Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan

International Journal of Biological Macromolecules xxx (xxxx) xxx

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Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan Xuebin Wang a, Wang Jincheng a,⇑, Song Shiqiang a, Rao Pinhua a, Wang Runkai a, Liu Shihui b,c,d a

College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, PR China Key Laboratory of Quality and Safety Regulating of Horticultural Crop Products, Ministry of Agriculture, Shanghai 201210 PR China c Shanghai Sunqiao Agricultural Science and Technology Co., Ltd, Shanghai, PR China d Hunan Agricultural University, Changsha 410128200000, PR China b

a r t i c l e

i n f o

Article history: Received 19 June 2019 Received in revised form 11 September 2019 Accepted 26 September 2019 Available online xxxx Keywords: Soil conditioner Microspheres Layer-by-layer Sustained release behavior Biodegradation

a b s t r a c t Fulvic acid (FA), as one type of soil conditioner, can enhance drought tolerance, improve nutrient ingestion, stabilize pH values of soil, and reduce fertilizer extracting behaviors. Moreover, it shows a good effect on increasing the permissiveness of plants to various environments, and the stress of salinity and drought is included. Therefore, it can be used as an ideal soil conditioner. In order to extend its effect, it may be loaded into the core-shell polymer microspheres. In this study, FA was added to calcium carbonate (CaCO3) microspheres, potassium alginate (PAL) and chitosan (CS) were assembled outside. Thus, PAL/CS/FA-CaCO3 microspheres were prepared using layer-by-layer self-assembly methods. The surface morphology, encapsulation efficiency and in vitro drug release characteristics were investigated. It was revealed that PAL/CS/FA-CaCO3 microspheres were formed with a particle size distribution between 2 and 6 lm due to self-assembly between PAL and CS. Drug release behavior analysis exhibited the sustained release processes were delayed to varying degrees due to different degradation degrees of CaCO3 microspheres at different pH values. Results also illustrated that these microspheres had a high loading capacity for FA, and possessed good biodegradability. Thus, PAL/CS/FA-CaCO3 microspheres exhibited promise for future applications compared with that of traditional soil conditioners. Ó 2019 Elsevier B.V. All rights reserved.

1. Introduction In recent years, due to years of frequent farming, excessive use of chemical fertilizers has led to soil alkalinity, soil hypoxia, water and microbial imbalances. The challenge of water resources and limited cultivable land resources severely restricts the continuable development of agriculture and has a significant impact on the future of mankind [1,2]. It is very urgent to adjust the physicochemical properties and chemical composition of the soil as well as nutrients. Soil conditioners may act to retain water and release nutrients, and have a beneficial effect on soil and plants. Fulvic acid (FA), a type of soil conditioner, offers many benefits to plants by improving drought tolerance, promoting nutrient absorption, stabilizing pH values of soil and reducing fertilizer loss [3]. At the same time, the dominant physiological mechanism of FA to enhance abiotic stress resistance of plant is rarely known. Current reports indicate that soil and foliar application of FA is beneficial for germination of crop seed, growth of seedling and ⇑ Corresponding author.

development of root [4,5]. Wang et al. reported that the application of FA can reduce the cadmium toxicity of lettuce [6]. In soil applications, FA humidification levels are suitable for increasing soil fertility, improving degraded soils, and repairing soils contaminated with metal or organic pollutants [7]. The impact of FA on the application properties of the soil at different pyrolysis temperatures has also been reported [8]. In addition, FA owns a characteristic of relatively low molecular weight and possesses many functional groups which are oxygen-rich and carbon-depleted [9]. However, FA is easy to lose moisture and may lose its effect in a short time. In order to maintain its long-term soil conditioning effects, it is necessary to use a carrier to provide a sustained release effect. Microcapsules are spherical or nearly spherical microcontainers or wrappers with a size of 1–10 lm. Microcapsules have been used primarily for drug delivery [10], maintaining enzyme activity [11], preserving probiotic survival [12], and others. Most importantly, microcapsules owned good sustained release properties. Vincekovic´ et al. reported a chitosan/alginate microcapsule containing both copper cations and Trichoderma viride for plant protection and nutrition [13]. Takei et al. reported that application

E-mail address: [email protected] (J. Wang). https://doi.org/10.1016/j.ijbiomac.2019.09.247 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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of soil conditioners coated with microcapsules to lactic acid bacteria can effectively remove root-knot nematodes in the soil [14]. Calcium carbonate (CaCO3) has received more attention because of its low cost, environmental friendliness and high stability [15]. In addition, its porous structure is advantageous for improving load efficiency [16]. The self-assembly process is typically accomplished by electrostatic adsorption on a CaCO3 microsphere template using a natural or a synthetic polymer [17–21]. Soil conditioners containing CaCO3 may be more favorable for the soil environment. Moreover, the adsorption ability of CaCO3 can make the release of the drug more slowly in the environment [22]. In this work, a novel type of microcapsules containing CaCO3 was prepared using carboxymethyl cellulose (CMC) which owned water retention properties as a regulator. In addition, potassium alginate (PAL) and chitosan (CS) were both used as shelf materials to form microcapsules by self-assembly electrostatic absorption process. This type of microcapsules had a high loading capacity for FA, owned good sustained release behavior, and possessed satisfactory biodegradability.

2.4. Preparation of PAL/CS/FA-CaCO3 microspheres These microspheres were prepared using layer by layer selfassembly method. 50 mL of 1 mg/mL FA was added to 10% (m/v) aqueous solution of CaCO3 microspheres (100 mL), and this mixture was dissolved and ultrasonically dispersed to obtain a uniform turbid liquid. Then, this turbid liquid was freeze-dried and stirred by ultrasound treatment. Subsequently, the dispersed liquid was added to 100 mL of 1% CS aqueous solution at pH = 5.5, stirred for 15 min, and the excess polyelectrolyte was removed by centrifugation (1000 rpm, 5 min). The centrifuge tube was collected and redistributed, and 100 mL of 1% PAL solution was added. This solution was stirred at 35 °C for 15 min, and the excess polyelectrolyte was removed by centrifugation (1000 rpm, 5 min). The CS and PAL solutions were used alternately until the required number of self-assembled layers was reached. Finally, the solution was crosslinked with 1% glutaraldehyde, and was then centrifuged and lyophilized to obtain the final FA drug-loaded microspheres, PAL/CS/FA-CaCO3. 2.5. Characterization

2. Materiails and methods 2.1. Materials


Potassium alginate (PAL, relative molecular weight M n ¼ 80776 g/mol, PDI = 3.097), chitosan (CS, relative molecular weight


Mn ¼ 52844 g/mol, PDI = 1.057), sodium carboxymethyl cellulose

2.5.1. Determination of particle size and zeta potential The size distribution and zeta potential were analyzed by NANO-ZS (Malvern Instruments Ltd., Malvern, UK). The zeta (f)potentials of CS and PAL during self-assembly of CaCO3 microspheres were measured at 25 °C using Zeta sizer Nano-ZS. These tests were performed in triplicate (n¼ 3) and the standard deviation (SD) was calculated.




(CMC, M n ¼ 49402 g/mol, PDI = 5.306), and anhydrous sodium carbonate were purchased from Shanghai Taitan Technology Co. Ltd (Shanghai, China). Potassium chloride was provided by Shandong Chemical Technology Co. Ltd. (Shandong, China). Fulvic acid (FA, 99% pure) was received from Hefei Basf Biotechnology Co. Ltd. (Anhui, China). Deionized water was used throughout this work.

2.2. Preparation of calcium carbonate microspheres Pure sodium carbonate (0.1 mol/L) and calcium chloride solutions (0.1 mol/L) were prepared separately. CMC (2.0 g) was added into the calcium chloride solution (100 mL) and let it completely dissolved. After ultrasonic mixing at room temperature for 5 min, the prepared sodium carbonate solution (100 mL) was slowly dropped into the mixture. Afterwards, stirring for 3 min (800 rpm), CaCO3 microspheres were obtained after centrifugation (8000 rpm, 5 min). These particles were washed with deionized water three times. Subsequently, CaCO3 particles were cured with absolute ethanol and dried at 60 °C in a vacuum oven for 3 h.

2.3. Preparation of PAL/CS/CaCO3 microspheres 0.5 mol/L (50 mL) potassium chloride solution was added to 2 mg/mL (50 mL) CS solution and 2 mg/mL (50 mL) PAL solution, respectively. The pH value was adjusted to 5.5 under intensely magnetic stirring. The above prepared CaCO3 microspheres were added to the CS solution to complete the first assembly at 35 °C. The mixture was then centrifuged 3 times (1000 rpm, 5 min), and the resulting sample was ultrasonically dispersed. Then, the mixture was added to the PAL solution and the second assembly was completed at 35 °C. The above procedure was repeated as needed, and the sample was crosslinked with 1% glutaraldehyde to obtain PAL/CS/CaCO3, a type of microcapsules with core-shell structure.

2.5.2. X–ray diffraction (XRD) XRD of the samples was analyzed using an X-ray powder diffractometer (D8 Advance, Bruker AXS, Germany). A PANalytical X’ Pert PRO X-ray diffractometer (40 kv, 40 mA) copper anode (copper Ka radiation k = 1.54187 Å) was used. This measurement was performed using a scanning range for 2h from 5 to 80°. 2.5.3. Optical microscope (OM) and scanning electron microscopy (SEM) observation The prepared samples were uniformly dispersed by ultrasonication method, and a small amount of the samples was dropped onto the glass slide. Then, the samples were compacted by a cover glass and excess water was removed, and were observed by OM. The surface morphology of all the particles was observed using a Hitachi S4800 SEM. This equipment was operated at an accelerating voltage of 5 kV. The samples were ultrasonically dispersed on a foil paper with ethanol, dried in a vacuum oven, and sputtered with gold before observation. 2.5.4. Fourier transform infrared spectroscopy (FTIR) FTIR was used to test the mixed microspheres, and the corresponding absorption spectra were obtained. These microspheres and KBr were mixed and compressed to measure their absorption spectra, and the spectra were collected in the wavenumber range of 400–4000 cm
1 with an OPD speed of 0.2 cm/s. 2.5.5. Energy dispersive spectrometer (EDS) EDS was conducted using a Hitachi S4800 scanning electron microscope, and an energy dispersive X-ray spectrometer (Horiba, Japan) was used to observe the morphology and measure the surface element distribution of the samples. 2.5.6. Thermogravimetric analysis (TGA) The thermal stability of the dried hybrid microspheres was investigated by TGA (Netzsch STA 449C) with a heating rate of

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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20 °C/min in a temperature range of 30–800 °C, and the atmosphere used was N2. 2.5.7. Brunauer–Emmett–Teller (BET) The specific surface area and average pore diameter of the samples with different degradation time were measured by nitrogen adsorption methods according to the BET and Barrett-JoyneHalenda (BJH) equations. The BET surface area was tested using a Micromeritics 3 Flex instrument at a relative N2 pressure.

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static interactions. After constructing LbL with a selected amount of (PAL/CS) double biopolymer layers, the polyelectrolyte was crosslinked with glutaraldehyde solution [20]. Then, the coated CaCO3 microspheres were redispersed in KCl aqueous solution prior to the measurement. The excess polyelectrolyte was removed by a centrifugation step after each adsorption process. The asprepared samples had a microsphere structure with biodegradable and sustained release properties. 3.2. Particle size distribution

2.6. Drug loading efficiency and sustained release behavior Using an UV–vis spectrophotometer, the total ultraviolet absorption of FA was measured by a spectrophotometer at 271 nm. Then, 0.1 g of PAL/CS/FA-CaCO3 microspheres was added to the dialysis bag after the ultrapure water was boiled. Phosphate buffer solution (PBS) was used as a solvent (pH value of PBS was 5, 6, 7, 8, respectively). 8 mL of PBS was added to the dialysis bag, sealed and immersed in a centrifuge tube of the corresponding 50 mL of PBS solution. The cumulative release rate (CRR) of FA was calculated by the equation as following:

CRRð%Þ ¼

! P C t  V total þ 0t
1 C t  V t  100% m0

where C t (mg/mL) and V t (3 mL) referred to the concentration and volume of the solution at time t. V total (50 mL) referred to the total volume fixed in solution, and m0 (mg) referred to the total weight of FA in the PAL/CS/FA-CaCO3 microspheres. 3 mL of the solution was taken from the centrifuge tube at intervals of 3 times, and after the completion, 9 mL of PBS solution was added to the centrifuge tube. Its absorbance was measured using an UV–vis spectrophotometer, and the blank controls were PAL/CS/FA-CaCO3 microspheres and their corresponding pH buffer solutions. 2.7. Drug biodegradation performance 0.1 g of the microspheres was placed in a solution in a 5 mL of centrifuged tube (pH = 5.0, 6.0, 7.0, 8.0, ultrapure water, 3 mL). They were placed in a shaker, and the reciprocating conditions were set at 37 °C for a predetermined time. Then, the samples were taken out, washed with deionized water, freeze-dried and vacuum dried to a constant weight. The BET surface area together with total pore volume of the soil conditioners were measured at constant temperatures, and SEM was combined to observe and analyze the biodegradation process. 3. Results and discussion 3.1. Preparation of PAL/CS microspheres for soil conditioner Fig. 1 presented preparation process of PAL/CS/FA-CaCO3 microspheres. First, using CMC as a regulator, CaCO3 microspheres were prepared by the reaction between Ca2+ and CO2
3 . The CMC on the surface of CaCO3 microspheres carried a large amount of negatively charged carboxy groups (COO
), which facilitated further assembly of the positively charged amino groups (NH+3) carried by the CS. It was not advantageous for the negatively charged soil conditioner molecules FA, therefore, the FA was absorbed by nanopores on the surface of CaCO3 microspheres. This was because the nanoporous structure of these microspheres provided a capillary force to load compounds regardless of their charge properties [23–24]. Multilayer accumulation was carried out from PAL and CS solutions by LbL technique. During this process, the electrophoretic mobility of CS and PAL suspensions was measured by zeta (f)potential to evaluate whether they were self-assembled by electro-

The particle size of the different microspheres was given in Fig. 2. In Fig. 2(a) and (b), it was shown that the particle size distribution of CaCO3 and self-assembled PAL/CS/CaCO3 microspheres was between 1 and 6 lm, and a discontinuity was exhibited after 4 lm. It was speculated that the microspheres were agglomerated. However, the distribution of PAL/CS/FA-CaCO3 microspheres was more evenly, and there was no agglomeration as shown in Fig. 2 (c). The particle size distribution was between 2 and 6 lm. It was deduced that PAL and CS were electrostatically and properly attracted, and more FA may be loaded. 3.3. Zeta (f)-Potential and XRD As shown in Fig. 3(A), the CaCO3 microspheres exhibited a high and negative surface charge of
37.9 ± 0.7 mV, mainly because the surface of CMC exhibited a negative charge. This can help them prevent accumulating in aqueous solution. Then, at the beginning of preparation of the core-shell particles, the change in charge was measured during assembly of the CS and PAL aqueous solutions at pH = 5.5. When the first CS layer was assembled onto CaCO3 particles, the potential was
15.4 ± 1.1 mV. When the second PAL layer was added, the potential of the particles was changed to
25.1 ± 1.6 mV. The particle potential was
14.8 ± 0. 9 mV when the third CS layer was assembled. In addition, the particle charge was
20.5 ± 0.8 mV when PAL as the fourth layer was assembled. The same trend for the assembly of the CS and PAL aqueous solutions at pH = 4 can be seen from Fig. 3(B). However, the f-potential value of the entire self-assembly process was always below zero, and there were no alternating positive and negative charges. This behavior may be resulted from the relatively low amount of primary amine groups in the protonated CS when the pH value of the solution was 5.5 during self-assembly process. However, the charge change was still quite obvious, and a zig-zag like trend of f-potential value was observed [25]. This demonstrated the assembly of PAL/CS membranes and preparation of core-shell (PAL/CS) particles were successfully achieved. Möhwald and Carla et al. previously reported the similar behaviors for depositing the same PAL/CS multilayer membranes on poly (DLlactic acid) (PDLLA) particles [26]. The corresponding XRD patterns (Fig. 3(C)) of the CaCO3 microspheres were identical to the standard card (No. 47-1743), indicating that the spherical structure of CaCO3 in the figure was calcite. It was well known that CaCO3 microspheres in the form of vaterite were unstable in aqueous solution, and they recrystallized in pure water within several hours into the most stable calcite phase at room temperatures [27–28]. It can also be confirmed by the characteristic peaks at 712 and 874 cm
1 in the FTIR spectra shown in Fig. 6 [29]. 3.4. Morphologies observation A simple and reproducible method was used to prepare spherical CaCO3 powders with a moderately narrow size distribution, mostly between the range of 1 and 6 lm, as shown in Fig. 4(a). This was in good accordance with their particle size distribution (Fig. 2).

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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Fig. 1. Scheme for fabrication process of PAL/CS/FA-CaCO3 microspheres.

Fig. 2. Particle size of different microspheres (a) CaCO3, (b) PAL/CS/CaCO3, (c) PAL/CS/FA-CaCO3.

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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Fig. 3. Zeta (f)-potential (A) pH = 5.5, (B) pH = 4; (C) XRD patterns of different microspheres. (a) CaCO3, (b) PAL/CS/CaCO3, (c) PAL/CS/FA-CaCO3.

The surface of CaCO3 microspheres was covered with CMC containing a large amount of negative charges. Thus, the same kind of charges was mutually exclusive and appeared as dispersed microspheres macroscopically. In Fig. 4(b) and (c), although the particles also had electrostatic repulsion, some microspheres may be agglomerated due to the viscous characteristics of the polymeric components, PAL and CS. SEM images of these particles were shown in Fig. 5. The shape of the prepared CaCO3 particles was close to a sphere as shown in Fig. 5(a). The surface morphology of the microspheres showed that the surface was very rough, and a large number of carbonate particles were combined to form a particular morphology. Fig. 5(b) showed the photos of PAL/CS/CaCO3 microspheres. The surface of the coated particles was found to be rougher than that of the initial CaCO3 micropores. This was because CS and PAL were selfassembled by electrostatic adsorption process. It can be clearly seen that a polyelectrolyte membrane was formed on the surface of CaCO3. Next, as shown in Fig. 5(c), FA was adsorbed by the CaCO3 microspheres, and then PAL and CS were electrostatically adsorbed to complete the self-assembly process. Thus, PAL/CS/FA-CaCO3 microspheres were formed. The drug loading efficiency reached 6.8%, and the encapsulation efficiency was 60.3% [24].

3.5. Fourier transform infrared spectroscopy FTIR spectra were used to assess the formation of PAL/CS/CaCO3 and PAL/CS/FA-CaCO3 microspheres. In Fig. 6A(c), a characteristic peak of C@O in alginic acid of PAL appeared, that is, a broad peak caused by asymmetric stretching vibration appeared at 1612 cm
1, and a narrow peak caused by symmetric stretching exhibited at 1428 cm
1 [30–31]. Due to the stretching vibration of CAO bonds, a wider absorption peak appeared at 1030 cm
1 [32]. The peaks at 1612 and 1656 cm
1 disappeared at the curves of PAL/CS/CaCO3 (Fig. 6B(b)) and PAL/CS/FA-CaCO3 (Fig. 6B(c)) microspheres, which were resulted from the interionic interactions between ACOO
and ANH+3 groups. The peaks around 1030 cm
1 also disappeared, indicating that the amino groups in CS and the carboxyl groups in PAL possessed electrostatic interactions [23– 24]. It was demonstrated that the prepared PAL/CS/FA-CaCO3 microspheres should own characteristic peaks of featured groups in both FA (Fig. 6A(b)) and PAL (Fig. 6A(c)). All three samples in Fig. 6B showed bands at 875 and 712 cm
1, indicating that a structure of calcite can be assigned [33]. This obtained crystal phase of CaCO3 was already confirmed by XRD (Fig. 3a–c) [23,34]. The characteristic peak of FA appeared at 1068 cm
1 in the curve of PAL/CS/

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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Fig. 4. OM images of different microspheres (a) CaCO3, (b) PAL/CS/CaCO3, (c) PAL/CS/FA-CaCO3, (d) comparative photographs between CaCO3, PAL/CS/CaCO3 and PAL/CS/FACaCO3.

Fig. 5. SEM images of different microspheres (a) CaCO3, (b) PAL/CS/CaCO3, (c) PAL/CS/FA-CaCO3.

FA-CaCO3. This illustrated that FA was successfully loaded into this type of composite microspheres. However, the peak intensity of FA at 1392 cm
1 was almost indistinguishable, which was mainly ascribed to its low content on the surface of the microspheres. Moreover, the strong symmetry and antisymmetric stretching vibrations in the soil conditioner microspheres mask the other characteristic peaks of the FA molecules.

3.6. Energy dispersive spectrometer EDS in combination with SEM allowed further analysis of the elemental species and corresponding content of the material domains. In order to further investigate whether FA was loaded in PAL/CS/FA-CaCO3 microspheres, EDS analysis of PAL/CS/CaCO3 and PAL/CS/FA-CaCO3 microspheres was carried out, and their

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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(A)

CMC and FA together with PAL and CS. At this stage, their respective decomposition temperatures were approximately the same, which can be considered as the fracture of the high molecular carbon chains. At the last stage of weight loss, the TGA curves dropped sharply from 620 to 730 °C, which were ascribed to the decomposition of calcium carbonate and were decomposed into CaO and CO2. Results also showed that the higher the calcium carbonate content, the better the thermal stability of the microspheres.

(a)

transmittance(%)

1656

(b) (c)

1392 1068

3.8. Drug sustained release behavior 1030

1612

(d)

1627 1428

4000

3500

3000

2500

2000

1500

1000

500

Wavenumber(cm-1)

(B)

transmittance(%)

(a)

712

(b) 1623

(c)

1157 1068

712

875 1438

4000

7

3500

3000

2500

2000

1500

1000

500

Wavenumber(cm -1) Fig. 6. FTIR spectra of (A) (a) pure CS, (b) pure FA, (c) pure PAL, (d) pure CMC; (B) (a) CaCO3 microspheres, (b) PAL/CS/CaCO3 microspheres, (c) PAL/CS/FA-CaCO3 microspheres.

images and data were shown in Fig. 7 and Table 1, respectively. Compared with the percentage of O element in the unloaded drug microspheres, 51.77%, as shown in Fig. 7a, this element of the FA loaded microspheres was increased to 54.64%, as shown in Fig. 7b. As noted, the surface elements of the microspheres had not changed much as the percentage of N and Ca content was almost unchanged. Referring to the molecular formula of FA, it was found that above increasing ratio was basically consistent with the ratio of FA. A conclusion can be made that the FA had been successfully loaded in PAL/CS/FA-CaCO3 microspheres. 3.7. Thermogravimetric analysis Fig. 8 presented the thermal stability of different microspheres. It was shown that, (a), the main components of hollow PAL/CS microspheres were organic polymer materials, and thus owned lowest thermal stability. For (b), (c) and (d), the weight loss of the three samples was divided into four stages. The initial weight loss started from 25 to 100 °C can be attributed to the dehydration of water physically adsorbed in the microspheres. The second mass reduction from 100 to 320 °C was equivalent to the combined water containing in CMC. The weight loss of the third stage between 320 and 620 °C was attributed to the decomposition of

As shown in Fig. 9, the drug loaded content increased rapidly during the first 24 h, and the CRR of FA in ultrapure water was 23.1%. At other pH values, the CRRs were between 37.1% and 42.2%. At pH = 5, the PBS buffer solution was equilibrated at 336 h; at pH = 6, it reached equilibrium at 312 h; at pH = 7 and pH = 8, they both reached equilibrium at 432 h. During the first few hours, the interactions between the electrostatic negative charges of PAL layer and the positive charges of CS promoted adsorption of FA onto the surface of PAL/CS/FA-CaCO3 microspheres. Meanwhile, the spontaneous deposition of FA onto the polyelectrolyte multilayers was a general phenomenon of polyelectrolyte capsules [35]. In addition, there was no obvious sudden release of PAL/CS/FA-CaCO3, thus achieving sustained release of FA. The sustained release profile of FA from PAL/CS/FA-CaCO3 microspheres should be ascribed to the fact that FA needed to first reach the PAL/CS membrane by diffusion, and then released from the pores of CaCO3 and membranes of PAL/CS. Drug release processes at different pH levels were delayed to varying degrees. This may be due to the different degradation degrees of CaCO3 microspheres at different pH values. A morphology analysis on the samples was performed after the sustained release process. Fig. 10(a) was a SEM image of PAL/CS/ FA-CaCO3 after slow release at pH = 5, which was considered to be the simultaneous corrosion in acid environment and the degradation in PBS solution. In Fig. 10(b), random micro-scale pores can be clearly seen at pH = 6, and the pore size was small. The pH value was less corrosive, Fig. 10(c), and some small microspheres from PAL and CS were formed on the surface. At pH = 8, many nanoparticles were adhered on the surface of microspheres as shown in Fig. 10(d), and these powders may be part of CaCO3 microspheres. In Fig. 10(e), under ultrapure water, the spherical appearance changed greatly, and PAL and CS were still adhered on the surface. The sustained release mechanism of FA in PAL/CS/FA-CaCO3 was studied using four mathematical models in which first order, Higuchi, Ritger-Peppas and parabolic diffusion were included:

Inð1
Mt =M1 Þ ¼
kt

ð1Þ

Mt =M1 ¼ kt1=2

ð2Þ

Mt =M1 ¼ ktn

ð3Þ

Mt =M1 =t ¼ kt
0:5 þ b

ð4Þ

where Mt/M1 is the release rate of FA at time t, k is the dynamic constant, n is the diffusion index (Fickian diffusion (n  0.43), non-Fickian or abnormal transmission (0.43 < n < 1.0)), b is a constant. Generally, the first-order equation (Eq. (1)) depicted a release process on account of ion exchange and depolymerization steps, where the dissolution rate relied on the drug content in the compound. The Higuchi equation (Eq. (2)) described the amount of drug diffusion into the receiving unit (the amount of liquid entering the buffer solution). The Ritger-Peppas equation (Eq. (3)) was used to explicate the diffusion and dissolution of drugs in the

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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Fig. 7. EDS patterns of C, N, O, Ca, and K of different microspheres (a) PAL/CS/CaCO3 microspheres, (b) PAL/CS/FA-CaCO3 microspheres.

PAL/CS layer. The value of n < 0.45 corresponded to drug diffusion control based on the ion exchange process; n > 0.89 was ascribed to the dissolution of PAL/CS/CaCO3; 0.45 < n < 0.89 was resulted from

the synergistic effect of drug diffusion and PAL/CS/CaCO3 dissolution [36–37]. The parabolic diffusion equation (Eq. (4)) clarified that the release process was dominated by the outer surface/edge

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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X. Wang et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx Table 1 EDS analysis results of different microspheres. Samples

Unn.c [wt.%]

C N O Ca

Norm.c [wt.%]

Atom.c [at.%]

(a)

(b)

(a)

(b)

(a)

(b)

23.93 5.72 52.56 26.44

18.86 5.24 49.49 25.01

22.03 5.26 48.37 24.34

19.13 5.32 50.19 25.36

31.40 6.43 51.77 10.4.

27.73 6.61 54.64 11.02

(a) PAL/CS/CaCO3 microspheres; (b) PAL/CS/FA-CaCO3 microspheres.

100

(b)

Weight(%)

90

(c) (d)

80

(a)

70 60 50 40 100

200

300

400

500

600

700

800

Temperature(°C) Fig. 8. TGA curves of different microspheres (a) hollow PAL/CS, (b) CaCO3, (c) PAL/ CS/CaCO3, (d) PAL/CS/FA-CaCO3.

Cumulative release rate (%)

100

80

60

40 pH=5 pH=6 pH=7 pH=8 ultrapure water

20

0 0

100

200

300

400

500

600

700

Time (h) Fig. 9. Drug sustained release profiles of PAL/CS/FA-CaCO3 microspheres obtained at different pH values.

diffusion process. In this equation, b was a constant, and its chemical meaning was not clearly resolved [38]. The k, n, R2 (variance) values gained from the fittings were summed up in Table 2. The results of these four kinetic models for fitting measurement data were shown in Fig. 11. From above results, the drug sustained release progress was not propitious to the same model, and this illustrated that the drug release mechanism was a complicated disintegration process [39]. In curve (a), it was successfully simulated by a parabolic diffusion model with a fairly high linear correlation coefficient (R2 > 0.99). This indicated that at pH = 5, the drug diffusion was primarily through the outer surface and the edge diffusion modes. In

curve (b), the Ritger-Peppas and Parabolic diffusion equations were compared. This illustrated that the drug can be depicted by the outer surface/edge diffusion as a rate controlling step and the FA ions diffused from the surface of the CaCO3 powders through the polymer membrane into the medium solution. In curve (c), almost four fitting equations can be applied, which meant that it was not a simple sustained release process but was carried out simultaneously at pH = 7. In curve (d), it best fitted the first-order release mechanism, n = 0.2401 (n < 0.45). However, in curve (e), the n value was 0.3924 (n < 0.45), except for the linear difference of Ritger-Peppas (R2 = 0.8176). The linear correlation of other equations was higher, and this illustrated that FA was released and diffused into the solution due to the concentration difference. During the whole release process, the dissolution of the membrane and the degradation of CaCO3 microspheres were hardly changed and can be neglected.

3.9. Drug biodegradation performance The morphology of different microspheres during the biodegradation process was observed by SEM. As shown in Fig. 12, the surface of the microspheres before biodegradation was covered with a thick layer of biopolymer. After one week of degradation, the microspheres with CaCO3 particles inside were clearly visible in Fig. 12(a) and (b). After three weeks of degradation, the overall morphology of PAL/CS/FA-CaCO3 was not completely destroyed ((c) and (d)). Some small cracks and erosion areas appeared on the surface, and the surface aggregation microspheres became larger. This was an intuitive indication that the PAL/CS/FA-CaCO3 began to biodegrade or hydrolyze. At the 5th week, the aggregated large microspheres began to degrade, and the surface biopolymer dissolved and showed obvious wrinkles ((e) and (f)). There were many micropores on the surface of PAL/CS/FA-CaCO3, which undoubtedly increased the degradation rate of PAL/CS/FA-CaCO3. In addition, it accelerated the entering amount of buffer solution PBS (pH = 5) into the PAL/CS/FA-CaCO3, and can improve its soil conditioning ability. Moreover, after seven weeks of biodegradation, the surface of PAL/CS/FA-CaCO3 was completely wrinkled and cracked ((g) and (h)). There were no accumulated microspheres, leaving only the entire microsphere structure. By the 9th week, the microspheres completely collapsed, only the collapsed structure was left, and the polymer membrane on the surface suffered severe erosion and turned into a network structure ((i) and (j)). Nitrogen adsorption-desorption measurements were employed to characterize the surface area and pore volume of PAL/CS/FACaCO3 degraded at various time periods (Fig. 12). Referring to Fig. 12(k)-(o), the cycles of N2 adsorption-desorption isotherm together with pore size distribution of PAL/CS/FA-CaCO3 microspheres were different, although they degraded in a similar degradation process. All isotherms belonged to type IV Brunauer– Deming–Derning–Teller (BDDT) classification [40], demonstrating continuous degradation of PAL/CS/FA-CaCO3. As summarized in Table 3, the BET specific surface area of PAL/CS/FA-CaCO3 was

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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X. Wang et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx

Fig. 10. SEM image of PAL/CS/FA-CaCO3 obtained after sustained release at different pH values (a) pH = 5; (b) pH = 6; (c) pH = 7; (d) pH = 8; (e) ultrapure water.

Table 2 Kinetic fitting parameters of PAL/CS/FA-CaCO3 at different pH values. Samples

pH = 5 pH = 6 pH = 7 pH = 8 Water

First-order

Higuchi

Ritger-Peppas

Parabolic diffusion

k

R2

k

R2

k

n

R2

k

b

R2

0.0031 0.0019 0.0053 0.0040 0.0034

0.9407 0.8163 0.9693 0.9936 0.9496

0.0309 0.0205 0.0360 0.0314 0.0376

0.9566 0.8723 0.9366 0.9857 0.9203

0.1500 0.1930 0.1666 0.1979 0.0568

0.2811 0.2094 0.2905 0.2401 0.3924

0.9881 0.9679 0.9663 0.9719 0.8176

0.1366 0.1674 0.1503 0.1969 0.1067

0.0072 0.0103 0.0074 0.0011 0.0064

0.9925 0.9863 0.9875 0.9826 0.9283

Fig. 11. Plots of different kinetic models for release of FA from PAL/CS/FA-CaCO3. (a) pH = 5; (b) pH = 6; (c) pH = 7; (d) pH = 8; (e) ultrapure water.

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

X. Wang et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx

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Fig. 12. SEM images of biodegradable morphology of different microspheres at pH = 5 within different time (a,b) 1st week, (c,d) 3rd weeks, (e,f) 5th weeks, (g,h) 7th weeks and (i,j) 9th weeks, (k–o) corresponding N2 adsorption-desorption isotherm and pore size distribution (inset) of PAL/CS/FA-CaCO3 microspheres.

Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247

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X. Wang et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx

Table 3 BET surface area and total pore volume of PAL/CS/FA-CaCO3 for different biodegradation time. Samples 2

SBET (m /g) VBJH (cm3/g)

1st week

3rd week

5th week

7th week

9th week

47.67 0.18

44.52 0.19

74.53 0.27

82.27 0.32

77.85 0.37

increased from 47.67 to 82.27 m2/g as the biodegradation time was increased from one week to seven weeks. When the degradation time was increased to nine weeks, the BET surface area was decreased to 77.85 m2/g. However, it was worth noting that the corresponding pore volume was increased from 0.18 to 0.37 cm3/ g, further indicating that the soil conditioner material was continuously degraded. Therefore, it was illustrated that PAL/CS/FACaCO3 was continuously biodegraded. These results showed that PAL/CS/FA-CaCO3 was a type of biodegradable, non-polluting, and environmentally friendly soil conditioner. 4. Conclusions In this work, a novel type of PAL/CS/FA-CaCO3 microspheres was prepared using layer-by-layer self-assembly methods. The surface morphology, encapsulation efficiency, in vitro drug release, and biodegradable characteristics were investigated. The drug sustained release profiles of PAL/CS/FA-CaCO3 demonstrated that the release mechanism of the microspheres was a complex and ion exchange process, and followed different release kinetics models. The biodegradation assays demonstrated that the PAL/CS/FACaCO3 microspheres exhibited different degradation modes at different pH values, and showed no potential to pollute the environment. In a word, the microspheres prepared in this study were non-toxic, having sustained release property, owning good biodegradation ability, and can be used as a new type of soil conditioners. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgments This work was supported by ‘‘Capacity Building Project of Some Local Colleges and Universities in Shanghai, China (No. 17030501200)”, ‘‘National Natural Science Funds, China (No. 51873103)”, ‘‘Talent Program of Shanghai University of Engineering Science, China (No. 2017RC422017)”, ‘‘Scientific and Technological Support Projects in the Field of Biomedicine, China (No. 19441901700)” and ‘‘First-rate Discipline Construction of Applied Chemistry, China (No. 2018xk-B-06)”. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2019.09.247. References [1] D. Arvor, I. Tritsch, C. Barcellos, N. Jégou, V. Dubreuil, Land use sustainability on the South-Eastern Amazon agricultural frontier: Recent progress and the challenges ahead, Appl. Geogr. 80 (2017) 86–97. [2] S. Sauer, Soy expansion into the agricultural frontiers of the Brazilian Amazon: The agribusiness economy and its social and environmental conflicts, Land Use Policy 79 (2018) 326–338. [3] H.Y. Suh, K.S. Yoo, S.G. Suh, Effect of foliar application of fulvic acid on plant growth and fruit quality of tomato (Lycopersicon esculentum L.), Hortic. Environ. Biotechnol. 55 (6) (2015) 455–461.

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Please cite this article as: X. Wang, J. Wang, S. Song et al., Preparation and properties of soil conditioner microspheres based on self-assembled potassium alginate and chitosan, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.247