub>COO)<sub>2</sub>, Ni(NO<sub>3</sub>)<sub>2</sub>‧6H<sub>2</sub>O, and Zn(CH<sub>3</sub>COO)<sub>2</sub>‧2H<sub>2</sub>O, respectively. Commercial uncontaminated soil (24 kilograms) was treated with 2.0 L of such salts aqueous solutions through four subsequent additions. The resulted mixture was stirred in a concrete mixer for three hours. Finally, the obtained contaminated soil was distributed into six pots (15 L each) and stored in the greenhouse under controlled conditions for 75 days. | |||
Moreover, four out of the six ''Cannabis sativa'' L. plants grown in pre-contaminated soil received irrigation water (0.50 L of water × 56 times) containing spirulina (added in powder form). As a proof of concept, two different concentrations were tested: 1.0 and 0.50 g•L<sup>−1</sup>. Overall, each experimental condition was tested in duplicate, involving a total of eight hemp plants (Table 1). | |||
{| | |||
| STYLE="vertical-align:top;"| | |||
{| class="wikitable" border="1" cellpadding="5" cellspacing="0" width="100%" | |||
|- | |||
| style="background-color:white; padding-left:10px; padding-right:10px;" colspan="3"|'''Table 1.''' Description of the experimental conditions indicating the corresponding strategies of control, as well as pre-contamination soil in presence and absence of spirulina. | |||
|- | |||
! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Experimental condition | |||
! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Description | |||
! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Plant ID | |||
|- | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|'''Control''' | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|Two plants are grown on uncontaminated soil as a reference | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|Ctrl | |||
|- | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|'''Pre-contaminated soil''' | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|Two plants are grown on pre-contaminated soil | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|C | |||
|- | |||
| style="background-color:white; padding-left:10px; padding-right:10px;" rowspan="2"|'''Pre-contaminated soil + spirulina''' | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|Two plants are grown on pre-contaminated soil with the addition of spirulina 1.0 g•L<sup>−1</sup> | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|CS1 | |||
|- | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|Two plants are grown on pre-contaminated soil with the addition of spirulina 0.50 g•L<sup>−1</sup> | |||
| style="background-color:white; padding-left:10px; padding-right:10px;"|CS0.5 | |||
|- | |||
|} | |||
|} | |||
Plants were constantly monitored until the harvest (over two months after the sowing) by recording three main morphometric parameters: (i) plant height (cm), i.e., height of stem from ground to apex; (ii) the number of leaf stages; and (iii) drum diameter (mm) (see ESI for the full list of the morphometric parameters, Table S1). The biomass (g) of stem and leaf samples was determined upon lyophilization; the sum of the biomass of the stem and leaf samples collected from the same plant was computed too and is herein referred to as “plant biomass.” The residual amount of heavy metals (mg•kg<sup>−1</sup>) contained in leaves, stems, and soil after the harvest was measured; for each plant, also the sum of the heavy metal content of leaves plus stems was calculated (see ESI for further details, Table S2). The shoots of plants were collected and transferred in refrigerated packaging with dry ice from the greenhouse to [[Laboratory|laboratories]] for further analyses. Samples of soil from each pot were collected after harvesting and kept in plastic bags at room temperature until analysis. Leaves and flowers were separated from stems, and both portions of the plant were firstly freeze-dried at –50 °C and 0.180 mbar for 72 hours in a lyophilizer (Christ Alpha 1–4 LSC, Martin-Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Then, the dried samples were mechanically ground in a blender, sieved through a mesh with a pore size of 0.5 mm, and stored at –20 °C until analysis. | |||
===Sample preparation and NMR analysis=== | |||
Revision as of 18:52, 11 September 2022
Full article title | A spectroscopic study to assess heavy metals absorption by a combined hemp-spirulina system from contaminated soil |
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Journal | Environmental Advances |
Author(s) | Musio, Biagia; Ahmed, Elhussein M.F.M.H.; Antonicelli, Marica; Chiapperini, Danila; Dursi, Onorfrio; Grieco, Flavia; Latronico, Mario; Mastrorilli, Piero; Ragone, Rosa; Settanni, Raffaele; Triggiani, Maurizio; Gallo, Vito |
Author affiliation(s) | Polytechnic University of Bari, Innovative Solutions S.r.l., International Centre for Advanced Mediterranean Agronomic Studies of Bari, ApuliaKundi S.r.l. |
Primary contact | Email: vito dot gallo at poliba dot it |
Year published | 2022 |
Volume and issue | 7 |
Article # | 100144 |
DOI | 10.1016/j.envadv.2021.100144 |
ISSN | 2666-7657 |
Distribution license | Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International |
Website | https://www.sciencedirect.com/science/article/pii/S2666765721001150 |
Download | https://www.sciencedirect.com/science/article/pii/S2666765721001150/pdfft (PDF) |
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This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed. |
Abstract
The efficiency of hemp (Cannabis sativa L.) in remediating sites contaminated with heavy metals has received great attention in recent years. The main advantage of this technology relies on its inherent sustainability, with a potential re-utilization of the significant amount of produced biomass, which acts as a valuable flow resource. In this study, a combined system consisting of Cannabis sativa L. (hemp) and the blue-green alga Arthrospira platensis (spirulina) was tested to clean up soils contaminated with cadmium, chromium, copper, nickel, lead, and zinc. The application of non-targeted nuclear magnetic resonance spectroscopy (NMR) methods combined with inductively coupled plasma atomic emission spectroscopy (ICP-AES) quantification provided an efficient strategy for detecting residual heavy metals within plant tissues and soil. Importantly, non-targeted metabolomic analysis helped to reveal the relationships between metabolites distribution in hemp tissues and the sequestered metals. It was demonstrated that hemp accumulates copper, chromium, nickel, and zinc preferentially in the leaves, while lead is distributed mainly in the stems of the plant. Moreover, it was found that, at higher concentrations, spirulina acts as a growth promoter, contributing to an increase in the final generated biomass. Results reported in this work indicate that the hemp-spirulina system represents a suitable tool for remediation of metal contaminated soils by modulating biomass production and metals uptake.
Keywords: non-targeted nuclear magnetic resonance, phytoremediation, phycoremediation, Arthrospira platensis, Cannabis sativa L., metal quantification
Graphical abstract:
Introduction
Dispersion of heavy metals in soils is an age-old problem deriving from both natural and anthropic sources. (Awa and Hadibarata, 2020) Among the anthropic contribution to soil contamination by metals, land application of treated wastewater, sewage sludge, fertilizers, and industrial activities are major concerns. (Vareda et al., 2019) Unbalanced amounts of heavy metals may cause perturbation of soil parameters with consequent toxic effects on plants, in the nearby water supplies, and, ultimately, in the whole food chain. (Arora et al., 2008; Kumar et al., 2019; Manzoor et al., 2018) Typically, elements such as copper (Cu), nickel (Ni), zinc (Zn), and chromium (Cr) are biologically essential for plant growth but become toxic for animals and plants when their concentrations exceed certain threshold levels. (Edelstein and Ben-Hur, 2018; Rizvi et al., 2020; Tiwari and Lata, 2018) Other heavy metals often found in contaminated soils, such as cadmium (Cd) and lead (Pb) are not essential for plants growth, and many studies have associated their presence with neurological and endocrinological toxicity for humans, along with carcinogenic effects. (Ali and Khan, 2019; Pratush et al., 2018; Rehman et al., 2018)
Since heavy metals are not biodegradable, they tend to accumulate in the environment, becoming a high risk for biota over several years after their introduction in an ecosystem. (Olsson et al., 1998; Tchounwou et al., 2012; Zwolak et al., 2019) The search for new solutions that can remediate soil contaminated by heavy metals is a critical prerequisite for the sustainable development of agriculture (Edelstein and Ben-Hur, 2018; ; Vardhan et al., 2019; Wuana and Okieimen, 2011), thus representing a topic of paramount importance. The most consolidated strategies to remediate such contaminated soils include physical and chemical approaches like isolation, through capping and subsurface barriers; immobilization, by solidification/stabilization, vitrification, and chemical treatment; physical separation; and extraction, by soil washing, pyrometallurgical extraction, in situ soil flushing, and electrokinetic treatment. (Dhaliwal et al., 2020; Gong et al., 2018; Gusiatin et al., 2020; Qin et al., 2020) However, alternative approaches are gaining greater attention as they combine cost-effectiveness, sustainability, low toxicity, and mobility decrease. They include bioaccumulation, phytoremediation (e.g., phytoextraction, phytostabilization, and rhizofiltration), bioleaching, and other biochemical processes in which living organisms such as plants or microbes are used to clean contaminants from an area.
In particular, phytoremediation is attracting the attention of the scientific community, since it has been demonstrated to be a cost-effective solution for the remediation of contaminated sites, and, in the meantime, to be a feasible method for bio-fixation of CO2, resulting in highly sustainable technology. (Awa and Hadibarata, 2020) The ability to absorb heavy metals generally depends on the biomass produced, as well as on the ability of the plant to accumulate and translocate heavy metals in its biomass. (Eid and Shaltout, 2016; Hernández-Allica et al., 2008; Pachura et al., 2016) According to recent scientific literature, a good candidate for phytoremediation of soil contaminated by heavy metals is the hemp plant. (Ahmad et al., 2016; Morin-Crini et al., 2019; Zielonka et al., 2020) Kompolti, also known as hemp, the non-psychoactive variety of Cannabis sativa L., is an annual dioecious high-yielding industrial crop, and it is mainly grown for its fibers and seeds, generally being used for textiles, clothing, insulation, biodegradable plastics, food, animal feed, and biofuel production. (Adesina et al., 2020; Crini et al., 2020; Schluttenhofer and Yuan, 2017; Vasantha Rupasinghe et al., 2020) Hemp possesses some characteristics that make it quite suitable for phytoremediation, such as high biomass, long roots, and a favorably short industrial life cycle of 180 days. Importantly, hemp demonstrates a strong capability to sequester heavy metals like cadmium, zinc, lead, nickel, copper, and chromium when they are present in contaminated soil and water. (Citterio et al., 2003; Galić et al., 2019; Piotrowska-Cyplik and Czarnecki, 2003; Zielonka et al., 2020)
Another attractive approach for the remediation of contaminated sites is the application of bioleaching technology, which uses direct metabolism or by-products of microbial processes to uptake heavy metals adsorbed onto the soil surface and to transform them so that the elements can be extracted when water is filtered through. Bioleaching has several advantages over conventional physical and chemical strategies, such as low cost, environmental sustainability, few hazardous characteristics of waste/sludge, low energy demand, and absence of toxic chemicals. (Bosecker, 1997; Drobíková et al., 2015; Mishra et al., 2005; Okoh et al., 2018; Rawlings, 2002; Sun et al., 2021)
Additionally, phycoremediation, which involves eukariotic algae and cyanobacteria in remediation processes, has been extensively applied to the treatment of wastewater. (Awa and Hadibarata, 2020) However, its application to the remediation of sediments and soils contaminated by heavy metals is less documented. Among the cyanobacteria, Arthrospira platensis possesses excellent chelating properties both towards heavy metals present in humans and towards those present in soil, water, and sludge. (Balaji et al., 2014; Bhattacharya, 2020; Konig-péter et al., 2015; Nalimova et al., 2005; Zinicovscaia et al., 2019, 2016) The dried biomass of Arthrospira platensis is commonly known as "spirulina," and it finds many applications in agriculture as a plant growth promoter, enhancing growth, increasing yield, and speeding up seed germination. (Tripathi et al., 2008; Wuang et al., 2016) Recently, the employment of this blue-green alga to uptake heavy metals in contaminated sites has been explored. (Cepoi et al., 2020; Wuang et al., 2016) The presence of a chloroplast-type ferredoxin in the active center has been reported as responsible for the chelating capability of spirulina (Tsukihara et al., 1978), whereby its efficiency is affected by many physical and chemical factors such as initial metal concentration, dosage, adsorption time, temperature, and pH. (Şeker et al., 2008)
The present study aims at both exploring the ability of the unreported combined use of hemp and spirulina to uptake six selected heavy metals (Cd, Ni, Cr, Pb, Cu, Zn) from artificially contaminated soil and investigating, under controlled plant growing conditions, their distribution into the plant tissues. Specifically, hemp was chosen as the main agent for biological remediation, and spirulina was added as an enhancer of both the plant growth and the translocation of heavy metals in the hemp. The application of a non-targeted nuclear magnetic resonance spectroscopy (NMR) approach combined with an estimation of the residual metals by inductively coupled plasma atomic emission spectroscopy (ICP-AES) into the cultivation soil and within the different tissues of the plant was applied in view of gathering useful information on the efficiency of the integrated hemp-spirulina system. Obtaining this information is crucial for the potential re-utilization of the hemp plant or shoots of it, after the phytoremediation stage, for alternative usages, like production of bio-materials for the textile, construction, and bio-fuel industries.
Materials and methods
Materials
3-(Trimethylsilyl)-2,2,3,3-tetradeutero-propionic acid sodium salt (TSP, CAS N. 24493-21-8, 99 %D, Armar Chemicals, Döttingen, Switzerland), hydrochloric acid (HCl, 37%, CAS N. 7647-01-0; ≥ 99.5%, Sigma-Aldrich, Milan, Italy), sodium oxalate (Na2C2O4, CAS N. 62-76-0; ≥99.5%, Sigma-Aldrich, Milan, Italy), sodium azide (NaN3, CAS N. 26628-22-8; ≥99.5%, Sigma-Aldrich, Milan, Italy), and deuterium oxide (D2O, CAS. N. 7789-20-0, 99.86 %D, Eurisotop, Saclay, France) were used for sample preparation. NMR tubes (Norell 509-UP 7) were provided by Norell, Landisville NJ, United States. The NMR samples were prepared using an automated system for liquid handling (SamplePro Tube, Bruker BioSpin).
The soil used during cultivation was Plagron Lightmix (Plagron, Ospel, The Netherlands), which had pH 6-7, electric conductivity (E.C. 1:5) 310-470 µS•cm−1 and (E.C. 1:1.5) 0.7-1.1 µS•cm−1, NPK (12-14-24) 1.5 kg•m−3, total N 180 g•m−3 (105:75, NO3:NH4), P2O5 210 g•m−3, K2O 360 g•m−3, dry matter 37% (of which 82% organic matter), and water retention of 6 mL•g−1 dry matter.
The soil was contaminated by Cd(NO3)2‧4H2O (CAS N. 10022-68-1, Carlo Erba Reagents, Milan, Italy), K2Cr2O7 (CAS N. 7778-50-9, Carlo Erba Reagents, Milan, Italy), Cu(SO4)‧5H2O (CAS N. 7758-99-8, Prolabo, Paris, France), Pb(CH3COO)2 (CAS N. 301-04-2, Carlo Erba Reagents, Milan, Italy), Ni(NO3)2‧6H2O (CAS N. 13478-00-7, Carlo Erba Reagents, Milan, Italy), Zn(CH3COO)2‧2H2O (CAS N. 5970-45-6, Carlo Erba Reagents, Milan, Italy).
Hydrochloric acid (HCl 37%, Merck, Darmstadt, Germany), nitric acid (HNO3 65%, Merck, Darmstadt, Germany), double-distilled water (DDW ≥ 99.5%), standard multi-elemental reference solution (Ag, Al, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sr, Tl, V, Zn [10 mg/l]) were used for heavy metals analysis by ICP-AES.
Algal strain and growth conditions
An Arthrospira platensis strain was cultivated by ApuliaKundi S.r.l. in open ponds (3,000 L per pond) under controlled greenhouse conditions with natural light at a temperature ranging from 22 to 28 °C.
The production cycle was monitored by turbidity measurements through a Secchi disk (200 mm diameter, Scubla, Remanzacco (UD), Italy). Once the disk was lowered into the algae suspension at 5÷6 cm and could not be seen anymore, the algae were collected. Overall, the production cycle lasted approximately three days. The collected algal biomass was filtered (40 μm filter), extruded, and cold dried at a temperature value lower than 38 °C. The study was conducted using the dried biomass in powder form, commonly named "spirulina."
Cultivation of Cannabis sativa L. (cv. Kompolti)
The cultivation of hemp by Enjoy Farm Soc. Coop. (Bitetto, Bari, Italy) took place indoors with controlled light and temperature conditions using a vegetative photoperiod of 18/6 h of light/dark for the first nine weeks, and a flowering photoperiod of 12/12 h of light/dark until harvest. (De Backer et al., 2012) Germination occurred 10 days after the seed sowing. After 50 days from germination, six plants were transplanted into 15 L pots filled with four kilograms of 75-days-old contaminated soil, in addition to two control plants transplanted into pots filled with four kilograms of uncontaminated soil. The pre-contaminated soil was prepared upon controlled addition into the soil of six different heavy metals, namely cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), nickel (Ni), and zinc (Zn). The choice of heavy metals and quantities used for contamination was based on the typical content of metals detected in different sites of the Apulia region of southern Italy, as provided by Agenzia Regionale per la Prevenzione e la Protezione dell'Ambiente (ARPA Puglia) and Acquedotto Pugliese S.p.A. Specifically, the pre-contaminated soil contained 16, 527, 200, 434, 357, and 448 mg•kg−1 of Cd(NO3)24H2O, K2Cr2O7, Cu(SO4)2‧5H2O, Pb(CH3COO)2, Ni(NO3)2‧6H2O, and Zn(CH3COO)2‧2H2O, respectively. Commercial uncontaminated soil (24 kilograms) was treated with 2.0 L of such salts aqueous solutions through four subsequent additions. The resulted mixture was stirred in a concrete mixer for three hours. Finally, the obtained contaminated soil was distributed into six pots (15 L each) and stored in the greenhouse under controlled conditions for 75 days.
Moreover, four out of the six Cannabis sativa L. plants grown in pre-contaminated soil received irrigation water (0.50 L of water × 56 times) containing spirulina (added in powder form). As a proof of concept, two different concentrations were tested: 1.0 and 0.50 g•L−1. Overall, each experimental condition was tested in duplicate, involving a total of eight hemp plants (Table 1).
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Plants were constantly monitored until the harvest (over two months after the sowing) by recording three main morphometric parameters: (i) plant height (cm), i.e., height of stem from ground to apex; (ii) the number of leaf stages; and (iii) drum diameter (mm) (see ESI for the full list of the morphometric parameters, Table S1). The biomass (g) of stem and leaf samples was determined upon lyophilization; the sum of the biomass of the stem and leaf samples collected from the same plant was computed too and is herein referred to as “plant biomass.” The residual amount of heavy metals (mg•kg−1) contained in leaves, stems, and soil after the harvest was measured; for each plant, also the sum of the heavy metal content of leaves plus stems was calculated (see ESI for further details, Table S2). The shoots of plants were collected and transferred in refrigerated packaging with dry ice from the greenhouse to laboratories for further analyses. Samples of soil from each pot were collected after harvesting and kept in plastic bags at room temperature until analysis. Leaves and flowers were separated from stems, and both portions of the plant were firstly freeze-dried at –50 °C and 0.180 mbar for 72 hours in a lyophilizer (Christ Alpha 1–4 LSC, Martin-Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Then, the dried samples were mechanically ground in a blender, sieved through a mesh with a pore size of 0.5 mm, and stored at –20 °C until analysis.
Sample preparation and NMR analysis
References
Notes
This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added.