Functionalized agarose hydrogel with in situ Ag nanoparticles as highly recyclable heterogeneous catalyst for aromatic organic pollutants

In the present research work, a highly recyclable catalyst of Ag-based agarose (HRC-Ag/Agar) hydrogel was successfully fabricated through a simple and efficient in situ reduction method without the aid of additional surface active agent. The interaction between the rich hydroxyl functional (–OH) groups in agarose and Ag can effectively control the growth and dispersion of Ag nanoparticles (NPs) in the HRC-Ag/Agar hydrogel and keep Ag NPs free from chemical contamination, which also guarantees the reusability of HRC-Ag/Agar hydrogel as catalysts. HRC-Ag/Agar hydrogel without freeze drying and calcination was investigated for their potential applications as highly active/recyclable catalysts in reducing aromatic organic pollutants (p-nitrophenol (4-NP), methylene blue (MB) and rhodamine B (RhB)) by KBH4. The optimal HRC-Ag/Agar-1.9 hydrogel can complete the catalytic reduction of 4-NP within 11 min. Moreover, HRC-Ag/Agar-1.9 hydrogel achieves the high conversion rate (> 99%) through ten catalytic runs. Similarly, HRC-Ag/Agar-1.9 hydrogel was able to achieve a reduction efficiency of RhB at 98% within 17 min and that of MB at 95% within 40 min. The advantages of simple synthetic procedure, no secondary pollution, strong stability and easily separated make the HRC-Ag/Agar hydrogel have great potential prospect for environmental applications.


Introduction
Aromatic organic pollutants are a class of structure containing benzene ring, such as phenol, polycyclic aromatic hydrocarbons and other heterocyclic dye molecules, which are highly toxic, have stable structure, are difficult to decompose naturally, are easy to cause serious environmental pollution (Awad et al. 2020;Zhang et al. 2021). In this regard, researchers are continuously trying to develop cost-effective and efficient catalyst to remove aromatic organic pollutants. However, the separation of product and catalysts has been a difficult problem for many scientists. Therefore, the design and preparation of novel heterogeneous, efficient and recyclable catalysts are extremely urgent.
In recent years, nanotechnology is emerging as a potential substitute for environmental protection and remediation (Salavati-Niasari et al. 2005, 2008Panahi-Kalamuei et al. 2015;Gholami et al. 2017). Nanomaterial-based catalysts have garnered increasing attention in wastewaters treatment applications due to their attractive catalytic capabilities for various organic compounds. New nanotechnology relies on the development of new functional materials; one kind of efficient method is to prepare nano-composite materials, and these may be the innovative combination of known  (Salavati-Niasari et al. 2002;Salavati-Niasari and Banitaba 2003;Salavati-Niasari 2005a, b). Embedding nanoparticles (NPs) into suitable support materials is a common method to obtain high activity and stability of composite materials (Chenab et al. 2020;Nasrollahzadeh et al. 2021;Uddin et al. 2021;Wagner et al. 2021). The synergies between the support materials and NPs are expected to provide the composite materials with superior functionality, increasing the mechanical strength of the composite while reducing the aggregation of NPs, enabling applications in various fields including catalysis, biosensing and environmental remediation. Ag NPs have attracted more attention due to their oxidation resistance, biocompatibility, stability and optical properties (Cai et al. 2022;Ghasemzadeh et al. 2021;Khan et al. 2019). Ag NPs and their composite catalysts are widely used in water treatment, agriculture, medicine, drug delivery, cosmetics, etc. However, the serious particle aggregation caused by the high specific surface energy of Ag NPs poses a barrier to the high activity, recyclability and commercial application of the catalyst. Therefore, improving the stability and longevity of Ag NPs during the catalytic reaction becomes the focus of practical application research (Basumatary et al. 2022;Tajbakhsh et al. 2016). Accordingly, the support plays a vital role in the preparation of composite catalysts with high stability. The homogeneous dispersion of Ag NPs on the supports can effectively prevent the aggregation between adjacent particles and improve its catalytic stability and activity. At the same time, the introduction of support can improve the utilization rate of Ag NPs (Tan Thi et al. 2021). In addition, to find a natural, cheap, stable structure of the supports to fix the catalyst is a more effective means from the angle of green chemistry (Du et al. 2020;Lu & Astruc 2020). The natural cross-linking hydrogels, such as activated agarose, are widely used as an effective support due to their excellent biocompatibility and biodegradability (Serwer 1983;Zarrintaj et al. 2018). Agarose is a linear polysaccharide that consists of alternating D-galactose and 3,6-anhydro-L-galactopyranose units linked by α-(1 → 3) and β-(1 → 4) glycosidic bonds, which can well form a semiflexible water-filled gel featured with abundant interconnected sub-micrometer pores (400-500 nm) and high water retention (> 90%) (Bertula et al. 2019;Felfel et al. 2019). The semi-flexible hydrogels formed by agarose have the advantages of high water retention rate, abundant sub-micrometer pore structure and abundant hydroxyl functional groups (-OH), which can offer a high density of charge carriers and ion migration sites (Khodadadi Yazdi et al. 2020;Yan et al. 2021).
Inspired by this, a highly recyclable catalyst of Ag-based agarose (HRC-Ag/Agar) hydrogel was produced via the in situ reduction of Ag ions without the aid of additional surface active agent during the crosslinking of agarose hydrogel. The interaction between the rich hydroxyl functional (-OH) groups in agarose and the Ag precursor ions can effectively control the growth and dispersion of Ag NP in the HRC-Ag/Agar hydrogel. The properties and structure of the HRC-Ag/Agar were studied by different techniques such as scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectra and Fourier transformed infrared (FTIR). A proposed mechanism for the reaction of agarose with Ag was provided. The catalytic process of HRC-Ag/Agar hydrogel without freeze drying and calcination for the degradation of p-nitrophenol (4-NP), methylene blue (MB) and rhodamine B (RhB) was further investigated to obtain the catalysts with both high catalytic activity and stability.

Preparation of HRC-Ag/Agar hydrogel and HRC-Ag/ Agar aerogel
A typical procedure for the synthesis of HRC-Ag/Agar-1.9 hydrogel was as follows. First, agarose powder (300 mg) was dispersed in deionized water (10 mL) at 95 °C water bath under stirring to form the agarose hydrosol. Then, the aqueous solution of AgNO 3 (196 μL, 300 mM) was added into the agarose hydrosol to form the agarose-Ag + hydrosol. After about 2 min of incubation, an aqueous solution of KBH 4 (98 μL, 3 M, fresh preparation) was added into the agarose-Ag + hydrosol with the 5:1 molar ratio of KBH 4 to AgNO 3 . After stirring for 2 min at 95 °C, the Ag/Agar hydrosol was left to cool down to room temperature to form the HRC-Ag/Agar hydrogel, in which the mass fraction of Ag NPs is 1.9 wt% (HRC-Ag/Agar-1.9 for short).
Similarly, the mass fractions of Ag NPs were changed from 1.9 to 1.0 wt% (HRC-Ag/Agar-1.0 for short) and 3.6 wt% (HRC-Ag/Agar-3.6 for short) when the volumes of AgNO 3 solution were changed from 196 μL to 98 μL and 392 μL, respectively, where the molar ratio of KBH 4 to AgNO 3 stays constant at 5.
The HRC-Ag/Agar aerogel can be obtained from HRC-Ag/Agar hydrogel via refrigerated at − 80 ℃ for 24 h and then freeze-dried in a vacuum environment for 48 h.

Characterization techniques
The scanning electron microscope (SEM) images of HRC-Ag/Agar aerogel were acquired with a field emission scanning electron microscope (Hitachi S-4800). Raman spectroscopy was performed using a Renishaw RM1000-Invia with laser excitation wavelength of 633 nm. The exposure time was 20 s. FTIR spectra were measured by an infrared micro-spectrometer (Thermo Scientific Nicolet iS50 (USA)). The phase structure of the as-prepared samples was analyzed by using X-ray diffraction (XRD) patterns on a Rigaku Ultima-IV system with Cu Kα radiation (λ = 0.15406 nm). X-ray photoelectron spectroscopy (XPS) measurements were carried out with a Thermo Fisher Scientific Escalab 250 XPS spectrometer, using Al Kα X-ray radiation for excitation; all core level spectra were referenced to the C1s neutral carbon peak at 284.6 eV. UV-vis absorption spectroscopy was conducted at room temperature on a T2602S spectrophotometer.

Catalytic reduction of 4-NP, MB and RhB
An aqueous solution of KBH 4 (200 μL, 300 mM) was added to the aqueous solution of 4-NP (4 mL, 0.1 mM) at room temperature with stirring. Then, HRC-Ag/Agar hydrogel (1 g, section thickness approx. 1 mm) was added into the mixture solution. After the addition of HRC-Ag/Agar hydrogel, the reaction solution was removed at different reaction times, and the UV-vis absorption spectroscopy was measured in the scanning range of 200-800 nm. The conversion rate was defined as:conversion rate = A 0 was the maximum absorbance at 395 nm, and A t was the absorbance measured at different reaction time (t); both the values of A 0 and A t were baseline corrected. The reduction of MB (4 mL, 0.1 mM) and RhB (4 mL, 0.1 mM) catalyzed by the HRC-Ag/Agar hydrogel (1 g, section thickness approx. 1 mm) in the presence of KBH 4 was carried out using the similar procedure.

Morphological, structural, chemical and surface properties
A schematic of the fabrication of HRC-Ag/Agar via in situ reduction of Ag ions without the aid of additional surface active agent during the crosslinking process of agarose hydrogel is shown in Fig. 1. The properties of agarose hydrogel are related to the mass fraction of agarose. In order to fully ensure the strength of hydrogel and facilitate the shaping of hydrogel in practical application, the mass fraction of agarose hydrogel in this work is selected as 3 wt %; 3 wt% agarose hydrosol was obtained by stirring agarose powder in deionized water at 95 ℃ for 5 min until complete dissolution. (Fig. 1a, b, and Fig. S1). The AgNO 3 aqueous solution was dispersed into agarose hydrosol, and a stable agarose-Ag + hydrosol (Fig. 1c) was formed based on the interaction between -OH groups and Ag + (Gopinath and Velusamy 2013;Xu et al. 2016). The Ag NP density in the HRC-Ag/Agar was readily controlled by adjusting the amount of AgNO 3 . Then, the agarose-Ag + hydrosol was treated with the aqueous solution of KBH 4 (Fig. 1d). The oxidation-reduction reaction is as follows: This treatment results in the formation and uniform dispersion of Ag NPs due to the interaction between the -OH groups in agarose and the Ag precursor ions (Song et al. 2011;Xie et al. 2007). Finally, the Ag/Agar hydrosol (Fig. 1e) was left to cool down to room temperature to form the HRC-Ag/Agar hydrogel (Fig. 1f).
The optical images of the prepared HRC-Ag/Agar-1.9 are shown in Fig. 1g-j. It can be seen from the overall and sectional view of the hydrogel (Fig. 1g) that HRC-Ag/ Agar-1.9 hydrogel is uniformly yellow-brown, and the Ag NPs are evenly distributed without any apparent color change caused by agglomeration of particles. In addition, it can be seen from Fig. 1h-j that the HRC-Ag/Agar-1.9 hydrogel obtained in this work owns the advantages of good plasticity. It can be uniformly attached to the surface of the container by structural forming process and peeled off to obtain hydrogel devices with complete structure. The HRC-Ag/Agar-1.9 hydrogels are structurally strong enough to be directly used as a reaction vessel (Fig. 1j). The catalysts can be separated from the reaction liquid by dumping directly, to realize the recycling of the catalyst and achieve the purpose of green chemistry.
The morphologies of the surfaces of the HRC-Ag/Agar-1.9 aerogels were investigated by SEM. Low magnification SEM image displayed in Fig. 2a indicated that the aerogels are characterized by rough surface compared with the SEM image of pure agarose (Fig. S2), porous three-dimensional (3D) network structure and irregular pore shape. As can be seen from high magnification SEM images shown in Fig. 2b, c, the nearly spherical Ag NPs with the average size of about 60 ± 10 nm were physically embedded or encapsulated in the HRC-Ag/Agar-1.9 aerogels. The combination of Ag NPs and agarose can minimize the loss and leaching of Ag NPs during the reaction process, as well AgNO 3 + KBH 4 → Ag + 1∕2B 2 H 6 + KNO 3 Fig. 1 Schematic illustration for the syntheses of HRC-Ag/ Agar hydrogel (a-f), the optical image of HRC-Ag/Agar-1.9 hydrogel (g-j) Fig. 2 SEM images (a-c) of as-prepared HRC-Ag/Agar-1.9 aerogels at different magnifications. EDS elemental mapping (d-f) and overlay (g) images of the elements (C, O, Ag) within the HRC-Ag/Agar-1.9 aerogels as the secondary contamination due to the difficulty in separating catalysts. The energy dispersive spectrometry (EDS) elemental mapping analysis further confirmed the existence of Ag NPs and showed that the distribution of Ag element in the HRC-Ag/Agar-1.9 is relatively uniform (Fig. 2d-g). The high uniformity of Ag NPs distribution indicates strong role of agarose in stabilization and distribution of Ag NPs in the hydrogel network. This could be due to the strong interactions of Ag NPs with -OH groups on the agarose chains, as indicated by XRD, XPS, Raman spectroscopy and FTIR.
The phase structures of agarose, HRC-Ag/Agar-1.0, HRC-Ag/Agar-1.9 and HRC-Ag/Agar-3.6 hydrogels were further characterized using XRD (Fig. 3A). The four peaks located at 2θ values of 38.1, 44.4, 64.4 and 77.3° can be indexed on the basis of the (111), (200), (220) and (311) crystallographic planes of cubic Ag NPs, respectively (Hu et al. 2018). Moreover, no Ag oxide peaks were observed, such as the peaks corresponding to 2θ values of 32.1, 46.1, 54.6 and 57.3°, indicating the absence of nonreduced AgNO 3 in the HRC-Ag/Agar (Sarkar et al. 2021). The weak reflections of immobilized Ag NPs are due to the encapsulation and fixation of Ag NPs by agarose and relatively low Ag content in the HRC-Ag/Agar hydrogels, while the reflection intensity of the peaks increases with the increase of silver content from 1.0 to 1.9, and 3.6 wt %. Furthermore, the wide diffraction peak of about 19.3° assigning to crystalline regions of the polysaccharide can also be found in the XRD patterns of agarose, which were formed by the hydrogen-bonding interactions (Hui et al. 2019;Qian et al. 2009). Obviously, this peak was widened and weakened in the patterns of HRC-Ag/Agar hydrogels, indicating the recombination had destroyed the original crystallinity of agarose. More precisely, the interaction between Ag NPs and -OH changes the intermolecular hydrogen and the crystalline regions of agarose. The above experimental results also preliminarily proved the interaction principle of Ag NPs with agarose as proposed in Fig. 1.
XPS was employed to further investigate the surface chemical state of Ag in the HRC-Ag/Agar-1.9 hydrogels. The XPS wide-scan spectra of agarose (Fig. 3B) showed that pure agarose mainly contains carbon (C) at 284.6 eV and oxygen (O) at 532.5 eV. In comparison with pure agarose, two new doublet peaks at 367.7 and 373.7 eV were observed in the XPS wide-scan spectra of HRC-Ag/ Agar-1.9 (Fig. 3B), which can be attributed to Ag3d 3/2 and Ag3d 5/2 , signals, indicating the successful attachment of Ag NPs with agarose. Figure 3C shows the Gaussian-Lorentzian fitting curves of the high-resolution Ag3d XPS spectra of HRC-Ag/Agar-1.9, which could actually be deconvoluted into only two characteristic peaks attributed to metallic Ag(0) NPs at 367.7 and 373.7 eV. Notably, Ag NPs tend to be oxidized if they are not well protected. The stable chemical valence state of the Ag NPs in HRC-Ag/ Agar-1.9 hydrogels was probably attributed to the good protection from agarose.
The physico-chemical characterization and the functional groups involved in its synthesis and stabilization process of the as-prepared HRC-Ag/Agar-1.9 were performed by Raman spectra and FTIR in Fig. 4. As shown in the Raman spectrum of the Agarose and HRC-Ag/ Agar-1.9 (Fig. 4A), the characteristics strong and sharp peak of C-H stretching vibrations was within the range of 2840-2940 cm −1 (Singh et al. 2014). Most notably, in the Raman spectrum of the HRC-Ag/Agar-1.9, a newly observed peak at 135 and 236 cm −1 representing the Ag-O stretching bond can be found (Nešović et al. 2018). This indicates the Ag-O stretching bond was generated through the oxygen atoms of hydroxyl functional groups (-OH) coordinating with the Ag atoms on the surfaces of the Ag NPs.
The mechanism for the reaction of agarose with Ag was further confirmed by FTIR. The FTIR spectra in Fig. 4B demonstrated the characteristic vibrational/bending characteristics of functional groups associated with the Agarose and HRC-Ag/Agar-1.9. The absorption peak of agarose at 1050 cm −1 due to C-O-H bending vibrations was weakened with the introduction of Ag NPs in the HR-Ag/Agar (Cao and Li 2021). The FTIR spectra changes of HRC-Ag/Agar-1.9 compared with agarose further indicated that the -OH group of agarose was effectively involved in the synthesis and stability of Ag NPs.

Catalytic reduction of 4-NP to 4-aminophenol (4-AP)
Ag NPs have been proved to show high catalytic activity in reducing relatively toxic aromatic organic pollutants to their relatively benign compounds (Gao et al. 2020;Sahoo et al. 2018). The catalytic activity of HRC-Ag/Agar was evaluated by reducing 4-NP to 4-aminophenol (4-AP), and the organic dyes (RhB and MB) to their leuco-forms.
The reduction of 4-NP to 4-AP occurred in the presence of KBH 4 is a thermodynamically viable while kinetically unfavorable reaction. The reaction does not occur even if it lasts for 24 h (Fig. S3) using pure agarose hydrogel as catalyst, while the right catalyst can influence the kinetic limit by accelerating the transfer of electrons from donor BH 4 − to acceptor 4-NP. The mechanism of reduction of 4-NP to 4-AP by KBH 4 in the presence of HRC-Ag/Agar is discussed in terms of the Langmuir-Hinshelwood (LH) model illustrated in Fig. 5 (Liu and Zhao 2009;Ningsih et al. 2022).
The catalysts prepared by hydrogel method usually need to undergo post-processing such as freeze drying and high temperature calcination to obtain dry matter as catalyst (Chi 2020). The post-processing method is relatively complicated and will increase the application cost. In order to further prove the advantages of the HRC-Ag/Agar hydrogels, the catalytic performance to 4-NP of HRC-Ag/Agar-1.9 hydrogel (Fig. 6A, 1 g, hydrogel slice with thickness of 1 mm) and HRC-Ag/Agar-1.9 aerogel obtained after freeze-drying (Fig. 6B, 30 mg, spongy block) was compared in detail. All the reduction processes were monitored by measuring the UV-vis absorption spectra of the reaction solution. Note that, the content of Ag in both states is roughly the same. In this work, the concentration of KBH 4 is much higher than 4-NP and can be considered as a constant, so the residual concentration (C t ) to initial concentration (C 0 ) is equal to the ratio of absorbance ratio of A t /A 0 . Therefore, the relationship between 4-NP conversion rate and reaction time can be obtained. As can be seen from Fig. 6C and Fig. S4, both HRC-Ag/Agar-1.9 hydrogel and HRC-Ag/Agar-1.9 aerogel can complete the catalytic reduction of 4-NP within 11 min, and their catalytic conversion rates are basically the same. The diffusion and transfer rate of pollutants affects the catalytic reaction rate by changing the kinetics. The construction of ultra-thin hydrogel slices and aerogel structures are both conducive to improving the contact area of pollutants. In addition, compared with aerogel, thin hydrogel slices are more conducive to rapidly balancing the concentration difference between local and main pollutants. Therefore, HRC-Ag/ Agar hydrogel can be directly used as the catalysts in the reaction process, which avoids the post-treatment process of freeze-drying, effectively reduces the cost and is conducive to the recycling of catalyst. Figure 7A shows the absorption spectra of the 4-NP reduction process by KBH 4 using HRC-Ag/Agar catalysts (All the HRC-Ag/Agar catalysts were kept in hydrogel state, with a total mass of 1 g, and the thickness of hydrogel slice was about 1 mm). The maximum absorption peak of 4-NP solution was located at 297 nm, which was red shifted to 395 nm after the addition of KBH 4 reducing agent. Then HRC-Ag/Agar catalyst was added to activate the hydrogenation catalytic reaction. With the progress of the reaction, the absorbance peak at 395 nm decreased with the disappearance of the yellow color of the solution, and a new absorption peak appeared simultaneously at 300 nm, which was caused by the product (4-AP) formed by the reduction of 4-NP. Note that the UV-vis absorption spectra in Fig. 7A showed only two isosbestic points at 270 and 308 nm. This result suggests that the catalytic reduction of 4-NP exclusively yielded 4-AP, without any other side products. The optical images corresponding to the color change are shown in the inset of Fig. 7A.
To select proper HRC-Ag/Agar catalysts, the catalytic activity of HRC-Ag/Agar with different Ag mass loading was investigated in detail. Figure 7B-D shows the timedependent absorption spectra of the 4-NP in the presence of HRC-Ag/Agar hydrogels with different Ag mass loading and KBH 4 . It is apparent that the peak at 395 nm decreased The optical image of HRC-Ag/Agar-1.9 hydrogel slice with thickness of 1 mm (A) and HRC-Ag/Agar-1.9 aerogel (B). Plot of conversion rate (C) against reaction time for HRC-Ag/Agar-1.9 hydrogel (black) and HRC-Ag/Agar-1.9 aerogel (red) rapidly and is completely wiped out within 13, 11 and 10 min, with the addition of HRC-Ag/Agar-1.0 hydrogels (Fig. 7B), HRC-Ag/Agar-1.9 hydrogels (Fig. 7C) and HRC-Ag/Agar-3.6 hydrogels (Fig. 7D), respectively. The rate of conversion of 4-NP to 4-AP increased with increasing of Ag content, which is due to the increase of catalytically active surface caused by the increase of the number density of Ag NPs. The cost of catalyst is also an important factor to be considered in addition to the conversion rate. By comparing the catalytic reaction processes of HRC-Ag/ Agar with different Ag contents, it was found that HRC-Ag/ Agar-1.9 can guarantee a higher conversion rate and the lower Ag mass loading can reduce the production cost. Therefore, HRC-Ag/Agar-1.9 was selected as the focus of this study.
Recyclability is an important factor to measure the usefulness of a practical catalyst, which can indicate the service life of the catalyst and whether the support can effectively prevent the loss of the active component. In this work, the recyclability was tested as follows: HRC-Ag/Agar-1.9 hydrogel (1 g, hydrogel slice with thickness of 1 mm) was used as catalyst for the catalytic reduction Fig. 7 A Control absorption spectra of 4-NP (black curve), 4-NP after the addition of KBH 4 (red curve), and after the catalytic process of 4-NP to 4-AP (blue curve). Insets show photographs of the reaction mixtures before (left) and after (right) the reduction of 4-NP by KBH 4 , catalyzed by HRC-Ag/Agar. Time-dependent absorption spectra of catalytic reduction reactions of 4-NP using HRC-Ag/Agar-1.0 hydrogels (B), HRC-Ag/Agar-1.9 hydrogels (C) and HRC-Ag/Agar-3.6 hydrogels (D) as catalysts, respectively of 4-NP. After 11 min of reaction, the reaction solution was poured out to leave the catalyst, and the catalyst was washed with deionized water, and then added to the fresh reaction mixture (4-NP + excess KBH 4 ), and the cycle was repeated ten catalytic runs. The UV-vis absorption spectra and conversion rate of 10 times recycled catalytic process with HRC-Ag/Agar-1.9 hydrogel were summarized in Fig. 8 and Table S1. All the characteristic peaks at 395 nm disappeared in all the 10 catalytic cycles, indicating that 4-NP has been completely reduced. Figure 8B depicts the conversion rate about each catalytic process, no significant decrease in catalytic activity was observed through the cycles and > 99% conversion rate were attained within 11 min for each cycle, which proved that HRC-Ag/Agar-1.9 hydrogels show great advantages in reusability of catalysts.
Consistent with the retention of catalytic turnover efficiency, there was no evidence of morphological changes apparent in SEM image ( Fig. 9) of the HRC-Ag/Agar-1.9 hydrogel after the ten cycles. This phenomenon illustrates that Ag attach to the agarose toughly via the interaction between Ag NPs and -OH groups as shown in Fig. 5, and the chemical reactant has no significant damage to the supports.

Catalytic reduction of organic dyes (RhB and MB)
Similar to the reduction of the 4-NP, the activity of the HRC-Ag/Agar-1.9 hydrogel as a catalyst for the reduction of the organic dyes RhB (Fig. 10A) and MB (Fig. 10B) by KBH 4 was further examined. The absorption peaks (550 nm for RhB, 660 nm for MB) successively decreased with reaction time after the addition of catalyst; 98% of RhB (within 17 min) and 95% of MB (within 40 min) were reduced to their leuco-forms. The corresponding optical images of color changes are shown in the inset of Fig. 10A, B. The UV-vis spectrum of 10 times recycled catalytic process of RhB with HRC-Ag/Agar-1.9 was shown in Fig. 10C, and > 95% conversion rate was attained within 17 min for each cycle (Fig. 10D). The above experimental results fully demonstrate that the as prepared HRC-Ag/ Agar hydrogel has a broad application prospect in the degradation of dye pollutants.

Conclusion
In summary, a simple and efficient method for the preparation of HRC-Ag/Agar hydrogel via in situ reduction of Ag ions without the aid of additional surface active agent during the crosslinking process of agarose hydrogels has been demonstrated successfully. The nearly spherical Ag nanoparticles with the average diameter of about Fig. 8 Recyclability of the HRC-Ag/Agar-1.9 hydrogel catalyst: (A) Absorbance spectra of 4-NP during ten cycles testing of the same HRC-Ag/ Agar-1.9 hydrogel, (B) measured conversion rate of 4-NP to 4-AP at each cycle. Sustainable catalytic conversion rate (> 99%) was achieved through ten catalytic runs Fig. 9 SEM image of HRC-Ag/Agar-1.9 hydrogel after 10 times recycled catalytic process 60 nm were formed and immobilized onto the agarose supports in the resulting HRC-Ag/Agar-1.9 hydrogel. It showed that HRC-Ag/Agar-1.9 hydrogel can be directly used as catalysts without freeze drying and calcination and showed good catalytic activity for the p-nitrophenol, methylene blue and rhodamine B reduction. Moreover, HRC-Ag/Agar-1.9 hydrogel can maintain the high conversion rate (> 99%) through ten catalytic runs due to the interaction between the rich hydroxyl functional groups in agarose and Ag which can effectively keep Ag nanoparticles free from chemical contamination. The materials established are economical, the recovery process of catalyst is easy and the procedure is useful with large-scale manufacturing of catalysts for environmental applications in the coming years.
Author contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JW, JZ, YS, XX, MC, PL, WY and YX. The draft of the manuscript was written by JW and YX. All authors commented Fig. 10 Time-dependent absorption spectra of catalytic reduction reactions of the organic dyes RhB (A) and MB (B) using HRC-Ag/ Agar-1.9 hydrogels as catalysts. Insets show photographs of the reaction mixtures before (left) and after (right) the reduction of RhB (A) and MB (B) by KBH 4 , catalyzed by HRC-Ag/Agar-1.9 hydrogels.
(C) Absorbance spectra of RhB during ten cycles testing of the same HRC-Ag/Agar-1.9 hydrogel. (D) Measured conversion rate of RhB to their leuco-forms at each cycle. Sustainable catalytic conversion rate (> 95%) was achieved through ten catalytic runs