Ulvan dialdehyde-gelatin hydrogels for removal of heavy metals and methylene blue from aqueous solution

Hydrogels based on the polysaccharide ulvan from the green macroalgae Ulva fenestrata were synthesized and evaluated as an adsorbent for heavy metals ions and methylene blue. Ulvan was extracted from Ulva fenestrata using diluted hydrochloric acid and recovered by precipitation with EtOH.

Carbohydrate Polymers 249 (2020) 116841 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Ulvan dialdehyde-gelatin hydrogels for removal of heavy metals and methylene blue from aqueous solution T Niklas Wahlströma, Sophie Steinhagenb, Gunilla Tothb, Henrik Paviab, Ulrica Edlunda,* a b Fiber and Polymer Technology, KTH Royal Institute of Technology, Teknikringen 56, SE-100 44, Stockholm, Sweden Department of Marine Sciences, Lovén Centre for Marine Sciences – Tjärnö, University of Gothenburg, SE-452 96, Strömstad, Sweden A R T I C LE I N FO A B S T R A C T Keywords: Ulvan Ulva Hydrogel Heavy metal Methylene blue Adsorption Hydrogels based on the polysaccharide ulvan from the green macroalgae Ulva fenestrata were synthesized and evaluated as an adsorbent for heavy metals ions and methylene blue Ulvan was extracted from Ulva fenestrata using diluted hydrochloric acid and recovered by precipitation with EtOH The extracted ulvan was converted into ulvan dialdehyde via periodate-oxidation and subsequently combined with gelatin yielding hydrogels The hydrogels showed good water-uptake capacity with a maximum swelling degree of 2400 % in water and 900 % in PBS buffer Adsorption tests of methylene blue showed a maximum adsorption capacity of 465 mg/g The adsorption data of methylene blue followed the pseudo-second order kinetics and agreed with the Langmuir adsorption isotherm The maximum adsorption capacity of heavy metal ions was 14 mg/g for Cu2+, mg/g for Co2+and mg/g for Ni2+and Zn2+ indicating that the hydrogels have a stronger affinity for Cu2+ than for Co2+, Ni2+, and Zn2+ Introduction Water pollution is a global environmental problem worldwide, especially in developing countries where it causes the death of a million people every year (Ebenstein, 2012) Many heavy metals are highly problematic water contaminants, often derived from manufacturing facilities such as metalworking operations, mining, and textile industries Emission of heavy metals is of great concern for both human health and the environment, due to toxicity and bioaccumulation in living organisms (Marcoveccio, Botte, & Freije, 2007; Phillips & Russo, 1978) Because of their toxicity to humans, several heavy metals and metalloids are listed in the Agency for Toxic Substances and Disease Registry (ATSDR)’s Substance Priority List (SPL) (Agency for Toxic Substances & Disease Registry (ATSDR), 2017) Dyes are other problematic water contaminants, typically originating from the textile industries (Mondal, 2008; Rastogi, Sahu, Meikap, & Biswas, 2008; Yaseen & Scholz, 2019) The emitted dyes are often resistant to photodegradation, biodegradation, and oxidation, so they will persist in the environment over time Dyes also discolor the seawater, which impedes the light penetration and retards the photosynthesis of water-living organisms Methylene blue is a common cationic dye used in the textile industry Methylene blue is harmful to humans at higher doses and can cause increased heart rate, vomiting, and shock (Ahmad & Kumar, 2010) For the above-mentioned reasons, the development of efficient ⁎ strategies for the removal of water contaminants such as heavy metals and dyes is an important field of research Research regarding development of more efficient water purification techniques is in line with the UN Sustainable Development Goals which targets universal and equitable access to safe and affordable drinking water for all Different strategies for removal of water contaminants are used industrially including physical adsorption, precipitation, ion-exchange resins, electrochemical methods and membrane filtration (Barakat, 2011) Among these methods, physical adsorption is one of the most commonly used Activated carbon is one of the most commonly used absorbents due to its high surface area and ability to adsorb a wide variety of contaminants from water including heavy metals and dyes (Barakat, 2011), however activated carbon is less efficient for heavy metals Organic hydrogels may offer a potent alternative Hydrogels are three-dimensional cross-linked polymer networks with a high water-uptake capacity The water-adsorption capacity of a hydrogel depends on factors such as porosity and the available amounts of binding sites or functional groups in the hydrogel The presence of polar functional groups such as −OH and −COOH in hydrogels also enables physical adsorption of heavy metal ions (Ngah & Hanafiah, 2008) Polysaccharides have gained high interest as a key-component in biobased hydrogels due to their hydrophilicity, high abundance, low cost, and environmental benefits Hydrogels based on polysaccharides have been evaluated as potential adsorbents for heavy metal ions or dyes Various Corresponding author E-mail address: edlund@polymer.kth.se (U Edlund) https://doi.org/10.1016/j.carbpol.2020.116841 Received 21 February 2020; Received in revised form 30 June 2020; Accepted 28 July 2020 Available online 06 August 2020 0144-8617/ © 2020 The Author(s) Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al polysaccharides have been evaluated, including cellulose (Dai & Huang, 2016; O’Connell, Birkinshaw, & O’Dwyer, 2008; Zhao et al., 2019; Zhou, Wu, Lei, & Negulescu, 2014), chitin (Bartczak et al., 2017; Wysokowski et al., 2014), starch (Yu et al., 2018), chitosan (Huang, Hsieh, & Chiu, 2015; Kandile & Nasr, 2009), pectin (Guilherme et al., 2010), and hemicelluloses (Ferrari, Ranucci, Edlund, & Albertsson, 2015; Sun, Liu, Jing, & Wang, 2015) Polysaccharides from the extracellular diatom Didymosphenia geminate has also been reported as an efficient adsorbent of heavy metals (Wysokowski et al., 2017) However, one issue with many commercial hydrogels is that the crosslinker is sometimes either toxic or derived from fossil-based sources (Hu, Wang, Xiao, Zhang, & Wang, 2019) For this reason, the development and implementation of biobased and environmentally friendly crosslinkers is an important field of research Gelatin is a polypeptide derived from collagen isolated from animal tissue such as porcine skin, and may serve as a biobased crosslinker for polysaccharide-based hydrogels Gelatin is non-toxic, biodegradable, and biocompatible, which makes it a promising crosslinker candidate The key strategy for crosslinking between gelatin and polysaccharides is a two-step process The polysaccharide is first oxidized with sodium periodate leading to the formation of aldehyde groups along the polysaccharide backbone The oxidized polysaccharide is then mixed with gelatin in aqueous solution which leads to covalent crosslinking between the aldehyde groups in the polysaccharide and the free amine groups in the lysine amino acid residues in gelatin This type of reaction is generally known as a Schiff-base reaction This strategy has been used in previous studies to synthesize hydrogels based on carboxymethylcellulose (Li, Ye, Li, Li, & Mu, 2016), pectin, (Gupta, Tummalapalli, Deopura, & Alam, 2014), pullulan (Zhang et al., 2019) and alginate (Sarker et al., 2014; Yuan et al., 2017) Sustainable marine biomass such as macroalgae is a so far largely unused source of biomass, which stands out as an alternative feedstock for polysaccharides aside land-based biomass Macroalgae offer several benefits compared with land-based biomass Macroalgae are a low-cost source of biomass that grows quickly and does not require the use of either irrigation or fertilizers, the latter being important in mitigating eutrophication Furthermore, the cultivation of macroalgae does not compete with valuable land areas (Kraan, 2013) The green macroalgae of the genus Ulva is one of the most popular edible seaweeds in the world It is widely distributed along the coastlines across the world Ulva spp are particularly suitable for cultivation due to its ability to thrive under many different growing conditions (Ye et al., 2011) However, the cultivation of Ulva spp is still quite limited It is mainly tank-cultivated (mainly in South Africa and Southeast Asia) rather than cultivated in the wild Aside from being used for food consumption, Ulva spp was recently identified as a potential feedstock for material applications due to its high content of polysaccharides (Taboada, Millán, & Míguez, 2010) Ulvan is such a polysaccharide Ulvan has a sulfated and quite complex structure consisting of a linear backbone with rhamnose (Rha), xylose (Xyl), glucuronic acid (GlcA), and iduronic acid (IdoA) as the main building blocks (Fig 1) The main repeating units in ulvan are β-D-GlcA (1 → 4)-α-L-Rha-3-sulfate, α-L-IdoA (1 → 4)-α-L-Rha-3-sulfate, β-D-Xyl (1 → 4)-α-L-Rha-3-sulfate, and β-D-Xyl-2sulfate (1 → 4)-α-L-Rha-3-sulfate Smaller amounts of other sugars such as galactose (Gal), mannose (Man), and glucose (Glc) can also occur The chemical composition of ulvan varies between different species and is also dependent on the cultivation conditions and the season of harvest (Lahaye & Robic, 2007; Lahaye, 1998; Quemener, Lahaye, & Bobin Dubigeon, 1997; Robic, Gaillard, Sassi, Lerat, & Lahaye, 2009; Robic, Rondeau-Mouro, Sassi, Lerat, & Lahaye, 2009; Wahlström et al., 2020) Ulvan has shown to exhibit both antioxidant (Morelli & Chiellini, 2010) and anticoagulant properties (Zhang et al., 2008) It has also shown high potential as a building block in hydrogels (Morelli & Chiellini, 2010; Morelli, Betti, Puppi, Bartoli et al., 2016; Morelli, Betti, Puppi, & Chiellini, 2016; Dash et al., 2018) Since ulvan can be extracted from the macroalgae Ulva which is one of the most widely Fig The characteristic disaccharide motifs in ulvan, a) β-D-GlcA (1 → 4)-αL-Rha-3-sulfate b) α-L-IdoA (1 → 4)-α-L-Rha-3-sulfate c) β-D-Xyl (1 → 4)-α-LRha-3-sulfate, and d) β-D-Xyl-2-sulfate (1 → 4)-α-L-Rha-3-sulfate distributed seaweeds in the world, ulvan has great potential as a valuable feedstock for the global and large-scale production of bio-based materials Like other seaweed-derived polysaccharides, ulvan also show affinity to heavy metal ions (Lahaye & Robic, 2007), which we hypothesize could merit ulvan as a possible candidate as a component in heavy metal adsorbing hydrogels Still, there are no reported studies regarding the use of ulvan in hydrogels for the adsorption of heavy metals or dyes Our aim was to investigate the potential use of ulvan as a building block in hydrogels for adsorption of heavy metals and dye from aqueous solution, using methylene blue as a model dye The hydrogels were prepared from oxidized ulvan (ulvan dialdehyde) and gelatin Previous studies have shown that ulvan is accessible to periodate-oxidation (de Carvalho et al., 2018) The devised strategy involves 1) to extract ulvan from the green macroalgae Ulva fenestrata, Linneaus 2)to oxidize the extracted ulvan to ulvan dialdehyde, and 3) crosslinking with gelatin in aqueous solution Our hypothesis is that ulvan dialdehyde will react with gelatin in a Schiff-base reaction leading to hydrogel formation We believe that the high content of free carboxylic groups in the formed hydrogels originating from the uronic acid moieties in ulvan dialdehyde could work as binding sites for both heavy metals and cationic methylene blue The formed hydrogels were thoroughly with respect to water-uptake capacity, mechanical properties, and adsorption capacity of heavy metals and methylene blue Correlations of properties and the gelatin:ulvan dialdehyde ratio were investigated Experimental 2.1 Chemicals and materials Hydrochloric acid (37 %, CAS nr: 7647.01-0), gelatin from porcine skin (Bloom-number 170–190, Mw = 40−50 kDa, pl = 7–9, CASnumber: 9000-70-8), methylene blue (CAS-number: 122965-43-9) Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Table Composition and appearance of ulvan dialdehyde-gelatin hydrogels Amount of gelatin (g) Amount of ulvan dialdehyde (g) Mass ratio gelatin: ulvan dialdehyde 0.200 0.050 80:20 0.200 0.085 70:30 0.200 0.133 60:40 0.200 0.200 50:50 0.133 0.200 40:60 500 mL round bottom flask, 2.4 g ulvan and 500 mL of deionized water was added The solution was stirred until complete dissolution of the ulvan To the solution was added 5.33 g of sodium periodate and the solution was allowed to stir at 20 °C under dark conditions for 48 h The reaction was quenched by the addition of 10 mL of ethylene glycol The solution was dialyzed against deionized water for 48 h and freeze-dried The product was recovered as a fluffy white powder with a mass of 1.60 g The sample was stored in a closed container at 20 °C until further use sodium (meta)periodate (99 %, CAS- number: 7790-28-5), ethylene glycol (99 %, CAS-number: 107-21-1), cupper nitrate trihydrate (98 %, CAS-number: 10031-43-3), cobalt nitrate hexahydrate (98 %, CASnumber: 10026-22-9), nickel nitrate hexahydrate (99 %, CAS-number: 13478-00-7) zinc nitrate hexahydrate (99 %, CAS-number: 10196-18-6) deuterium oxide (99 atom% D, CAS-number: 7789-20-0), 2,4 dinitrophenylhydrazine (98 %, CAS-number: 119-26-6) and PBS buffer tablets (MDL-number: MFCD00131855) were obtained from Sigma Aldrich Ethanol (99 %, CAS nr: 64-17-5) was purchased from VWR Chemicals All chemicals where used as received Ulva fenestrata, previously known as Ulva lactuca (Hughey et al., 2019), was collected from Inre Vattenholmen (58°52′37.4″N 11°6′52.1″E) and brought back to the lab within h of collection Collected Ulva fenestrata individuals were rinsed several times in natural seawater to remove grazers and loose epiphytes The seaweeds were placed into cultivation tanks (90 L) under a neutral light cycle (16 h daylight, h darkness) at a light intensity of 140 μE m−2 s-1 The light source was an INDY66 LED 60 W 4000 K 6000 lm The seaweeds continuously received filtered seawater that was passed through μm filters No additional medium or chemicals were added to the water The natural seawater used in the flow-through system was pumped in from the bay outside the Tjärnö Marine Laboratory (58°52′36.4″N 11°6′42.84″E) Thus, the salinity and temperature fluctuated depending on the prevailing weather and seasonal conditions During the cultivation period (March to May 2019), the salinity ranged from 11 to 25 PSU and the temperature increased from to 19 °C The biomass used during our experiments was molecularly identified by DNA barcoding Sequences of the tufA gene unequivocally identified the algal tissue as Ulva fenestrata Molecular identification followed the protocol described by Steinhagen, Karez, and Weinberger (2019) 2.2.3 Preparation of ulvan dialdehyde-gelatin hydrogels The hydrogels were prepared in 10 mL glass vials Five different hydrogels were prepared using different mass ratios of ulvan dialdehyde and gelatin to investigate how the relative ratio between ulvan dialdehyde and gelatin affects the hydrogel properties Three replicates of each hydrogel were prepared The compositions of the hydrogels are given in Table For example, the sample denoted GU-80−20 was prepared by dissolving 0.050 g ulvan dialdehyde in mL 0.01 M PBS buffer (pH = 7.4) at 80 °C In a separate container, 0.20 g gelatin was dissolved in mL 0.01 M PBS buffer (pH = 7.4) at 60 °C Both solutions were cooled to 20 °C before mixing The ulvan dialdehyde solution was added dropwise to the gelatin solution under vigorous stirring The glass vials were sealed and placed on a shaking board at 20 °C overnight to ensure complete gelation of the samples The formed hydrogels were removed from the glass vials by breaking the vials The gels were allowed to dry in air at 20 °C for 48 h in a fume-hood 2.3 Characterization techniques 2.3.1 Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) FTIR spectra of ulvan, ulvan dialdehyde, and the dried hydrogels were recorded between 4000-600 cm−1 at 20 °C with a spectral resolution of cm−1 using a Perkin-Elmer Spectrum 100 FTIR with a triglycine detector and equipped with an attenuated total reflectance crystal accessory (ATR Golden Gate) from Graseby Specac LTD (Kent, England) Corrections were made for atmospheric water and carbon dioxide The obtained spectra were calculated as an average of 64 scans The data were analyzed using PerkinElmer Spectrum software 2.2 Synthesis of ulvan dialdehyde-gelatin hydrogels 2.2.1 Extraction and purification of ulvan from Ulva fenestrata Ulvan was extracted from Ulva fenestrata according to a protocol from our previous study (Wahlström et al., 2020) Briefly, freeze-dried Ulva fenestrata (50 g) was grinded to a fine powder using a Bosch MKM6003 coffee miller (Clas Ohlson, Sweden) and suspended in L of 0.01 M HCl (pH = 2) The mixture was heated to 90 °C for h under continuous stirring The solution was cooled to 20 °C and centrifuged at 5000 rpm for The supernatant was collected and freeze-dried The freeze-dried sample was re-dissolved in 200 mL of deionized water and dialyzed against deionized water for 48 h After dialysis, the ulvan was precipitated by addition of 800 mL absolute EtOH The mixture was centrifuged at 5000 rpm for and the supernatant was discarded The precipitated ulvan was washed with absolute EtOH and air-dried overnight The product was recovered as a white fluffy powder Yield: 9.0 g (18 % (w/w) of the starting biomass) The sample was stored in an airtight container at 20 °C until further use 2.3.2 Nuclear magnetic resonance spectroscopy (13C-NMR) The ulvan and the ulvan dialdehyde fractions were analyzed by 13CNMR Freeze-dried samples (80 mg) were dissolved in 1.0 mL of D2O, and NMR spectra were recorded at 20 °C on a Bruker DMX-500 NMR spectrometer at 500 MHz Spectra were calculated as an average of 16384 scans and the data were analyzed using MestReNova software 2.3.3 Thermal gravimetric analysis (TGA) The thermal stability of the hydrogels was estimated using a Mettler Toledo TGA/DSC Approximately mg of each sample was heated in alumina cups from 40 °C to 800 °C at a heating rate of 10 °C/min under an N2 atmosphere using a flow rate of 50 mL/min The residual water content in the dried samples were calculated as the mass loss in the temperature interval 40−110 °C The data were processed and 2.2.2 Synthesis of periodate-oxidized ulvan (ulvan dialdehyde) The synthesis of ulvan dialdehyde was performed as described by de Carvalho et al (2018), with the below described modifications To a Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al glucuronic acid and iduronic acid was used as the reference The data were processed and analyzed using Chromelon 7.1 analyzed using STARe software From the TGA data, the residual water content in the dried hydrogels was estimated by measuring the weightloss of the samples between 40−110 °C 2.3.8 Rheological behavior of ulvan dialdehyde-gelatin hydrogels Hydrogels were prepared as cylindrical discs with a thickness of 0.2 cm and a diameter of 0.8 cm The storage modulus (G’) and the loss modulus (G’’) of the hydrogels were measured by a TA Discovery Hybrid (DHR-2) rheometer equipped with a 0,8 cm stainless steel Peltier plate at 20 °C The applied strain was set to 0.3 % and the oscillating frequency range was from to 100 rad/s A solvent trap containing deionized water was used during all the measurements to prevents drying of the gels The loss tangent tan(δ) of the hydrogels was calculated with Eq (1) tan(δ) expresses the ratio of the energy loss to the energy stored which provides information about the overall viscoelasticity of the hydrogels 2.3.4 Field-emission scanning electron microscopy (FE-SEM) The morphology of the hydrogels was observed by ultra-high-resolution field emission scanning electron microscopy (FE-SEM) using a Hitachi S-4800 operating at kV The hydrogels were swollen in deionized water for 48 h after which they were frozen in liquid nitrogen and freeze-dried overnight The dried samples were then attached to the sample supports using double-sided adhesive carbon tape and sputtercoated with a nm Pt/Pd layer using a Cressington 208HR under an inert atmosphere 2.3.5 Estimation of aldehyde group content in ulvan dialdehyde The content of aldehyde groups in ulvan dialdehyde was determined colorimetrically by using 2, dinitrophenylhydrazine (DNPH) as described in a previously reported protocol (Tummalapalli & Gupta, 2015) with some modifications First, 2,4 dinitrophenylhydrazine (20 mg) was dissolved in a mixture of 0.3 mL 98 % H2SO4, 3.0 mL EtOH and 6.7 mL H2O To this solution was added 50 mg ulvan dialdehyde and the solution was allowed to react for h at room temperature The solution was filtered through 0.45 μm PTFE filters The absorbance of the solution before and after reaction with ulvan dialdehyde was measured at 370 nm with UV–vis spectroscopy UV–vis spectra were recorded on a UV–vis spectrophotometer (UV-2410) The data was processed using UVProbe The content of aldehyde group was calculated from the decrease in absorbance of the solution after reaction with ulvan dialdehyde G′′ G′ tan(δ ) = (1) 2.3.9 Swelling behavior of ulvan dialdehyde-gelatin hydrogels The swelling behavior of the hydrogels was tested separately in deionized water and 0.01 M PBS buffer Dried hydrogels (0.1−0.3 g) were immersed in 100 mL distilled water or 100 mL PBS buffer and allowed to swell for 48 h at 20 °C The swollen hydrogels were weighed after wiping off excess water with a paper towel The degree of swelling (SD) was calculated using Eq (2) SD (%) = me − m × 100 m0 (2) where me is the mass of the swollen hydrogel and m0 is the initial mass of the dry hydrogel before swelling 2.3.6 Size-exclusion chromatography (SEC) The molecular weights of the ulvan before and after oxidation was measured by SEC as described in our previous study (Wahlström et al., 2020) Dried samples of ulvan and ulvan dialdehyde (20 mg) were completely dissolved in 10 mL of 10 mM NaOH Before injection, the samples were filtered through 0.20 μm PTFE filters The HPLC system was equipped with a WPS-3000SL autosampler, an LPG-3400SD gradient pump, three PSS Suprema columns with pore sizes of 30 Å, 1000 Å and 1000 Å in series (300 × mm, 10 μm particle size) together with a guard column (50 × mm, 10 μm particle size) and a Waters-410 Refractive Index Detector (Wafers, Milford, CA, USA) The mobile phase was 10 mM NaOH and the temperature was kept at 40 °C during the run Pullulan samples with molecular weights averages (Mw) ranging from 342 g/mol to 708,000 g/mol were used as reference samples 2.3.10 Heavy metal adsorption of ulvan dialdehyde-gelatin hydrogels The heavy metal adsorption test was made for Cu2+, Co2+, Ni2+and 2+ Zn Specimens of dry hydrogel (10 mg each) were placed in mL of heavy metal solution containing 30 mg/L (30 ppm) of each heavy metal The pH of the solution was 6.5 Adsorption tests were also done with solutions containing 150 mg/L (150 ppm) of each heavy metal, and for a solution containing 30 mg/L (30 ppm) of each heavy metal where the pH value was adjusted to The samples were left on a shaking board at 20 °C for 24 h after which the hydrogels where removed from the solution The heavy metal mass concentration in each solution, before and after adsorption, was measured by induced coupled plasma optical emission spectroscopy (ICP-OES, iCAP 6500 Thermo Scientific) All heavy metal solutions were diluted 10 times prior to the measurements The percent of adsorption was calculated by using Eq (3) 2.3.7 High-performance anion exchange chromatography with pulsed amperiometric detection (HPAEC-PAD) The composition of monosaccharides and uronic acids of the extracted ulvan were measured by HPAEC-PAD as described in our previous study (Wahlström et al., 2020) Ulvan was hydrolyzed using a modified method described in a previous study (De Ruiter, Schols, Voragen, & Rombouts, 1992) A dried ulvan sample (1.0 mg) was placed in an oven-dried Pyrex tube and mL of M HCl solution in methanol (dried with Na2SO4) was added to the tube The tube was sealed and heated to 100 °C for h in a heating block The solution was neutralized by adding 200 μL of pyridine to the reaction tube, cooled to 20 °C and the solvent was evaporated under flowing N2 gas Hydrolysis was performed by adding mL of M trifluoroacetic acid (TFA) to the dried sample, and the solution was heated to 120 °C for h in a heating block followed by cooling to room temperature The composition of monosaccharides and uronic acids was determined using a HPAEC-PAD (HPAEC-PAD, ICS-3000 Dionex) The column was a CarboPac PA1 (4 × 250 mm) column The eluent was pumped at 1.5 mL/min using a program starting with 0.10 M NaOH and increasing to 0.16 M NaOH and 0,19 M NaAc during the run A mixture of arabinose, rhamnose, galactose, glucose, xylose, mannose, A (%) = c − ce × 100 c0 (3) where ce is the mass concentration (given in mg/L) of heavy metals remaining in the solution after adsorption and c0 is the initial heavy metal mass concentration (given in mg/L) The adsorption capacities (qe) of the hydrogels (given as the amount in mg of adsorbed heavy metal per gram of hydrogel) were calculated using Eq (4) qe = (c0 − ce )⋅V m (4) where ce is the mass concentration (in mg/L) of heavy metals remaining in the solution after adsorption, c0 is the initial heavy metal mass concentration (in mg/L), V is the volume of the solution, and m is the mass of the hydrogel 2.3.11 Methylene blue adsorption of ulvan dialdehyde-gelatin hydrogels The adsorption of methylene blue was tested using an aqueous solution containing either 30, 300, or 1500 mg/L of methylene blue Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al mechanical and thermal properties of the hydrogels were investigated Adsorption tests at higher methylene blue mass concentrations (2000 mg/L and 3000 mg/L) were performed with the sample named GU-4060 (Table 1) Dried hydrogels (10 mg each) were placed in mL methylene blue solution and the solutions were placed on a shaking board at 20 °C for 24 h, after which the hydrogels where removed from the solution The mass concentration of methylene blue in each solution, before and after adsorption, was measured by UV–vis spectroscopy by measuring the absorbance at 668 nm UV–vis spectra were recorded on a UV–vis spectrophotometer (UV-2410) The data was processed using UVProbe software The percent of adsorption and adsorption capacities where calculated using Eqs (3) and (4) 3.1 Characterization of extracted ulvan and oxidized ulvan (ulvan dialdehyde) Ulvan was extracted from the green macroalgae Ulva fenestrata using diluted hydrochloric acid and the supernatant was dialyzed to remove salts and other low-molecular weight compounds The ulvan was recovered from the supernatant by precipitation with EtOH No further purification steps of the ulvan were performed This method was used in our previous study, yielding a ulvan fraction containing 76.8 % (w/w) carbohydrates (Wahlström et al., 2020) The ulvan was recovered as a white solid and the overall yield was 18 % (w/w) from the starting biomass The chemical structure was confirmed with FTIR and 13 C-NMR Monosaccharide and uronic acid analysis with HPAEC-PAD showed the presence of rhamnose, glucuronic acid, iduronic acid and xylose which are all typical building-blocks in ulvan Glucose and trace amounts of mannose was also detected The presence of glucose may be due to co-extracted starch (Lahaye & Robic, 2007) The monosaccharide and uronic acid composition of the extracted ulvan is shown in Supporting Information, Table S1 The extracted ulvan was converted to ulvan dialdehyde by oxidation with sodium periodate The reaction scheme is shown in Supporting Information, Fig S1 This reaction introduces aldehyde groups along the backbone of ulvan More specifically, the reaction takes place at the uronic acid moieties in ulvan by conversion of the vicinal diols into aldehyde groups The reaction took place under dark conditions to prevent the decomposition of the periodate solution upon exposure to light One side-effect of periodate oxidation is that the reaction also leads to depolymerization of the polysaccharide due to cleavage of the glycosidic bonds To evaluate the change in molecular weight before and after periodate oxidation, the molecular weight of ulvan was measured before and after oxidation The extracted ulvan before oxidation showed a high molecular weight (Mw = 761 500 g/mol) However, the molecular weight of the oxidized ulvan (ulvan dialdehyde) was significantly lower (MW = 10 400 g/mol) indicating that the ulvan was depolymerized to a large extent during the oxidation This is in line with a previous study which showed that ulvan undergoes a significantly reduction in molecular weight upon periodate-oxidation (de Carvalho et al., 2018) The total content of aldehyde groups in ulvan dialdehyde was estimated colorimetrically by 2,4 dinitrophenylhydrazine according to a previously reported protocol (Tummalapalli & Gupta, 2015) The aldehyde content was estimated to 1.58*10−3 mol of aldehyde groups per gram of sample Ulvan and ulvan dialdehyde were analyzed by FTIR and 13C-NMR spectroscopy to confirm if the periodate-oxidation was successful The FTIR spectra show many characteristic peaks for polysaccharides The broad peak at 3400-3300 cm−1 corresponds to OHe stretching and the peak at 2900 cm−1 corresponds to C–H stretching The peaks at 1620 cm−1 and 1420 cm−1 correspond to symmetric and asymmetric stretching of carbonyl groups in the uronic acid moieties The strong peaks around 1070−1030 cm−1 corresponds to C-O-C stretching The peaks at 1215 cm−1 and 845 cm−1 corresponds to S=O stretching and C-O-S stretching of sulfate groups (Robic, Bertrand, Sassi, Lerat, & Lahaye, 2009) By comparing spectra of ulvan and ulvan dialdehyde it is clear that a new peak appears at 1725 cm−1 in the spectrum of ulvan dialdehyde (curve b, Fig 2, left) which corresponds to C]O stretching of carbonyl groups in aldehyde, confirming that the oxidation reaction was successful However, the ulvan dialdehyde sample still has a band at 3300 cm−1, which indicates that there are unreacted OH groups left This can be explained by the fact that the different building blocks in ulvan show different reactivity towards sodium periodate A previous study showed that the periodate oxidation of ulvan mainly take place at the uronic acid moieties, while the non-sulfated rhamnose and xylose residues are more resistant towards periodate oxidation (de Carvalho et al., 2018) which leads to unreacted OH groups 2.3.12 Kinetic studies and adsorption isotherms for methylene blue adsorption Adsorption kinetic studies were performed with the sample named GU-40-60 (Table 1) Each sample specimen (45 mg each) was placed in 90 mL of 30 mg/L methylene blue solution The methylene blue concentration was measured after 30 min, h, h, h, h, and 24 h The pseudo-first order model (Eq (5)) and the pseudo-second order model (Eq (6)) were used to describe the adsorption (Zhou et al., 2017) ln(qe − qt ) = ln(qe ) − k1 t (5) t t = + qt qe k2 qe2 (6) where t is the time, k1 is the pseudo-first order rate constant, and k2 is the pseudo-second order rate constant qt is the adsorption capacity (in mg/g) at a specific time and qe is the adsorption capacity after 24 h k1 was estimated by plotting ln(qe-qt) as a function of t and the slope of the line was calculated k2 was estimated by plotting t/qt as a function of t and estimate the slope and intercept of the line The adsorption isotherm of the methylene blue adsorption was estimated with the sample GU-40-60 by placing 10 mg dry hydrogel specimens in separate containers containing 10 mL methylene blue solution with starting mass concentrations of 300, 1500, 2000, and 3000 mg/L, respectively After 24 h, the hydrogels were removed from the solution and the adsorption capacities (qe) were calculated with Eq (4) Two adsorption isotherms were evaluated, the Langmuir isotherm, Eq (7) and the Freundlich isotherm, Eq (8) ce c = e + qe qm KL qm ln(qe ) = ln(KF ) + (7) ln(ce ) n (8) where qe is the adsorption capacity after 24 h, ce is the mass concentration (in mg/L) of methylene blue remaining in the solution after 24 h, qm is the theoretical maximal adsorption capacity KL and KF are the Langmuir and Freundlich constants, respectively, and n is a constant which is related to the heterogeneity of the hydrogel KL was estimated by plotting ce/qe as a function of ce and estimating the slope and intercept of the line KF was estimated by plotting ln(qe) as a function of ln(ce) and estimate the intercept of the line (Zhou et al., 2017) Results and discussion We developed a method for the fabrication of hydrogels based on gelatin and the polysaccharide ulvan extracted from the green macroalgae Ulva fenestrata collected along the Swedish west coast The hydrogel preparation was a three-step process where the ulvan was first extracted from Ulva fenestrata using diluted hydrochloric acid(aq) The isolated ulvan was further oxidized to ulvan dialdehyde with sodium periodate After purification and drying, the ulvan dialdehyde was crosslinked with gelatin in PBS buffer, producing hydrogels The adsorption capacity of heavy metals and methylene blue as well as the Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Fig FTIR spectra (left) and 13 C-NMR spectra (right) of ulvan, a) before periodate oxidation, and b) after periodate oxidation (ulvan dialdehyde) The 13C-NMR spectrum of ulvan (curve a, Fig 2, right) shows many of the characteristic signals for ulvan A table of assigned peaks are given in the Supporting Information, Table S4 The strong signal at 17 ppm corresponds to the carbon in the methyl group in rhamnose moieties The anomeric region (100−105 ppm) shows the characteristic signals for the anomeric carbons in rhamnose, glucuronic acid and iduronic acid The weak signal around 175 ppm corresponds to carbons in the carboxyl groups located at the uronic acid moieties (de Carvalho et al., 2018) By comparing the spectrum of ulvan and ulvan dialdehyde, some significant differences can be observed No signals for aldehyde groups in the range 170−200 ppm were observed in the spectrum for ulvan dialdehyde However, new signals in the range 88−91 ppm appeared in the 13C-NMR spectrum of ulvan dialdehyde which indicates the presence of hemiacetals or gem-diol groups (de Carvalho et al., 2018) Previous studies have shown that periodateoxidized polysaccharides such as ulvan dialdehyde tend to form hemiacetals or gem-diols (hydrated aldehydes) in aqueous solution (Amer et al., 2016; de Carvalho et al., 2018; Fan, Lewis, & Tapley, 2001; Jejurikar et al., 2012) due to the high reactivity of the aldehyde groups formed during periodate oxidation The main reason why the aldehyde peak was observed in the FTIR spectrum and not in the 13C-NMR spectrum is probably because the aldehyde peak is only visible if the sample is free from water (Amer et al., 2016; Fan et al., 2001; Jejurikar et al., 2012) The FTIR spectra was recorded on freeze-dried samples which were substantially free from water and therefore, the aldehyde peak is visible On the other hand, the 13C-NMR spectra were recorded with an aqueous solution of ulvan dialdehyde which may have led to formation of hemiacetals and gem-diols resulting in the appearance of the peaks at 88−91 ppm instead of aldehyde peaks around 170−200 ppm The overall conclusion from the FTIR and 13C-NMR analysis is that the aldehydes groups were formed during the periodate oxidation of ulvan dialdehyde, but the aldehyde groups are in equilibrium with the corresponding hemiacetals and gem-diols in aqueous solution react with the primary amine groups (-NH2) in gelatin originating from the lysine amino acid residues The reaction leads to the formation of covalent C]N bonds between ulvan dialdehyde and gelatin which is known as imine bonds or Schiff-bases Since the crosslinking reaction took place in PBS buffer at pH = 7.4, and the pKa value of the amine groups in lysine is approximately 10.5, the amine groups will be partially protonated (-NH3+) under these reaction conditions, so another possible crosslinking mechanism is physical crosslinking between the NH3+ groups in gelatin and the COO− groups in ulvan dialdehyde Physical crosslinking between the NH3+ groups in gelatin and the SO3− groups in ulvan dialdehyde is also possible Finally, gelatin itself can form a gel in aqueous solution by physical entanglement of the of the gelatin molecules The hydrogel network formed in the reaction between ulvan dialdehyde and gelatin could therefore possibly contain two types of crosslinks and also regions consisting of entangled gelatin chains FTIR spectroscopy was used to investigate if a Schiff-base reaction occurred during the reaction between ulvan dialdehyde and gelatin The FTIR spectra of the hydrogels show characteristic peaks for both ulvan dialdehyde and gelatin (Fig 3) The broad peak at 3400-3300 cm−1 corresponds to OHe stretching and the peak at 2900 cm−1 corresponds to C–H stretching The peaks at 1620 cm−1 and 1420 cm−1 corresponds to symmetric and asymmetric stretching of carbonyl groups in the uronic acid moieties of ulvan dialdehyde The bands at 1215 cm−1 and 845 cm−1 correspond to S=O stretching and C-O-S 3.2 Characterization of the chemical structure of ulvan dialdehyde-gelatin hydrogels Hydrogels were synthesized by mixing ulvan dialdehyde and gelatin in PBS buffer at pH = 7.4 Gelation of the samples occurred after ∼ h, but the samples were left for 16 h to ensure complete crosslinking All the hydrogels appeared as soft and pliable materials that could be easily removed from the reaction containers and handled without damaging the gels (Table 1) The hydrogels became weaker in the water-swollen state but did still not break when handled even after 48 h of swelling The suggested crosslinking reaction is shown in Supporting Information, Fig S2 The aldehyde groups (−CHO) in ulvan dialdehyde Fig FTIR spectra of hydrogels, a) pure gelatin, b) GU-80-20, c) GU-70-30, d) GU-60-40, e) GU-50-50, f) GU-40-60, g) pure ulvan dialdehyde Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al of gelatin shows only one degradation peak at 320 °C corresponding to cleavage of peptide bonds and degradation of gelatin (Sarker et al., 2014) TGA was also used to measure the thermal stability of the hydrogels The TGA curves and corresponding DTG curves are shown in Supporting Information, Figs S5–S6 The DTG curves of the hydrogels show four main degradation peaks The peak around 100 °C corresponding to evaporation of bounded water, the peak at 200 °C corresponding to depolymerization and degradation of ulvan dialdehyde, the peak at 320 °C corresponding to cleavage of peptide bonds and degradation of gelatin, and the carbonization peak at 700 °C Mass loss below 100 °C is due to evaporation of bound water and not due to degradation of the hydrogel Therefore, the hydrogels are thermally stable up to around 200 °C where the ulvan dialdehyde chains starts to degrade From the TGA curves, the residual water-content of the dried hydrogels were estimated to 5% (w/w) The values were calculated by calculating the mass loss of the hydrogels in the temperature interval 40−110 °C stretching of sulfate groups in ulvan dialdehyde (Robic, Bertrand et al., 2009) The peaks at 3100 cm−1 and 1540 cm−1 correspond to N-H stretching and bending in amine groups in gelatin, respectively (Sarker et al., 2014) No clear peak for C]N stretching in Schiff-bases around 1650 cm-1 was observed, probably due to overlap with the carbonyl peak at 1620 cm−1 Furthermore, no peak for aldehyde groups around 1725 cm−1 was observed for the samples GU-80-20 and GU-70-30 indicating that the aldehyde groups in ulvan dialdehyde was consumed during the crosslinking reaction which suggest that a Schiff-base reaction did occur between ulvan dialdehyde and gelatin However, a shoulder peak around 1725 cm-1 was observed for the samples GU-6040, GU-50-50 and GU-40-60 probably due to unreacted aldehyde groups in ulvan dialdehyde This is an indication that the consumption of aldehyde groups in ulvan dialdehyde is incomplete at higher ulvan dialdehyde concentrations 3.3 Rheological behavior of ulvan dialdehyde-gelatin hydrogels The mechanical stability of the hydrogels during stress was evaluated by performing a frequency-sweep experiment All samples show typical behavior for crosslinked networks indicated by the storage modulus (G’) always being higher than the loss modulus (G’’) and the value of tan(δ) is below during the whole frequency-sweep (Fig 4) This is an indication that the elastic response from the hydrogels dominates over the viscous response, which is characteristic for hydrogels Fig also shows that the storage modulus increases with increasing angular frequency indicating that the elastic response from the hydrogels becomes more and more dominant over the viscous response with increasing frequency The storage modulus is strongly dependent on the hydrogel composition By comparing the storage modulus for the samples, GU-80-20, GU-70-30, GU-60-40, and GU-50-50 (Fig 4) with their compositions (Table 1), it can be observed that the storage modulus increases as the mass percentage of ulvan dialdehyde increases One possible explanation is that when the content of ulvan dialdehyde increases, the content of aldehyde groups increases, which increases the number of chemical crosslinks between ulvan dialdehyde chain leading to a higher storage modulus However, the sample with the highest mass percentage of ulvan dialdehyde, GU-40-60 had a lower storage modulus than the samples GU-60-40 and GU-50-50, probably due to that the fact that the sample GU-40-60 contained a lower amount of gelatin (Table 1) which give rise to fewer crosslinks and a lower storage modulus Overall, the data from the rheology experiments suggests that the crosslinking reaction between ulvan dialdehyde and gelatin was successful 3.5 Swelling-behavior of ulvan dialdehyde-gelatin hydrogels The water-uptake capacity of the hydrogels was investigated by swelling in deionized water for 48 h (Fig 6) The sample GU-80-20 had the lowest swelling capacity (SD = 1000 %) and the sample GU-40-60 had the highest (SD = 2400 %) By comparing the hydrogel compositions in Table with the corresponding SD values, it is clear that SD increases as the mass percentage of ulvan dialdehyde in the hydrogel increases One possible explanation is that by increasing the amount of ulvan dialdehyde in the hydrogel, we also increase the number of available binding sites for water molecules, which leads to a higher driving force for water uptake and therefore a higher swelling The swelling behavior was also investigated in PBS buffer by swelling the hydrogels in 0.01 PBS buffer for 48 h Clearly, the hydrogels had lower SD in PBS buffer than in deionized water, which was expected given the higher ionic strength of the PBS solution compared with deionized water GU-80-20 had the lowest swelling capacity (SD = 300 %) and GU-40-60 had the highest (SD = 900 %) The same trend was observed for swelling in deionized water The observed SD in this study is within the range that was reported in previous studies on swelling of ulvanbased hydrogels in PBS buffer (Morelli, Betti, Puppi, Bartoli et al., 2016; Morelli, Betti, Puppi, Chiellini et al., 2016; Morelli & Chiellini, 2010; Dash et al., 2018) Likewise, the reported values of SD are within the range for what has been reported for oxidized polysaccharides crosslinked with gelatin (Sarker et al., 2014; Yuan et al., 2017) 3.4 Morphology and thermal behavior of ulvan dialdehyde-gelatin hydrogels 3.6 Adsorption properties of ulvan dialdehyde-gelatin hydrogels The ulvan dialdehyde-gelatin hydrogels were tested as a potential absorbent for the dye methylene blue Dried hydrogels were immersed in individual methylene blue solutions with starting mass concentrations of 30, 300, and 1500 mg/L, respectively Adsorption tests with higher methylene blue mass concentration (2000 mg/L and 3000 mg/L) were performed with the sample named GU-40-60 The hydrogels were removed from the solution after 24 h and the remaining methylene blue mass concentration was quantified with UV–vis spectroscopy The percent of adsorptions and the adsorption capacities were calculated using Eqs (3) and (4) and are reported in Fig GU-80-20 had the lowest adsorption capacity and GU-40-60 had the highest (Fig 7) By comparing the gel compositions in Table with the corresponding adsorption capacities, it is clear that the adsorption capacity increases as the mass percentage of ulvan dialdehyde in the hydrogel increases One possible explanation is that by increasing the amount of ulvan dialdehyde in the hydrogel, we also increase the number of available COO− and SO3− groups, which could act as binding sites for the cationic methylene blue More available binding sites in the hydrogel will result in higher adsorption It is also evident The morphology of the hydrogels was studied by SEM The hydrogels were swollen in deionized water for 48 h after which they were frozen in liquid nitrogen and immediately freeze-dried overnight to preserve the morphology All samples exhibit a similar morphology consisting of many small pores separated by thin walls (Fig 5) This porous structure is believed to be convenient for penetration of water and small molecule into the hydrogel network leading to efficient water uptake and adsorption of dye and heavy metals The thermal stability of extracted ulvan, ulvan dialdehyde and gelatin was measured by TGA The TGA curves and corresponding DTG curves are shown in Supporting Information, Figs S3–S4 The TGA curves for ulvan and ulvan dialdehyde look similar, indicating that the oxidation of ulvan did not have any impact on the thermal stability The DTG curves of ulvan and ulvan dialdehyde show three degradation peaks, one small peak around 100 °C corresponding to evaporation of bound water, one major peak at 200 °C corresponding to depolymerization and degradation of ulvan, and one small peak at 700 °C, probably due to carbonization of the degradation products The DTG curve Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Fig Rheological behavior of hydrogels showing the storage modulus (G’), loss modulus (G’’) and tan(δ) as a function of angular frequency, a) GU-80-20, b) GU-7030, c) GU-60-40, d) GU-50-50, e) GU-40-60 which will strongly influence the overall adsorption capacity Hence, the adsorption capacities of hydrogels prepared from different polysaccharides cannot be directly compared The adsorption kinetics of methylene blue in ulvan dialdehyde hydrogels was investigated by measuring the adsorption capacity of the sample GU-40-60 as a function of time The hydrogel was placed in 30 mg/L methylene blue solution and the adsorption capacity was measured after 30 min, h, h, h, h, and 24 h As shown in Fig 8a, the adsorption capacity increases quickly during the first h After h, the absorption capacity increases much more slowly Between h and 24 h, the adsorption capacity of the hydrogel is almost constant The pseudofirst order and the pseudo-second order kinetic model were used to investigate the kinetics of the methylene blue adsorption (Fig 8b-c) By comparing Fig 8b and c, it is clear that the adsorption data fits very well with the pseudo-second order kinetic model (Fig 8c) From Fig 8b-c, the kinetic parameters in Eqs (5) and (6) were calculated The kinetic parameters are given in Table S2, Supplementary information The adsorption isotherm for methylene blue adsorption was also evaluated Two different adsorption isotherms were evaluated: the from Fig that the adsorption capacity also depends on the starting concentration of the methylene blue solution For example, the adsorption capacity of the sample GU-40-60 increased from 15 mg/g to 465 mg/g as the concentration of the methylene blue solution increases from 30 mg/L to 3000 mg/L This is most probably because a more concentrated solution of methylene blue induces a stronger driving force for adsorption On the other hand, the percent of adsorption decreased from 98 % to 30 % as the methylene blue concentration increases from 30 mg/L to 3000 mg/L, which indicated a limited number of binding sites in the hydrogels The adsorption capacity of the sample GU-40-60 did not change when the methylene blue concentration was increased from 2000 mg/L to 3000 mg/L indicating that 465 mg/g is the maximum adsorption capacity The maximum adsorption capacity (465 mg/g) obtained in this study is in the range of what has been reported for polysaccharide-based hydrogels in previous studies (Table 2) (Dai & Huang, 2016; Liu, Zheng, & Wang, 2010; Sun et al., 2015; Wang, Wang, & Wang, 2013; Zhou et al., 2014) However, the adsorption capacity of a hydrogel depends on factors such as the number of available binding sites Different polysaccharides may differ largely in chemical structure and number of available binding sites Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Fig SEM-images of ulvan dialdehyde-gelatin hydrogels at x200 magnification, a) GU-80-20, b) GU-70-30, c) GU-60-40, d) GU-40-60 The ulvan dialdehyde-gelatin hydrogels were also tested as a potential absorbent for heavy metals (Fig 10) Dried hydrogels were immersed in a solution containing four different heavy metals, Cu2+, Co2+, Ni2+, and Zn2+ for 24 h The adsorption capacity was tested at two different initial mass concentrations (30 mg/L and 150 mg/L) and two different pH values (3 and 6.5) Adsorption tests at pH = 6.5 was performed without adjusting the pH after dissolving the metal salts to the solution to assess the adsorption capacity when no other ions are present in the solution However, heavy metal polluted industrial effluents are typically more acidic and adsorption tests were hence also conducted at pH = to assess the hydrogels performance under more acidic conditions The adsorption of Cu2+ was constantly higher than the adsorption of Co2+, Ni2+, and Zn2+, so the hydrogels seems to have a stronger affinity towards Cu2+ ions, in agreement with a previous study on ulvan (Lahaye & Robic, 2007) The hydrogels have almost equal affinity towards Co2+, Ni2+, and Zn2+ The adsorption of heavy metals is overall lower at pH = than at pH = 6.5 One possible explanation is that the carboxylic groups in the uronic acid moieties in ulvan dialdehyde are protonated to a higher extent at pH = than at pH = 6.5 The carboxylic groups act as binding sites for heavy metal ions The interaction between the carboxylic groups and the heavy metal ions in solution will be weaker when the carboxylic groups are protonated At pH = 3, the amine groups in gelatin will also be protonated to a higher extent The protonated amine groups (NH3+) could cause electrostatic repulsion of the heavy metal ions, which prevents heavy metal adsorption at lower pH values Consequently, the affinity to heavy metal ions will be lower at pH than at pH 6.5 By comparing the adsorption capacities of the hydrogels, we can see that the sample GU-80-20 had the lowest adsorption capacity and the sample GU-40-60 had the highest The hydrogels reached a maximum adsorption capacity of 14 mg/g for Cu2+, mg/g for Co2+, mg/g for Ni2+, and mg/g for Zn2+ (Fig 10, bottom-right) which is in the lower range of what was previously reported in previous studies for polysaccharide-based hydrogels (Ferrari et al., 2015; Guilherme et al., 2010; Kandile & Nasr, Fig The degree of swelling (SD) of ulvan dialdehyde-gelatin hydrogels in deionized water (white bars) and in 0.01 PBS buffer (grey bars) The values are given as mean values ± standard deviation of three replicates Langmuir isotherm, Eq (7), and the Freundlich isotherm, Eq (8) The Langmuir isotherm model assumes that the surface of the hydrogel is homogenous and that every binding site can adsorb one molecule of methylene blue It also assumes that only a monolayer of methylene blue is adsorbed on the surface (Liu et al., 2010) The Freundlich adsorption isotherm model assumes that the surface of the hydrogel is heterogeneous and it also allows multilayer adsorption (Liu et al., 2010) By comparing Fig 9a and b, it is clear that the Langmuir isotherm model (Fig 9a) gives a much better linear fitting to the data than the Freundlich isotherm model (Fig 9b) This is an indication that the adsorption of methylene blue onto ulvan dialdehyde-gelatin hydrogels follows the Langmuir isotherm model which indicates that only one monolayer of methylene blue is adsorbed The calculated adsorption isotherm parameters are given in Table S3, Supplementary information Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Fig The percent of adsorption (left) and the adsorption capacities (right) of ulvan dialdehyde-gelatin hydrogels for the adsorption of methylene blue at different starting mass concentrations Data are given as the mean values from triplicate measurements Conclusion Table Comparison between the maximum adsorption capacity (qe) of methylene blue obtained in this study and the adsorption capacity of other polysaccharidebased hydrogels Hydrogel composition qe (mg/g) Reference Ulvan dialdehyde/gelatin Cellulose/sepia ink Chitosan/polyacrylic acid Xylan/polyacrylic acid/Fe3O4 Alginate/organo-illite smectite Cellulose nanocrystals/polyacrylamide 465 138 1800 438 1843 326 This work Dai and Huang (2016) Liu et al (2010) Sun et al (2015) Wang et al (2013) Zhou et al (2014) This study involved the preparation of hydrogels based on gelatin and the polysaccharide ulvan extracted from the green macroalgae Ulva fenestrata Ulvan was extracted from Ulva fenestrate, converted to ulvan dialdehyde by oxidation with sodium periodate, and then crosslinked with gelatin in a Schiff-base reaction leading to the formation of hydrogels Hydrogels with different mass ratios of ulvan dialdehyde and gelatin were prepared to investigate how the composition affects the hydrogel properties The hydrogels were tested as a potential absorbent for heavy metals and the dye methylene blue Other important parameters such as morphology, swelling behavior, mechanical properties, and thermal stability were also investigated The mass ratio of ulvan dialdehyde and gelatin in the hydrogel had a huge impact on the hydrogel properties The degree of swelling of the hydrogels increases as the mass percentage of ulvan dialdehyde in the hydrogel increases The highest recorded degree of swelling was 2400 % and 900 % in deionized water and 0.01 M PBS buffer, respectively The adsorption capacity of methylene blue and heavy metals was improved by a higher mass percentage of ulvan dialdehyde in the hydrogel The hydrogels adsorbed up to 465 mg/g of methylene blue The adsorption of 2009; Huang et al., 2015; O’Connell et al., 2008; Sun et al., 2015; Yu et al., 2018; Zhao et al., 2019) However, the hydrogels prepared in these studies were based on either cellulose, chitin, chitosan or hemicellulose which chemical structures differs largely from ulvan dialdehyde in terms of functional groups and monosaccharide composition As discussed previously, the adsorption capacities of hydrogels prepared from different polysaccharides cannot be directly compared since they may differ largely in terms of chemical structure and number of binding sites etc Fig a) The adsorption capacity of methylene blue as a function of time, b) linear fitting curve for the pseudo-first order adsorption kinetic model, c) linear fitting curve for the pseudo-second order adsorption kinetic model 10 Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Fig Linear fitting curves of a) Langmuir adsorption isotherm model, and b) Freundlich isotherm model Fig 10 The percent of adsorption (left) and the adsorption capacities (right) of ulvan dialdehyde-gelatin hydrogels for the adsorption heavy metal solution at different pH values and starting mass concentrations Data are given as the mean values from triplicate measurements thermally stable up to 200 °C Future work will involve development of methods for improving the adsorption properties methylene blue follows the pseudo-second order kinetics and the adsorption data was in good agreement with the Langmuir adsorption isotherm indicating monolayer adsorption The adsorption of heavy metal ions reached a maximum of 14 mg/g for Cu2+, mg/g for Co2+, mg/g for Ni2+, and mg/g for Zn2+ Rheology measurements showed typical behavior for hydrogels indicated by G’ > G’’ for the whole frequency sweep TGA analysis showed that the hydrogels were CRediT authorship contribution statement Niklas Wahlström: Conceptualization, Data curation, Methodology, Formal analysis, Validation, Investigation, Visualization, 11 Carbohydrate Polymers 249 (2020) 116841 N Wahlström, et al Writing - original draft, Writing - review & editing Sophie Steinhagen: Methodology, Data curation, Formal analysis, Writing - original draft, Writing - review & editing Gunilla Toth: Conceptualization, Methodology, Supervision, Funding acquisition, Writing - review & editing Henrik Pavia: Funding acquisition, Methodology, Supervision, Writing - review & editing Ulrica Edlund: Conceptualization, Methodology, Data curation, Supervision, Funding acquisition, Writing - review & editing Jejurikar, A., Seow, X T., Lawrie, G., Martin, D., Jayakrishnan, A., & Grøndahl, L (2012) Degradable alginate hydrogels crosslinked by the macromolecular crosslinker alginate dialdehyde Journal of Materials Chemistry, 22(19), 9751–9758 Kandile, N G., & Nasr, A S (2009) Environment friendly modified chitosan hydrogels as a matrix for adsorption of metal ions, synthesis and characterization Carbohydrate Polymers, 78(4), 753–759 Kraan, S (2013) Mass-cultivation of carbohydrate rich 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block in hydrogels for adsorption of heavy metals and. .. both heavy metals and cationic methylene blue The formed hydrogels were thoroughly with respect to water-uptake capacity, mechanical properties, and adsorption capacity of heavy metals and methylene. .. mL methylene blue solution and the solutions were placed on a shaking board at 20 °C for 24 h, after which the hydrogels where removed from the solution The mass concentration of methylene blue