Sporopollenin exines microcapsules, derived from the naturally occurring spores of Lycopodium clavatum, have been loaded in-situ with humic acid sodium salt-Zinc (HA-Zn) complex. The chemical treatment method utilised to prepare the sporopollenin microcapsules from raw spores was discussed and the resulted sporopollenin microcapsules were characterised using SEM, TGA and FTIR. Metal complexes of the sodium salt of humic acid and zinc ion were prepared using different protocols and in-situ loaded into the pre-treated sporopollenin microcapsules. The resulted complex was characterised before and after the encapsulation process using FTIR, TGA and XRD techniques. The morphology of the empty and loaded sporopollenin was not altered. Infrared spectroscopy revealed an increase in the absorption for COO – vibrations at 1583 and 1384 cm –1 in the FTIR spectra of HA-Zn complex compared to that of the original sodium salt of humic acid, indicative of bonding of the metal ions in hydrated form to the carboxyl or phenolic hydroxyl groups or both of the sodium humate molecules. TGA results of the HA-Zn complex loaded sporopollenin showed that around %15 of residual HA-Zn was successfully encapsulated indicative of the efficiency of the protocol used. We showed also that biodegradable magnetite nanoparticles can be surface modified with HA and encapsulated into sporopollenin. The resulted biosorbents microcapsules can be used for enhanced magnetic removal of either heavy metals or HA from different aqueous media.
Sporopollenin (SP), the biopolymer shell of pollen grains of higher plants, is a highly resilient yet poorly characterised material which has been described as “one of the most extraordinary resistant materials known in the organic world” [
The paramagnetic iron oxide magnetite nanoparticles have attracted intriguing attentions in several environmental and biomedical applications owing to their biocompatibility [
Scheme 1. Typical structure for Humic Acid (HA).
humic substances. The cumulative level of heavy metals (such as nickel, zinc, and cadmium) in water significantly affects the human health and environment [
Much effort has been devoted to developing efficient adsorbents with suitable chemical compositions, microstructures, and surface functionalities for the removal of heavy metals and organic pollutants from different aqueous media. The synergetic adsorption of heavy metals and humic substances is very challenging. This is mostly owing to their competitive adsorption onto most adsorbents. Therefore, the motivation of the current study was to develop suitable adsorbents having high adsorption capability, low toxicity and biocompatibility. Several materials, such as activated carbon, silica gel, resins, clays, functionalised sporopollenin, and biosorbents have been studied for the removal of heavy metal ions via sorption technique, which was extensively used [
Raw Lycopodium clavatum pollens powder was purchased from Fagron, UK. A stock
Scheme 2. Encapsulation of magnetic HA-metal complexes inside sporopollenin exines.
solution of 0.1 M solution of heavy metals ZnCl2, CdCl2, NiCl2, PbCl2 (purchased from Merck) was prepared by dissolving the appropriate amount of salt in double distilled water. Humic acid sodium salt having a molecular weight range of (2000 - 500,000), ferrous sulphate FeSO4∙7H2O, ferric chloride FeCl3, ammonium hydroxide (33%) and potassium iodide were purchased from Sigma-Aldrich. Double distilled water was used throughout all experiments.
The infrared spectra were obtained in the 650 - 4000 cm−1 range by a Perkin-Elmer 100 FTIR spectrometer. Samples were ground with anhydrous potassium bromide (spectrosol grade) to obtain disks to a ratio of 1/9 (w/w). FTIR spectra were a result of 3 scans against a background. Thermogravimetric analysis (TGA) curves were obtained on a Setaram TG Analyzer/Setsys analyser (EXSTAR S11 7300 at a temperature range of 298 - 1073). Transmission electron microscope (TEM) samples were prepared by dropping a diluted suspension of magnetite nanoparticles onto 400-mesh carbon-coated copper grids with the excessive solvent immediately evaporated using Hitachi H-800 TEM (Hitachi, Japan) at an operating voltage of 200 kV. Scanning electron microscope (SEM) analysis obtained using JSM-5400 LVJEOL (Japan). A platinum coating of was deposited on either empty or loaded SP samples by using an auto fine coater JFC-1600 (JEOL, Japan) at 20 mA for 1 minute. Images were taken with an acceleration voltage of 5 kV at various magnifications. X-ray diffraction (XRD) analysis was carried out using Philips X-ray diffractometer PW 1370; Co. Wrist Action. Metal ion concentrations in the solutions were measured using a flame atomic absorption spectrophotometer (Contr AA 300, Analytik je). Optical images were taken by Nikon microscope fitted with Lainsy digital camera (5 MP, Egypt) and the images were processed with microvision software.
To take advantages of large cavity volume of the core of the spores, the core cytoplasm and the intine layer should be removed to extract the robust sporopollenin shell to be ready for encapsulation process. We extracted sporopollenin from Lycopodium clavatum pollen by suspending 100 g raw dry pollen powder in 800 mL acetone and stirred under reflux for 5 hours [
We have used two methods for preparation of magnetite nanoparticles: 1) the conventional method for co-precipitation of ferric and ferrous ions in alkaline medium and 2) the newly developed method by reacting only ferric ions as starting material and KI solution in alkaline medium [
Samples of HA-sodium salt (20 mg) were suspended in aqueous solutions (40 cm3) of the desired metal ion such as (Zn+2, Cd+2, Ni+2, Pb+2). The formed suspensions were mechanically stirred for 24 hrs at room temperature and the solid complex materials (referred to as HA-Zn for complexation with Zn+2) were separated by filtration and then lyophilised. The infrared spectra of HA, HA-Zn samples were measured in KBr pellets using FTIR spectrophotometer in the region 4000 - 400 cm−1 with the accumulation of 32 scans per sample.
There are different protocols and methods that demonstrate the extraction of empty sporopollenin from their raw pollen grains. These extraction protocols can involve either harsh chemical treatments utilising strong acids and bases at raised temperatures or using mild conditions. It has been suggested that the hydrolysis of ester groups of the SP microparticles can occur under these harsh conditions resulted in a slight change to their structure [
Several analytical techniques have been used to completely determine the chemical structure of different sporopollenin species. However, their full chemical structure is yet incomplete and requires further studies. Nevertheless, some studies indicated that sporopollenin is mainly an aliphatic polymer with phenolic and aromatic groups or conjugated side chains [
In this section morphology of the surface sporopollenin after extracting the genetic materials from their cores is presented. Figures 1(a)-(d) show SEM images for the empty sporopollenin after the chemical extraction process from their raw pollens. It can be seen in the first place that the average particle size of the treated sporopollenin is around 27 μm, with nearly monodisperse distribution for this plant species with a rough surface which is one of the advantages of these natural particles. The native reticulate microstructure and ornamentation is retained for the treated SP particles as seen in the close-up SEM image in
components of the spore, including proteins, lipids, nucleic acids and polysaccharides. This indicative of the great resists of these SP against strong acids bases and several chemical attacks. Bohne et al. [
Since humic substances contain several known functional groups such as carboxyl (-COOH), hydroxyl (-OH), amine (-NH2) and others, they exhibited great affinities to cations of different heavy metals. It was reported that the main functional groups found in HA (the main fraction of humic substances) are carboxyl, phenolic hydroxyls and alcoholic hydroxyls [
It was reported that the origin of HA substances and their pre-treatments affects the sorption capacity of metal ions [
antisymmetric (vasCOO−) stretching vibrations, respectively, of carboxylate groups, indicative that the HA-Zn complex of humic substances is formed primarily via metal-carboxylate bonds. The participation of other functional groups (phenolic hydroxyls, diketone groups) in the complexing of metals would be difficult to identify on the basis of FTIR analysis [
It was reported that HA solutions with an initial pH > 7 are more suitable for formation of HA complexes than solutions having pH 7.0 [
In line with the above interpretations, the use of neutral HA solutions will make phenolic OH groups unavailable for both dissociation and cation exchange. Therefore, the complex formation utilising the neutral HA solutions can be considered incomplete and takes place merely via electrovalent bonding by COO- groups [
We demonstrate for the first time that metal-ion complexes can be encapsulated within the empty cores of the natural sporopollenin microparticles with or without biocompatible magnetite nanoparticles. This can be achieved using different encapsulation, complexation and capping protocols. As was mentioned earlier, active substances can be encapsulated into empty sporopollenin either via passive diffusion through their nano-channels or by diffusion through the trilete scar of these microparticles for those materials that are larger in size than the nano-channels [
The FTIR spectra of empty sporopollenin are shown in
FTIR Region (cm−1) | Suggested groups from FTIR of empty sporopollenin |
---|---|
4000 - 2000 | § 3416 cm−1 (medium, broad) indicates the presence of hydroxyl OH groups. § 2925 cm−1 (strong, sharp) and 2855 cm−1 (strong, sharp but less intense). Likely to be due to CH2 stretching frequencies for saturated carbons. |
1750 - 1560 | § All showed broad peak in this region which indicates the presence of C=O containing groups (peak at 1712 cm−1). § No absorption around 1750 - 1735 cm−1, the region characteristic of ester. |
1200 - 1000 | § broad, variable ethers (C-O ν) at 1138 cm−1 |
848 | § medium aromatic (C-H) out of plane wagging. |
empty SP as shown in
In
The interactions of heavy metal ions with raw and surface modified sporopollenin have been previously studied [
Figures 6(a)-(d) shows optical and SEM images of sporopollenin suspension loaded with HA-Zn complex. The SP/HA-Zn suspension in pure water was observed using an optical microscope and the image is shown in
the loaded SP microparticles and it can be clearly seen that the main surface decoration is intact after loading with HA-Zn complex. We noticed the formation of very small aggregations having a diameter of 1 μm or less which might be some of the formed HA-Zn complex nanoparticles deposited inside the surface network of the SP comparing to the clean surface of empty SP (
The thermogravimetric analysis (TGA) provides useful information on thermal stability and the amount of the materials encapsulated. The TGA of the empty and HA-Zn loaded Sp microcapsules are shown in
corresponds to the decomposition of both SP and HA organic materials. The fourth weight loss of 20.5% observed in 471˚C - 601.6˚C range can be attributed to the decomposition of remaining SP grains. It can be concluded from the TGA analysis of
We have further extended our investigation by attempting to encapsulate the magnetite coated HA into the SP biosorbents ensuring a greener way of uptake and recover the adsorbed heavy metal ions from different media. In this respect, different protocols can be used to achieve this new encapsulation green process. We have coated the magnetite nanoparticles first with HA-Na molecules then loaded them into the empty SP microparticles to be ready for metal ion uptake experiments. We have used two different methods for preparation of magnetite nanoparticles; the first was based on the classical co-precipitation of ferric and ferrous ions in basic medium and the second is a newly developed method based on the use only ferric ions as starting substance in a single reaction [
It was anticipated that at pH ranging between 8 and 14 the complete precipitation of Fe3O4 would take place with a stoichiometric ratio of 2:1 (Fe3+/Fe2+) in oxygen free conditions according to the thermodynamics of the reaction represented in Equation (4) [
response of the magnetite/HA-Zn encapsulated SP microparticles biosorbents to the external magnetic field confirming the successful encapsulation of magnetite/HA-Zn nanoparticles into the SP. The tested uptake recovery of Zn++ ions from tap water using this new magnetic biosorbents was around 97%. Further and full sorption study is under way in our laboratory using these new solid natural materials.
To further confirm the successful coating of magnetite nanoparticles with HA-Na molecules, XRD and FTIR were performed for the coated magnetite.
We have shown that naturally, robust and low cost, sporopollenin exines microcapsules derived from the naturally occurring spores of Lycopodium clavatum can be loaded with HA-metal complexes or HA-coated magnetite nanoparticles for the first time. This has enabled us to take the advantages of utilising three green and biocompatible materials namely; sporopollenin, humic substances and magnetite nanoparticles. Humic acid substances showed high affinity to the metal ion studied with a high recovery percentage from tap water. The formation of the HA-Zn complex was confirmed using FTIR analysis and was successfully loaded into empty sporopollenin which also has been confirmed by FTIR and TGA analysis. Surface morphology of the SP before and after metal ion complex encapsulation did not show any significant change but some complexes nanostructures were observed inside the reticulate microstructure of the treated SP biosorbents. Interestingly, we showed also for the first time that biodegradable HA coated magnetite nanoparticles, prepared with KI via single ferric ion reaction method, can be encapsulated into empty sporopollenin. The resulted new biosorbents
microcapsules can be used for enhanced magnetic removal of either heavy metals or HA substances from different aqueous media. Using different metal cations for complexation with other biologically functional molecules (as a model drug) inside the sporopollenin microcapsules can have a wide range potential clinical applications such as using metal cation-organic complexes as antimicrobial microcapsules. This allows the fabrication of benign encapsulates that would not release hazardous substances to the environment. We have demonstrated a proof of concept in this study; although, it can be applied to different metal-ion complexes, other metal oxide nanoparticles and different naturally occurring microcapsules which we believe will open up interesting analytical, pharmaceutical and medical applications.
A.K.F. Dyab, Abdallah, E.M., Ahmed, S.A. and Rabee, M.M. (2016) Fabrication and Characterisation of Novel Natural Lycopodium clavatum Sporopollenin Microcapsules Loaded In-Situ with Nano-Magnetic Humic Acid-Metal Complexes. Journal of Encapsulation and Adsorption Sciences, 6, 109-131. http://dx.doi.org/10.4236/jeas.2016.64009