pH-Responsive Eco-Friendly Chitosan-Chlorella Hydrogel Beads PDF

water Article pH-Responsive Eco-Friendly Chitosan–Chlorella Hydrogel Beads for Water Retention and Controlled Release of Humic Acid Hao Li 1,2 , Jin Wang 1,2 , Yu Luo 1,2, *, Bo Bai 1,2, * and Fangli Cao 3 1 Key Laboratory of Subsurface Hydrology and Ec...

water Article pH-Responsive Eco-Friendly Chitosan–Chlorella Hydrogel Beads for Water Retention and Controlled Release of Humic Acid Hao Li 1,2 , Jin Wang 1,2 , Yu Luo 1,2, *, Bo Bai 1,2, * and Fangli Cao 3 1 Key Laboratory of Subsurface Hydrology and Ecological Effect in Aird Region of the Ministry of Education, Chang’an University, Xi’an 710054, China; [email protected] (H.L.); [email protected] (J.W.) 2 School of Water and Environment, Chang’an University, Xi’an 710054, China 3 SCEGC Installation Group Company Ltd., Xi’an 710054, China; [email protected] * Correspondence: [email protected] (Y.L.); [email protected] (B.B.) Abstract: For improving the mechanical strength of controlled release fertilizer (CRF) hydrogels, a novel material of Chlorella was employed as a bio-based filler to prepare chitosan–chlorella hydrogel beads with physical crosslink method. Here, the synthesis mechanism was investigated, and the chitosan–chlorella hydrogel beads exhibited enhanced mechanical stability under centrifugation and sonication than pure chitosan hydrogel beads. Chlorella brought more abundant functional groups to original chitosan hydrogel, hence, chitosan–chlorella hydrogel beads represented greater sensitivity and controllable response to external factors including pH, salt solution, temperature. In distilled water, the hydrogel beads with 40 wt% Chlorella reached the largest water absorption ratio of 42.92 g/g. Moreover, the mechanism and kinetics process of swelling behavior of the chitosan– chlorella hydrogel beads were evaluated, and the loading and releasing of humic acid by the hydrogel


beads as a carrier material were pH-dependent and adjustable, which exhibit the potential of chitosan–
 chlorella hydrogel beads in the field of controlled release carrier biomaterials. Citation: Li, H.; Wang, J.; Luo, Y.; Bai, B.; Cao, F. pH-Responsive Keywords: hydrogel; controlled release; swelling; Chlorella; pH sensitivity Eco-Friendly Chitosan–Chlorella Hydrogel Beads for Water Retention and Controlled Release of Humic Acid. Water 2022, 14, 1190. https:// 1. Introduction doi.org/10.3390/w14081190 Fertilizer is an indispensable part of modern agriculture. The proper application of Academic Editor: Adriana fertilizers can maintain soil fertility and improve crop yields. However, during the volatiliza- Bruggeman tion and leaching process, fertilizer losses have greatly reduced the utilization efficiency, Received: 27 February 2022 increased agricultural costs and threatened environmental pollution, especially in develop- Accepted: 5 April 2022 ing countries. Recently, controlled release fertilizers have received considerable attention Published: 8 April 2022 worldwide with the potential to achieve a partial synchronization of nutrient release and physiological need of plants which can effectively prevent fertilizer losses. Polymer, Publisher’s Note: MDPI stays neutral using as the initial material of hydrogel, has been proven to be an effective prolonged- with regard to jurisdictional claims in release carrier for the advantage of absorbing a high amount of water and release it over published maps and institutional affil- a long period of time in combination with a fertilizer [3–5]. The combination of hydrogel iations. and fertilizer, like a “mini reservoir”, reduces both the environmental impact of fertilizer and evaporation losses. However, a large amount of polymers such as poly(acrylic acid), poly(acrylamide), and copolymer are difficult to biodegrade. Consequently, seeking Copyright: © 2022 by the authors. biodegradable, abundant, and low-cost natural polymers for controlled release fertilizers Licensee MDPI, Basel, Switzerland. have become a priority for engineers. This article is an open access article Chitosan, the product of deacetylation of the natural polysaccharide chitin, is a com- distributed under the terms and monly used biomaterial composed of β-(1-4)-2-acetamido-D-glucose and β-(1-4)-2-amino- conditions of the Creative Commons D-glucose units, with a pKa value varying from 6.3 to 6.5 [7,8]. Chitin, mainly derived from Attribution (CC BY) license (https:// crustaceans such as shrimp and crab, is one of the most plentiful biopolymers on Earth , creativecommons.org/licenses/by/ and hence crab and shrimp shell waste provides a sufficient source for the industrial pro- 4.0/). duction of chitosan. There are three types of reactive functional groups in chitosan, an Water 2022, 14, 1190. https://doi.org/10.3390/w14081190 https://www.mdpi.com/journal/water Water 2022, 14, 1190 2 of 17 amino/acetamido group as well as both primary and secondary hydroxyl groups at the C-2, C-3, and C-6 positions, respectively. The amino group is responsible for differences in structure and physico-chemical properties due to its intra/inter-molecular hydrogen bonds. The chemical and physical properties of chitosan depend on the molecular weight (MW), degree of deacetylation (DDA), and degree of crystallinity, extent of ionization/free amino group. The physico-chemical properties, which include viscosity, solubility, ad- sorption on solids, elasticity, tear strength, and bio-functional activities, are dependent on the molecular weight of the polymer concerned. The molecular weight of chitosan also affects the crystal size and morphological character. The drug release from chitosan hydrogels is related to the molecular weight of chitosan. Generally, as the molecular weight of chitosan increases, the rate of drug release into the biological medium decreases. With exceptional biocompatibility and biodegradability, chitosan has proven to be an effec- tive prolonged-release carrier. For example, Wu and colleagues prepared colon-targeted chitosan/alginate hydrogel beads by using chitosan and sodium alginate as cross-linking agents and used hydrogel beads for a colon-targeted release of doxorubicin hydrochloride (DOX). However, the limited chain flexibility and poor mechanical strength of chi- tosan hydrogels have imposed difficulties on the wide application of chitosan. Generally, cross-linking techniques with crosslinkers and inorganic fillers are effective methods to improve the mechanical strength of chitosan-based hydrogels. Shawky prepared a variety of composite microspheres of chitosan with carbon nanotubes as fillers to improve the removal of mercury (II) from water by chitosan. Some studies have also combined montmorillonite with chitosan to enhance the adsorption capacity of the material. However, these introduced substances were difficult to degrade and are not environmen- tally friendly. Bio-based fillers have the advantages of abundant sources, low cost, and biodegradability, and their use will not create new environmental problems. Bio-fillers obtained from agricultural waste such as sugarcane bagasse, soy protein, wheat straw, and rice husk are also used to reinforce polymers because of their abundance and as an economical solution for waste management. In addition, the physical properties of the bio-based filler, such as hydrophobicity, are also factors to be considered. If the filler and the matrix are not compatible, modification of the filler needs to be considered. Therefore, in order to improve the performance of chitosan hydrogels, it is essential to select bio-based fillers which are biodegradable, compatible with chitosan, and provide excellent mechanical performance. Chlorella is a spherical, unicellular microalga with a diameter of 3–8 µm. It grows by photosynthesis and can be found in both freshwater and seawater, with many species. Chlorella is one of the most widely cultured microalgae species because it is nutrient-rich, ideal for production, fast-growing, and requires only sunlight and nutrients to grow and reproduce in a short period of time, which provides a wide and inexpensive source of Chlorella. Structurally, Chlorella is comprised of the cell wall, cell membrane, cytoplasm, chloroplast, nucleus, vacuoles, and mitochondria. It has a remarkably robust cell wall, mainly composed of cellulose, hemicellulose, proteins, and lipids , which have ample functional groups such as hydroxyl (-OH), carboxyl (-COOH), amino (-NH2 ), and amide groups (-CONH2 ). These hydrophilic functional groups would form a large number of hydrogen bonds with chitosan, resulting in stronger van der Waals and electrostatic effects, making the chemical bonding interactions between Chlorella cells and chitosan stronger than inorganic fillers. Moreover, the tough cell wall of Chlorella has excellent tensile strength, which can provide the required mechanical performance for chitosan hydrogels. In addition, the cell wall of Chlorella not only protects cells from external invasion but also has semi-permeable property, that is, each cell presents as a hollow structure that allows water molecules to permeate through. The space inside the Chlorella cells may act like a miniature reservoir to accumulate water, thereby improving the water retention properties of chitosan hydrogel beads. On the other hand, natural fibers which are often used as bio-based fillers, such as cotton, jute, hemp, and flax require fertile land for cultivation. Therefore, one of the advantages of Chlorella is that it does not compete with agricultural Water 2022, 14, 1190 3 of 17 crops for valuable arable land. To our knowledge, there are few studies using Chlorella as a bio-based filler to enhance the properties of natural polymers. The awareness about the environment and sustainability has led to the demand for alternative petroleum-based polymer products, such as natural polymers reinforced with bio-based fillers. Inspired by these, Chlorella has been selected as a novel material to employ as a bio-based filler to reinforce polymer performance. In this paper, chitosan–chlorella hydrogel beads have been manufactured through the physical crosslinking method. Con- sidering that the cellular structure and biochemical properties of Chlorella can strengthen the mechanical strength and water absorption properties of chitosan hydrogels, we investi- gate the relevant properties of the prepared chitosan–chlorella hydrogels. Similarly, the surface structure of the chitosan–chlorella hydrogel beads was investigated with scanning electron microscopy (SEM), and their structure was characterized with Fourier transform infrared spectroscopy (FTIR). Moreover, we discuss the swelling mechanism and kinetics to study the swelling process in water. Finally, in order to evaluate the feasibility of hydrogel beads for controlled release fertilizers, their pH and temperature stimuli-responsive release behavior for the slow release of humic acid was investigated. 2. Materials and Methods 2.1. Experimental Materials Food-grade Chlorella powder was purchased from Shengqing Biotechnology Co., Ltd. (Xi’an, China). Chitosan (degree of deacetylation ≥ 0.90 and Mw = 700,000–800,000) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) Analytic-grade glacial acetic acid, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2 ), magnesium chloride (MgCl2 ), aluminum chloride (AlCl3 ), sodium hydroxide (NaOH), hydrochloric acid (HCl), etc., were provided by Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) Humic acid with a purity of no less than 90% was purchased from Shanghai Yuanye biotech Co., Ltd. (Shanghai, China). 2.2. Synthesis of Chitosan–Chlorella Hydrogel Beads A 1 g amount of chitosan was added to 50 mL 2% (v/v) acetic acid solution, stirring constantly to dissolve the chitosan. Then, a certain amount of Chlorella powder was added to the chitosan solution and stirred continuously for 1 h. The above blended solution was dropped into a 2 M NaOH solution using a syringe needle with a diameter of 0.6 mm. This results in the formation of alkaline hydrogel beads. The formed chitosan–chlorella beads were left in NaOH solution for another 3 h for gelation. The beads were separated from the NaOH solution with a metal sieve and washed repeatedly until the solution pH became neutral. Then, the resulting sample was dried at room temperature to obtain the chitosan–chlorella hydrogel beads. 2.3. Materials Characterization The dried chitosan and chitosan–chlorella hydrogel beads were ground into pow- der. Then, 1 mg of the powder was blended with 100 mg of KBr in an agate mortar and pressed into flakes after grinding evenly. Afterwards, FTIR infrared spectra were mea- sured at 4000–450 cm–1 wavenumber in a Nicolet FTIR spectrometer. Surface morphology, shape, and size of the chitosan–chlorella hydrogel beads were investigated by scanning electron microscope. 2.4. Degree of Swelling Measurements A known weight of dry hydrogel sample was submerged in 250 mL swelling media (distilled water, various salt solutions, and buffer solutions with desired pH) and remained at room temperature for 4 h until the swelling equilibrium was reached. The sample was Water 2022, 14, 1190 4 of 17 removed and absorbed the excess water with filter paper and weighed. The degree of swelling was obtained by the following formula: We − Wi Sd = (1) Wi where We and Wi are the weights of the hydrogel beads’ swelling equilibrium and initial weights, respectively. In other words, the degree of swelling is calculated according to the grams of water absorbed per gram of sample. 2.5. Determination of Mechanical Stability The mechanical stability of chitosan–chlorella hydrogel beads was judged by the response of the samples to ultrasonic vibration and centrifugation at high speed. The dried samples were left in distilled water for 4 h to reach swelling equilibrium. Then, a certain mass of swelling hydrogel beads was centrifuged at 4000 rpm or ultrasonified at 40 kHz, respectively. The mass of samples retained was weighed at distinct time intervals (t; min). The weight retention rate was obtained by the following formula: Wa Weight retention (%) = × 100 (2) We where We and Wa are the wet weights of hydrogel beads before and after centrifugation or ultrasonification, respectively. 2.6. Loading and Slow-Release Efficiency for Humic Acid To study the potentiality of chitosan–chlorella hydrogel beads as controllable loading and release carriers, humic acid was selected as a model fertilizer. Humic acid was loaded into the hydrogel beads with 40 wt% Chlorella content, so as to explore its feasibility in the controllable loading and release system. The 0.5 g dried hydrogel beads were placed in 50 mL 30 ug/mL humic acid with pH from 4 to 12 and then shaken in a constant temperature shaker at 120 rpm for 30 min at 30 ◦ C. The humic-acid-loaded hydrogel beads were oven dried at 30 ◦ C after filtration separation. The concentration of humic acid solution in the supernatant was calculated by absorbance at 259.6 nm wavelength with a 752 UV-vis spectrophotometer. The loading efficiency of humic acid was calculated by the following equation: C0 − C1 Loading efficiency (%) = × 100 (3) C0 where C0 and C1 are the concentrations at the initial and end of the loading of humic acid, respectively. The humic-acid-loaded samples were immersed in distilled water with various pH and shaken in a constant temperature shaker at 120 rpm for 3 h. The concentration of humic acid in the mixed solution was determined at intervals, and the release efficiency of humic acid was calculated by the following equation: 2C2 Release efficiency (%) = × 100 (4) C0 − C1 where C2 is the concentration of humic acid after release. 3. Results and Discussion 3.1. Synthesis and Characterization of Chitosan–Chlorella Hydrogel Beads The preparation mechanism of the chitosan–chlorella hydrogel beads is shown in Figure 1. Chitosan, with a pKa value of about 6.3, can be dissolved in dilute acid, but not in water or alkaline medium because the amino group (-NH2 ) in the structural unit can be reacted with acid readily and converted into soluble protonated amino group (-NH3 + ).  Water 2022, 14, 1190 5 of 17 Therefore, an acidic solution was often used as the solvent for dissolving chitosan. In this study, a 2% acetic acid solution was used to dissolve chitosan. After a long time stir- ring, chitosan powder was completely dissolved in acetic acid solution, during which the hydroxyl, amino, and N-acetylamino groups distributed on the chitosan macromolecule chain interact to form various intra- and intermolecular hydrogen bonds. As a result, the molecular chains of chitosan were intertwined under the action of hydrogen bonding and van der Waals forces, thus forming a three-dimensional network structure. Meanwhile, the -NH2 group of glucosamine monomer on chitosan molecular chains were protonated into positively charged -NH3 + groups in acidic environment, and electrostatic repulsion between -NH3 + groups expanded the three-dimensional network. Chlorella powder was subsequently added into chitosan solution, the expansion caused by electrostatic repulsion made it easier for Chlorella cells to disperse and embed into the chitosan network uniformly. Then, the chitosan–chlorella mixture solution was dripped into sodium hydroxide solu- tion through a needle. At alkaline conditions, -NH3 + groups were deprotonated, and the solubility of chitosan was reduced and precipitated out of the solution, resulting in the formation of hydrogel beads. During the synthesis process, the changing process from high viscosity droplets to hydrogel beads can be clearly observed, and these changes in its shape Water 2022, 14, x FOR PEER REVIEWand morphology confirmed the formation of hydrogel. Therefore, it can be concluded that 6 the Chlorella cells were successfully encapsulated into the chitosan cross-linked network and the chitosan–chlorella hydrogel beads were synthesized. Figure1. 1. Figure Mechanism Mechanism of formation of formation of chitosan–chlorella of chitosan–chlorella hydrogel hydrogel beads. beads. The FTIR spectra of chlorella, chitosan beads, and chitosan–chlorella hydrogel beads are shown in Figure 2. For chitosan, the peaks at 1659 and 1326 cm−1 are attributed to the –NH2(a) deformation vibration and C-N stretching vibration, respectively. The characteristic absorption band at 3362, 1598, and 1095 cm−1 are attributed to O–H hydroxyl lative transmittance (%) group, N–H angular deformation, and C–O–C in glycosidic linkage, respectively. For 1326 1598 1659 (b) Chlorella, the broad peak at 3301 cm−1 is due to the stretching vibration of intermolecular 3362 1095 1401 1241 1079 1546 (c) 3301 1658 16 107 15 34 Water 2022, 14, 1190 6 of 17 hydroxyl, and the peak at 1079 cm−1 is attributed to the C-O stretching vibration, which indicate the presence of abundant hydroxyl groups on Chlorella. Furthermore, the characteristic absorption band at 1658, 1546, 1401, and 1241 cm−1 are from C=O stretching vibration (amide I), N-H bending vibration (amide II), and C-N absorption band (amide III) in the amide, respectively. In the FTIR spectra of chitosan–chlorella hydrogel beads shown in curves c and d, hydrogel beads with 20 wt% and 40 wt% Chlorella retained most of the characteristic peaks of chitosan and Chlorella. However, the absorption peaks of chitosan at 3362, 1095 cm−1 and Chlorella at 3301, 1079 cm−1 shifted in different degrees. Meanwhile, the absorption peaks of chitosan at 1659 and 1326 cm−1 were also shifted. The shift in the characteristic peaks indicated that in the three-dimensional network of hydrogel beads, chitosan and Chlorella cells were connected and entangled together mainly through Figure 1. Mechanism intermolecular of formation hydrogen bondsof chitosan–chlorella between hydrogelgroups, amino and hydroxyl beads. which result in a stable three-dimensional network. (a) Relative transmittance (%) 1326 1598 1659 (b) 3362 1095 1401 1241 1079 1546 (c) 3301 1658 1660 1072 1594 3425 (d) 1068 1643 1592 3421 4000 3500 3000 2500 2000 1500 1000 Wavenumber (cm−1) Figure 2. FTIR spectra of (a) chitosan, (b) Chlorella, (c) 20 wt% chitosan–chlorella hydrogel beads, and Figure 2. FTIR spectra of (a) chitosan, (b) Chlorella, (c) 20 wt% chitosan–chlorella hydrogel beads, (d) 40 wt% chitosan–chlorella hydrogel beads. and (d) 40 wt% chitosan–chlorella hydrogel beads. Figure 3 provides the particle size image and SEM micrographs, which deliver the Figure 3 provides information on the size, the particle shape, and size surfaceimage and SEM topography micrographs, of the hydrogel beads. which deliver th It can be seen from information on Figure 3a that the size, the diameter shape, and surface of swollen chitosan–chlorella topography hydrogel of the hydrogel beadsIt can b beads. seen from Figure 3a that the diameter of swollen chitosan–chlorella hydrogelmm. had an average size of 3 mm, while the average diameter of dried beads is about 0.9 beads had The increase of particle size indicated that the beads have a certain swelling ability. It an average size of 3 mm, while the average diameter of dried beads is about 0.9 mm. Th can be seen that Chlorella cells are clustered with a diameter of 3–4 microns in Figure 3b. increase of particle size hydrogel The chitosan–chlorella indicatedbeads that shown the beads have3d in Figure a certain swelling had a rougher ability. surface andIt can b seen that Chlorella exhibited cellsshrinkage more severe are clustered resultingwith fromadrying diameter of 3–4 compared microns to the chitosan inhydrogel Figure 3b. Th chitosan–chlorella beads with more hydrogel beads shown perfect spherical shape ininFigure Figure3c.3d had a rougher However, the shrunkensurface and exhib beads itedcould morestillsevere recovershrinkage to full spherical shape after resulting fromswelling, dryingwhich was attributed compared to the to the three- chitosan hydroge dimensional network of hydrogel and stability of Chlorella cells. Figure 3e,f beads with more perfect spherical shape in Figure 3c. However, the shrunken beads could were the surface microtopography images of chitosan hydrogel beads and chitosan–chlorella hydrogel still recover to full spherical shape after swelling, which was attributed to the three-di beads, respectively. Chitosan–chlorella hydrogel beads had more pores on the surface, with mensional network the average diameterof hydrogel andbeing of these pores stability aboutof0.2 Chlorella µm. This cells. Figure porous 3e,f were structure broughtthe surfac microtopography images of about excellent adsorption chitosan properties hydrogel to the hydrogelbeads beads.and chitosan–chlorella In conclusion, landfilling ofhydroge beads, respectively. Chlorella Chitosan–chlorella cells enhanced the performance ofhydrogel beads hydrogel beads and had more exerted thepores on the advantages of surface composite materials.  with the average diameter of these pores being about 0.2 μm. This porous struc brought about excellent adsorption properties to the hydrogel beads. In conclusion, l Water 2022, 14, 1190 filling of Chlorella cells enhanced the performance of hydrogel beads and7 ofexerted 17 the vantages of composite materials. Figure3.3.(a)(a) Figure Particle Particle size size imageimage of chitosan–chlorella of chitosan–chlorella hydrogel hydrogel beads beads swelling swellingand equilibrium equilibrium dried; and dried; (b) Clusters of Chlorella cells; (c) SEM micrographs of chitosan hydrogel beads; (b) Clusters of Chlorella cells; (c) SEM micrographs of chitosan hydrogel beads; (d) SEM micrographs (d) SEM crographs of of chitosan–chlorella chitosan–chlorella hydrogel beads; hydrogel beads; (e) surface (e) surface microstructure of microstructure of thebeads; the chitosan hydrogel chitosan hyd gelsurface (f) beads; (f) surface microstructure microstructure of the chitosan–chlorella of the chitosan–chlorella hydrogel beads. hydrogel beads. 3.2. Mechanical Stability of Chitosan–Chlorella Hydrogel Beads 3.2. Mechanical Stability of Chitosan–Chlorella Hydrogel Beads The mechanical stability of the chitosan–chlorella hydrogel beads was measured by the wetThe mechanical weight retention stability of the chitosan–chlorella under ultrasound hydrogel and centrifugation. Figure beads 4a shows thewas water measure the wet weight retention of samplesretention underChlorella with different ultrasound contentand centrifugation. after a certain time of Figure 4a shows the w centrifugation. The van derofWaals retention samplesforces anddifferent with hydrogenChlorella bonding,content which are widely after present a certain time between of centrifuga hydrogels and water molecules, determined the water retention The van der Waals forces and hydrogen bonding, which are widely present properties of hydrogels.between In the first 20 min, the water retention rate decreased sharply, and then gradually slowed drogels and water molecules, determined the water retention properties of hydrogels down. The reason for this phenomenon was that, the hydrogel beads contained more In the bound weakly first 20water min,that thewas water retention easier lost, andrate withdecreased the increasesharply, and or of centrifugal then gradually slo ultrasonic down. time, theThe reasonoffor proportion thiswater bound phenomenon wastothat, that is difficult thebecame separate hydrogel beads higher, contained m resulting weakly in a slowerbound water that dehydration rate.was easier After lost, and with centrifugation for 60the increase min, of centrifugal the weight retention ofor ultras chitosan time, the hydrogel beadsof proportion containing bound waterChlorella wasishigher that thantothat difficult Chlorella,higher, withoutbecame separate and resu the higher the content of Chlorella in the beads, the higher the weight retention in a slower dehydration rate. After centrifugation for 60 min, the weight retention of of beads. In other words, the presence of Chlorella enhanced the mechanical stability of hydrogel beads. tosan hydrogel beads containing Chlorella was higher than that without Chlorella, and The cross-linking of Chlorella with the chitosan network has strong hydrogen bonding higher the the interactions, content of Chlorella hydrogen in the bonds served as abeads, the higher crack bridge the to retain an weight retention intact structure and of bead other words, stabilize the presence the deformations of Chlorella For of centrifugation. enhanced example,the Cong mechanical stability of hydr et al. have concluded beads. that the The cross-linking intertwined of Chlorellanetwork hydrogen-bonding with theofchitosan hydrogelnetwork has strong could effectively relaxhydrogen the b locally applied stress and dissipate the crack energy to improve the mechanical ing interactions, the hydrogen bonds served as a crack bridge to retain an intact struc strength of hydrogel beads. On the other hand, the natural toughness of the cell wall of Chlorella and stabilize the deformations of centrifugation. For example, Cong et al. have conclu is resistant to the compressive forces brought about by centrifugation, preventing the that thebeads hydrogel intertwined from beinghydrogen-bonding severely compressed. network of hydrogel could effectively relax locally applied stress and dissipate the crack energy to improve the mechanical stre of hydrogel beads. On the other hand, the natural toughness of the cell wall of C rella is resistant to the compressive forces brought about by centrifugation, preventin hydrogel beads from being severely compressed. Similar results were obtained from the ultrasonic experiments of chitosan–chlo hydrogel beads as shown in Figure 4b. After ultrasonication for 60 min, water reten  of the chitosan hydrogel network. Therefore, the hydrogen bond cross-linked network formed by chitosan with Chlorella could better withstand ultrasonic fragmentation and keep the stability of beads. In summary, filling Chlorella into the hydrogel network with the effect of intermolecular forces can effectively reduce the water loss and improve the Water 2022, 14, 1190 mechanical stability. 8 of 17 (a) (b) Figure4.4.Weight Figure Weightretention retention of of chitosan–chlorella chitosan–chlorella hydrogel hydrogel beads beadscentrifuged centrifugedatat4000 4000rpm rpm(a)(a)and and ultrasonified at 40 kHz (b). ultrasonified at 40 kHz (b). 3.3. Similar Swellingresults Behavior of Chitosan–Chlorella were obtained from theHydrogel Beads ultrasonic experiments of chitosan–chlorella hydrogel beads as shown 3.3.1. Equilibrium in Figure Swelling Ratio at4b.Various After ultrasonication pH Solutions and for 60 min, water Pulsatile retention of Behavior the samples with higher Chlorella content was significantly higher than those with lower It is well known that the swelling behavior of hydrogels is significantly related to the Chlorella contents. This indicated that the hydrogel beads formed by Chlorella with chitosan pH of the swelling medium [34,35]. Therefore, the variety of the swelling ratio of chitosan– were more stable than the pure chitosan hydrogel beads in mechanical properties. The chlorella hydrogel beads based on pH change was investigated, and the swelling medium hydrophilic functional groups on the surface of Chlorella cells were linked together by was buffer solutions with pH ranging from 2 to 10 at room temperature. In this study, the strong hydrogen bonding interactions with -OH and -NH2 groups on chitosan; thereby, the influence of ionic strength during the swelling process was avoided by using HAc-NaAc crosslinking of Chlorella cells and chitosan network increased the density of the crosslinked buffer solution, and the pH was controlled only by the dropwise addition of HCl or NaOH network and improved the physical strength of chitosan–chlorella hydrogel beads. In solution. other words, the presence of abundant hydrogen bonds reinforced the skeletal structure Figure 5a demonstrates the swelling behavior of hydrogel beads with different con- of the chitosan hydrogel network. Therefore, the hydrogen bond cross-linked network tents of Chlorella in the buffer solution of pH from 2 to 10. The chitosan–chlorella hydrogel formed by chitosan with Chlorella could better withstand ultrasonic fragmentation and beads reached maximum swelling at pH 6~8, and the order of the swelling ratios for sam- keep the stability of beads. In summary, filling Chlorella into the hydrogel network with ples the withofdifferent effect Chlorellaforces intermolecular content canwas: 40 wt%reduce effectively > 30 wt% > 50 wt% the water loss>and 20 wt% improve> 0 wt%. the When the Chlorella mechanical stability. content was lower than 40 wt%, the swelling degree increased with the increase in the Chlorella content. This is because the Chlorella cell may serve as a site for the storage 3.3. of water Swelling in the Behavior cross-linked network, of Chitosan–Chlorella andBeads Hydrogel the water molecules enter the intracel- lularEquilibrium 3.3.1. of Chlorella after entering Swelling Ratiotheatcross-linked Various pH network Solutionsby osmosis. and This Pulsatile suggests that the Behavior presence of Chlorella enhanced the swelling capacity of the hydrogel It is well known that the swelling behavior of hydrogels is significantly beads. When the pH related to is lower than 6, the swelling degree increases with the increase in the the pH of the swelling medium [34,35]. Therefore, the variety of the swelling ratio ofpH value. However, when the pH exceeds chitosan–chlorella 8, thebeads hydrogel swelling baseddegree on pHstarted changeto was go down. The reason investigated, and for the this result swelling was that medium waswhen bufferthe pH value solutions with pHof the external ranging from 2solution was temperature. to 10 at room lower than the pKa In this study, the influence of ionic strength during the swelling process was avoided by using HAc-NaAc buffer solution, and the pH was controlled only by the dropwise addition of HCl or NaOH solution. Figure 5a demonstrates the swelling behavior of hydrogel beads with different contents of Chlorella in the buffer solution of pH from 2 to 10. The chitosan–chlorella hydrogel beads reached maximum swelling at pH 6~8, and the order of the swelling ratios for samples with different Chlorella content was: 40 wt% > 30 wt% > 50 wt% > 20 wt% > 0 wt%. When the Chlorella content was lower than 40 wt%, the swelling degree increased with the increase in the Chlorella content. This is because the Chlorella cell may serve as a site for the storage of water in the cross-linked network, and the water molecules enter the intracellular of Chlorella after entering the cross-linked network by osmosis. This suggests that the presence of Chlorella enhanced the swelling capacity of the hydrogel beads. When the pH is lower than 6, the swelling degree increases with the increase in the pH value. However, when the pH exceeds 8, the swelling degree started to go down. The reason for this result was that when the pH value of the external solution was lower than the pKa (approximately 6.3) of  crease of swelling degree. Conversely, at high pH (above about 8), the -NH3+ on chitosan was deprotonated to amino group, the influence of electrostatic attraction on crosslinking network is weakened. Besides, the hydrogen bonding induced by -OH and -NH2 and the “screening effect” of excess Na+ in external solution are also the reasons for the decrease Water 2022, 14, 1190 of swelling degree. 9 of 17 In order to study the swelling reversibility of beads, the chitosan–chlorella hydrogel beads with optimal water absorption were selected to be alternately immersed in solutions atchitosan, two pHchitosan’s values of amino 6 and 10 to study groups werepH-dependent protonated, thus swelling reversibility. electrostatic It between attraction can be seen from + -NH3the Figure dilates the5b that the swelling cross-linked networkof of the the chitosan–chlorella hydrogelwhich chitosan–chlorella hydrogel, beadsleads is pH-de- to pendence and reversible an enhancement withHowever, in swelling. a relatively thequick speed. hydrogen bondWhen the beads formed by -OHwere in a buffer on chitosan hinderedwith solution the expansion pH 6, theyof the network. swelled becauseInofaddition, chitosan electrostatic can bedue repulsion dissolved to aminoin acidic protona- solution. The corrosion of the chitosan–chlorella hydrogel beads by the external tion and shrinks at pH 10 due to deprotonation leads to the disappearance of electrostatic solution destroys the repulsion. Thecrosslinking network results showed the structure and leads to behavior swelling–deswelling the decrease of swelling degree.hy- of chitosan–chlorella Conversely, + drogel beadsat exhibited high pH (above good about 8), the -NH3reversibility pH-dependent on chitosanand was could deprotonated to amino be repeated many group, times. the influence of electrostatic attraction on crosslinking network is weakened. Besides, the hydrogen bonding induced by -OH and -NH2 and the “screening effect” of excess Na + in external solution are also the reasons for the decrease of swelling degree. (a) (b) Figure Figure5.5. (a) (a) Swelling ratioof Swelling ratio ofchitosan–chlorella chitosan–chlorella hydrogel hydrogel beads beads in buffer in buffer solutions solutions withwith various various pH; pH; (b) Reversible (b) Reversible swelling swelling of chitosan–chlorella of chitosan–chlorella hydrogel hydrogel beads beads with 40with wt%40Chlorella wt% Chlorella contentcontent at pH 6 at pH and6 pH and10. pH 10. 3.3.2. In order Effect oftoVarious study the Saltswelling reversibility Solutions on Waterof beads, the chitosan–chlorella hydrogel Absorbency beads with optimal water absorption were selected to be alternately immersed in solutions The swelling degree of chitosan–chlorella hydrogel beads influenced by different cat- at two pH values of 6 and 10 to study pH-dependent swelling reversibility. It can be ions can be summarized from Figure 6a. The water absorption of the beads in salt solution seen from the Figure 5b that the swelling of the chitosan–chlorella hydrogel beads is was significantlyand pH-dependence lower than that reversible withina distilled relativelywater. In the When quick speed. swellingtheof ionicwere beads hydrogels, in a which is a common phenomenon, it is usually due to anion–anion electrostatic buffer solution with pH 6, they swelled because of electrostatic repulsion due to amino repulsion caused by theand protonation charge screening shrinks at pH effect 10 dueoftocations and osmotic deprotonation leadspressure difference between to the disappearance of the inside and outside of the hydrogel network. As can be seen electrostatic repulsion. The results showed the swelling–deswelling behavior of from Figure 6a, the chitosan– swelling chlorella ratio of hydrogel hydrogel beads good beads exhibited in different salt solutions pH-dependent wereand reversibility ranked couldfrom highest to be repeated lowest as KCl > NaCl > MgCl2 > CaCl2 > AlCl3. This may be due to the complexation of many times. polyvalent metal cations and hydroxyl groups on chitosan and the influence of ionic 3.3.2. Effect of Various Salt Solutions on Water Absorbency strength. The swelling degree of chitosan–chlorella hydrogel beads influenced by different cations can be summarized from Figure 6a. The water absorption of the beads in salt solu- tion was significantly lower than that in distilled water. In the swelling of ionic hydrogels, which is a common phenomenon, it is usually due to anion–anion electrostatic repulsion caused by the charge screening effect of cations and osmotic pressure difference between the inside and outside of the hydrogel network. As can be seen from Figure 6a, the swelling ratio of hydrogel beads in different salt solutions were ranked from highest to lowest as KCl > NaCl > MgCl2 > CaCl2 > AlCl3. This may be due to the complexation of polyvalent metal cations and hydroxyl groups on chitosan and the influence of ionic strength. Water 2022, 14, x FOR PEER REVIEW 11 of 18 Water 2022, 14, 1190 10 of 17 (a) (b) (c) Figure Figure6. 6. (a) (a) Water absorbencyofofchitosan–chlorella Water absorbency chitosan–chlorella hydrogel hydrogel beads beads in solutions in salt salt solutions (1 mmol/ L); (1 mmol/L); (b) (b) Influence of salt concentrations on the equilibrium swelling degree of chitosan–chlorella hydrogel hy- Influence of salt concentrations on the equilibrium swelling degree of chitosan–chlorella drogel beads beads with 40with wt% 40 wt% Chlorella Chlorella content; content; (c) (c) Salt factors Salt sensitivity sensitivity factors of chitosan–chlorella of chitosan–chlorella hydrogel beadshydro- gel beads with 40 wt% Chlorella content at a concentration with 40 wt% Chlorella content at a concentration of 10 mmol·L. − of 1 10 mmol∙L −1. 3.3.3. The swelling Effect of chitosan–chlorella of Various Temperature hydrogel on Waterbeads in different concentrations of NaCl, Absorbency CaCl2 , and AlCl3 salt solutions was studied as shown in Figure 6b. Two conclusions were Figure obtained 7 demonstrates from Figure 6b: First, thethe effect of theratio swelling temperature on swelling of the hydrogel of chitosan–chlorella beads decreased with hydrogel beads. It is obvious that the swelling degree decreased increasing concentrations of saline solution. This phenomenon was attributed to the fact significantly with the increasing that when the temperature. concentration When of thethesalttemperature was low solution increased, (20~40 °C), the the concentration swelling difference of rate of the chitosan–chlorella the ions between the internal and external hydrogel beads saline with solution of the hydrogel high content network of Chlorella wasbecame significantly smallerthan higher and the thatosmotic without pressure difference Chlorella. Whendecreased. The highwas the temperature salt concentration high (50, 60 resulted °C), the swell- in high external osmotic pressures, and in order to ing rate of hydrogel beads decreased significantly due to the breakage counteract the increaseof in external bonds. hydrogen osmotic pressure, water molecules diffused from the inside to the outside of the hydrogel At lower temperature, the presence of Chlorella makes it easier for water molecules to dif- network, ultimately leading to a decrease in water uptake at equilibrium. Second, the fuse water into the hydrogel. absorption abilityTherefore, of hydrogels theinswelling ratio various salt of hydrogel solution beadsconcentration at the same with high Chlorella content from high is stronger. to low wasWhenNa+ > Ca the2 +temperature increased > Al3 +. The data in Tableto 60 °C, Chlorella 1 showed loststrength that the ionic its bioactivity atishigh ordered as Al3 > Ca2 > Na under the same concentration of the salt solution. Thathigh temperature,+ resulting + + in the swelling ratio of the hydrogel beads with is, Chlo- rella content degree the swelling decreasing of the to the same hydrogel beadslevel as thatwhen decreased withthe lowionic Chlorella strengthcontent. The presence of the external of Chlorella cells in the hydrogel network enhanced the water retention property to a cer- tain extent, which indicated that Chlorella was feasible as a filling biomaterial. Water 2022, 14, 1190 11 of 17 salt solution increased. The influence of ionic strength on the swelling of hydrogel can be calculated through Flory’s equation : 2 (i/2Vu I 1/2 ) + (1/2 − Xi )/V1 S5/3 ≈ (5) Ve /V0 where S is the degree of swelling, i/Vu is the electric charge density of hydrogel, I is the ionic strength of external solution, (1/2 − Xi )/V 1 is affinity of the hydrogel and the swelling medium, and Ve /V 0 is the cross-link density. Table 1. Effect of salt solutions on swelling ratio. Solution (10 mmol/L) Ionic Strength 1 (mol ion/dm3 ) Se (g/g) NaCl 0.01 15.25 CaCl2 0.03 12.52 AlCl3 0.06 9.12 1 I = 12 Σ(Ci Zi2 ), where I is the ionic strength of the saline solution, Ci is each ionic concentration of the saline solution, and Zi is the charge number of the corresponding ion. In order to contrast the sensitivity difference of chitosan–chlorella hydrogel beads to different swelling media, a dimensionless salt sensitivity factor f for 10 mmol·L−1 salt solution was designed and could be calculated by the following formula : Sg f = 1− (6) Sd where Sg and Sd are the water absorption in given fluid and in distilled water, respectively. Figure 6c shows the dimensionless salt sensitive factor of chitosan–chlorella hydrogel beads in various salt solutions with the same concentration of 10 mmol/L. The higher the f value becomes, the greater the salt sensitivity is observed, and the lower the swelling degree in the corresponding fluid. As shown in Figure 6c, the f value is related to the charge number of the metal cation. More specifically, the salt sensitivities of different multivalent saline solutions are in the order of monovalent > divalent > trivalent cations. The AlCl3 solution exhibit the strongest salt sensitivity, and here, the anti-swelling effect of ionic crosslinking plays a more critical role compared to the charge shielding effect. 3.3.3. Effect of Various Temperature on Water Absorbency Figure 7 demonstrates the effect of the temperature on swelling of chitosan–chlorella hydrogel beads. It is obvious that the swelling degree decreased significantly with the increasing temperature. When the temperature was low (20~40 ◦ C), the swelling rate of the chitosan–chlorella hydrogel beads with high content of Chlorella was significantly higher than that without Chlorella. When the temperature was high (50, 60 ◦ C), the swelling rate of hydrogel beads decreased significantly due to the breakage of hydrogen bonds. At lower temperature, the presence of Chlorella makes it easier for water molecules to diffuse into the hydrogel. Therefore, the swelling ratio of hydrogel beads with high Chlorella content is stronger. When the temperature increased to 60 ◦ C, Chlorella lost its bioactivity at high temperature, resulting in the swelling ratio of the hydrogel beads with high Chlorella content decreasing to the same level as that with low Chlorella content. The presence of Chlorella cells in the hydrogel network enhanced the water retention property to a certain extent, which indicated that Chlorella was feasible as a filling biomaterial. Water 2022, 14, x FOR PEER REVIEW 12 of 18 Water 2022, 14, 1190 12 of 17 Figure 7. Swelling ratio of chitosan–chlorella hydrogel beads in distilled water at various temperatures. Figure 3.4.7. Swelling Swelling Kinetics ratio of chitosan–chlorella in Distilled Waterhydrogel beads in distilled water at various tempera- tures. The time-dependent swelling process of the chitosan–chlorella hydrogel beads with various Chlorella contents in distilled water was given in Figure 8a. The swelling degree 3.4. Swelling Kinetics in Distilled Water of samples increased rapidly at first, and then the rate of increase gradually reduced. The The state final time-dependent of swelling swelling equilibriumprocess of the chitosan–chlorella was achieved after approximatelyhydrogel beads 50 min. withthe During various Chlorella contents in distilled water was given in Figure 8a. The swelling swelling process, water needed to continuously overcome the osmotic pressure inside degree of samples increased the hydrogel. Water 2022, 14, x FOR PEER REVIEW Therapidly higher at thefirst, and then difference the rate of of osmotic increasethe pressure, gradually reduced. faster the The water diffused 13 of 18 finalinto state theofhydrogel swelling beads. equilibrium As thewas achieved hydrogel beadsafter approximately swelled 50 min. During by water absorption, the the osmotic swelling process, pressure water between difference needed tothe continuously overcome inside and outside the beads of the osmotic pressure inside continuously the decreased, hydrogel. and the The higherrate swelling the difference of osmoticand gradually decreased pressure, finallythe fasterequilibrium. reached the water diffused into the hydrogel beads. As the hydrogel beads swelled by water absorption, the osmotic pres- sure difference between the inside and outside of the beads continuously decreased, and the swelling rate gradually decreased and finally reached equilibrium. To investigate the swelling kinetics mechanism of hydrogels in distilled water, the pseudo-first-order and pseudo-second-order models were employed to fit the experi- mental data. The pseudo-first-order swelling kinetic model was based on the assumption that adsorption is controlled by diffusion processes, and the adsorption ratio was depend- ent on the number of remaining adsorption sites. This swelling kinetic model could be given by the following equation : K1t ln( Se − St ) = ln S e − (7) 2.303 While the pseudo-second-order kinetics model could be expressed as the following formula:(a) (b) t 1 t = + (8) St K 2 S e2 Se where Se (g/g) is the equilibrium swelling ratio, St (g/g) is the swelling ratio at contact time t (min), and K1 and K2 are the pseudo-first-order rate constant (min−1) and the pseudo- second-order rate constant (g·mg−1·min−1), respectively. As a result, the fitted curves of the swelling process obtained by the pseudo-first- order and pseudo-second-order kinetic models were presented in Figure 8b,c. The values of K1, K2, Se, and the correlation coefficients (R2) are given in Table 2. The R2 value of the pseudo-second-order kinetics model was closer to one. Furthermore, the Se value calcu- lated by the pseudo-second-order kinetic model were closer to experimental data than those obtained by the pseudo-first-order model. It means that the pseudo-second-order model fits the data better than the pseudo-first-order model. Thus, this swelling process better fits (c)the pseudo-second-order kinetic model. Figure Figure (a)(a) 8. 8. Swelling Swellingprocess processof ofchitosan–chlorella hydrogelbeads chitosan–chlorella hydrogel beadsinindistilled distilled water;