The variations in the metabolic composition of the aqueous extracts of leaves and stems were investigated by performing an OPLS-DA.A clear separation between the two classes of samples along the first two components was obtained and the analysis of the Splot allowed for the detection of the most significant x-variables and,thus,the metabolites contributing to such clustering.Specifically,the buckets having values of VIPpredictive >1 and values of p1 >|0.5| were analyzed more deeply.The spectral regions containing signals related to glucose,fructose,and galactose moieties contributed significantly to the clustering of the stems.Also,the buckets containing the signals assigned to the organic acids were characterized by significant values of VIP and p1.An analogous trend was observed for the buckets related to the signals assigned to γ-aminobutyric acid at 1.89–1.93 ppm and 3.01–3.05 ppm,and glutamine at 2.13–2.17 ppm.Interestingly,the clustering of the leaves was affected by some regions of the spectra containing very broad signals at 0.88,1.29,2.23,and 5.31 ppm ,presumably attributable to the lipids of the cell membranes and/or walls.The comparison of the 1D 1 H NOESY spectra of the leaves collected from control plants ,vigorous plants ,weak plants indicated that such broad signals were predominant in vigorous plants.Such broad signals were not observed in the aqueous extracts of stems.Also,the buckets containing the signals of ethanol at 1.17–1.25 ppm contributed relevantly to the grouping of the leaves towards the stems.These data could be explained by the fact that the leaves and,particularly the leaves sampled from vigorous green plants,have active photosynthetic processes and intact cell structures,grow racks system whereas stems of weakest plants begin to enrich with lignin and have less active photosynthesis.
The spectral regions containing these broad signals correlated positively with the calculated amounts of residual copper in the leaves.Under physiological conditions,Cu is found in two common forms,Cu preferentially binding sulfur-containing compounds having a thiol or a thioether group,and Cu ,that coordinates mainly with oxygen or imidazole nitrogen groups.The main functions of Cu are the transport of electrons in mitochondria and chloroplasts,the control of the cellular redox state ,and the remodeling of the cell wall,which is one of the major Cuaccumulation sites in hyper accumulating plants.Moreover,Cu leaf content is inversely associated with the intensity of the buckets relating to glucose and fructose and ethanol.In weakest plants,sucrose,the main soluble component of the phloem sap and translatable product of photosynthesis might be hydrolyzed into the two constituting monomers and fermented by yeast and/or bacteria producing ethanol.In a second task,the effect of the treatment with spirulina during the cultivation of hemp towards the remediation of contaminated soil was investigated.Thus,a deeper look was taken at the higher components of the OPLS model described in paragraph 3.1.Indeed,a good clustering of samples according to the treatment was visible in the t[2] vs t[5] scores plot.The second component and the fifth one explained together 11.75% of the x-variance predictive of the y-variance ,suggesting that the treatment with spirulina affected slightly the metabolic composition of the plant.Such evidence is relevant to make possible a re-utilization of the hemp,which does not change its metabolome during the phytoremediation stage.Specifically,the samples collected from plants grown in contaminated soil and the samples obtained from plants treated with a higher concentration of spirulina tended to cluster at values of t[2] >0.Conversely,the samples derived from plants treated with a lower concentration of the blue-green alga and the control ones were distributed preferentially at values of t[2] <0.A further piece of evidence was that samples C and the samples CS1 tended to separate into two clear groups along t[5].Analysis of the loading plot suggested that the uptake of all the metals,except for lead,contributed relevantly to the clustering of the plants treated with a higher dose of spirulina.Also,the average plant biomass was generally higher in contaminated plants compared to the uncontaminated ones.In addition,the plants that grew in contaminated soil and were irrigated with a higher dose of spirulina presented a higher value of biomass,confirming the activity of these cyanobacteria to promote plant growth.
The average plant contents of Ni ,Zn ,and Cd were greater in contaminated plants grown in soils added with spirulina than those grown without it.To get deeper insights into the effects deriving from the dose of spirulina,OPLS was applied to CS1 and CS0.5 samples.The observations,namely CS1 and CS0.5 ,separated along the second predictive component P2,explaining about 9.5% of the predictive x-variance.The distribution was strongly affected by the part of the plant the samples derived from,leaves or stems,with a noticeable clustering along the first predictive component P1.The corresponding loading plot showed important dose-dependent variations.Indeed,nickel,zinc,copper,and chromium were more abundant in samples treated with a higher dose of spirulina.Generally,as the concentration of spirulina increased,the residual metal content increased both in the leaves and in the stems ,whereas the Cr content increased in leaves and decreased in stems.While no dose-effect was observed for Cd,noticeable variations were exerted by increased amounts of spirulina on the quantities of Pb incorporated into the plant.Specifically,the average content of Pb in the plant was higher in CS0.5 than in CS1,suggesting that a higher amount of the blue-green alga contained in the soil might compete with hemp to sequester this metal.A piece of further evidence was that the average soil residual of all heavy metals resulted higher when it was irrigated with a higher concentration of spirulina ,suggesting that the cyanobacteria stationing in the soil may exert a strong action of metal chelation,avoiding leaching of them during the watering stage and acting as a reservoir for hemp to absorb the chelated metals.Such results find fundamentals in the reported ability of Arthrospira platensis to adsorb and accumulate all the six metals here tested through various mechanisms.Very small effects were observed on the metabolic profile when samples CS1 were compared with CS0.5.Interestingly,according to the loading plot ,the buckets at 2.66 and 2.74 ppm,containing the signals assigned to citric acid,contributed importantly to the observed grouping between the samples treated with the two different doses of spirulina.Specifically,the signals of citric acid were more intense in samples treated with a higher dose of spirulina.Considering that citric acid is one of the main chelating agents in plants,this evidence may support the hypothesis that,at a higher concentration,the cyanobacteria can chelate the heavy metals sequestering them into the soil.
As a result,less quantity of heavy metal is available for the plant to uptake,and,thus,more chelating agents inside the plant are in the free form not coordinating any metal.Industrial hemp is an annual crop grown for its seed and fibers.The three main global producers of hemp are China,Europe,and North America.Hemp is found in countless daily products: foods ,beverages ,cosmetics,and personal care products ,textiles ,papers and cardboard ,jewelry and fashion,leisure products ,animal feed,animal litter,mulching and horticulture,detergents,bio-fuel,building materials,insulation,composites ,etc.Hemp is also the object of numerous fundamental studies for innovative potential applications in bio-pesticides,biotechnology,pharmacy and medicine,paints,energy production,and wastewater treatment.Hemp shives,as valuable co-products produced during the process of extracting fiber from hemp straw,are constituted from the xylem tissue of the stem.Long considered as a by-product of the industry,used for plant mulch or animal bedding ,hemp shives are now used for house insulation,energy,and fuel production.There is also an interest in using these co-products as adsorbent materials to complex environmental pollutants since this resource is abundant,cheap,and easily burned.The last one is interesting in the context of recovery of adsorbed metals and their revalorization for example.Moreover,these materials have a highly reactive lignocellulosic macromolecular structure capable of interacting with other substances.Finally,the industry also needs to find new outlets because the volume of shives produced annually is constantly increasing.Recently,hemp fibers have been studied as adsorbent for metal removal and for the preparation of activated carbons for similar applications.However,work on hemp in shives/hurds form for environmental applications is rare.Furthermore,the identification of interactions involved in liquid-solid adsorption phenomena is often carried out using empirical mathematical models that allow the modeling of experimental results and the drawing of conclusions from the assumptions of these models.Another approach is to couple different microscopic and spectroscopic techniques to obtain information on these mechanisms.This study is part of the European research project FINEAU which brings together academics and industrialists focused on the potential use of hemp shives to recover metals from industrial effluents aiming their valorization.Two types of materials were targeted in this study by comparing shives washed with water with those chemically modified using polycarboxylic agent ,and with shives washed with water and activated with sodium carbonate.
The last one was the most efficient biosorbent of copper in our previous study among investigated raw shives and those treated with H2O,H3PO4,or KOH.The chemical treatment with Na2CO3 was carried out on a laboratory scale by copying the process already used by one of the industrialists of the project on other cellulosic materials.The objective of these two modifications is to purify and increase the capacity of raw shives towards metals,rolling flood tables while presenting high selectivity and performances independently of the pH and ionic strength representing two important issues for wastewater from the surface treatment industry.Recently,Mongioví et al reported that in the context of copper recovery by SHI-C and SHI-BTCA samples,these two materials had similar high adsorption capacities and fast adsorption kinetics,while their adsorption behaviors were very different.For example,the pH of the solution after copper adsorption onto the SHI-BTCA sample increased significantly,while that of the SHI-C sample varied much less,suggesting different interactions between these shives samples and the adsorbed copper.In addition,the amounts of sodium released into the solutions after an exchange with metal ions increased strongly in the case of the SHI-BTCA sample due to the presence of COONa groups in this material,also confirming different adsorption mechanisms.This study has two objectives: the first is to characterize the surface state of the materials before and after copper adsorption and the second is to assess the interactions between the materials and the metal ions involved in the adsorption process using microscopic and spectroscopic tools,namely computed nano-tomography ,energy-disperse X-ray spectroscopy,X-ray photoelectron spectroscopy ,Fourier-transform infrared spectroscopy,Raman spectroscopy,and X-ray absorption near-edge structure spectroscopy.Three different treatments were applied to SHI-R samples.A water washing ,an activation using sodium carbonate ,and a grafting reaction using 1,2,3,4-butanetetracarboxylic acid were carried out to study their role in the performance of samples which were further used as adsorbents for copper presents in aqueous solution.To prepare SHI-W,SHI-R samples were simply washed with water for 2 days at room temperature.After that,they were filtered and dried in an oven at 80◦C until a constant mass was obtained.
The SHI-C shives were prepared by treating SHI-W samples in 1 M Na2CO3 for 4 h at 40◦C.Thereafter,the samples were washed extensively with water until a neutral pH was obtained and dried in an oven at 80◦C until a constant mass was obtained.To obtain SHI-BTCA,SHI-W samples were pre-treated with 1 M NaOH for 3 days at ambient temperature under mechanical stirring.The shives suspension was then filtered on a filtering funnel and thoroughly washed with distilled water until the water was colorless.After drying at 60◦C in a ventilated oven for 15 h,the samples were immersed in an aqueous solution containing 100 g/L of BTCA and 30 g/L of NaPO2H2 for 15 h at ambient temperature under mechanical stirring.After draining,shives were spread on the bottom of a glass crystallizer put in a ventilated oven at 160◦C for 90 min.They were suspended in distilled water overnight under stirring,filtered,thoroughly washed with distilled water,and dried in a ventilated oven at 60◦C for 15 h.The shives were finally activated by converting the acidic form of carboxylic groups into their basic carboxylate form by immersing materials in a NaHCO3 solution under mechanical stirring for 24 h.SHI-BTCA samples were filtered,washed with distilled water until neutral pH was reached,and dried in a ventilated oven at 60◦C for 15 h.The interactions between copper and hemp-based samples were assessed by using different microscopic and spectroscopic techniques such as computed nano-tomography ,energy-disperse X-ray spectroscopy,Fourier-transform infrared spectroscopy,Raman spectroscopy,X-ray photoelectron spectroscopy ,and X-ray absorption near-edge structure spectroscopy.Nano-CT images given in Fig.3a show the typical cross-section of raw hemp shives.Namely,different tissues such as the woody part constituting the main part of the shives and some remaining pith on the right side can be observed.The woody part is made of different cell types: i) isolated or grouped in groups of two or three vessels which have a quite thin cell wall and a diameter of approximately 50-150 µm; ii) the fibers with a thick cell wall,an irregular polygonal cross-section and a diameter of only a few µm and iii) the rays which are oriented in the radial direction and are generally composed of one cell in width.