Sustainable Process for Separating Chitin and Simultaneous

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Sustainable Process for Separating Chitin and Simultaneous Synthesis of Carbon Nanodots from Shellfish Waste using 2 % Aqueous Urea Solution Ramamoorthy Devi, and Raghavachari Dhamodharan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b00877 • Publication Date (Web): 14 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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ACS Sustainable Chemistry & Engineering

Sustainable Process for Separating Chitin and Simultaneous Synthesis of Carbon Nanodots from Shellfish Waste using 2 % Aqueous Urea Solution Ramamoorthy Devi and Raghavachari Dhamodharan *$ Department of Chemistry Indian Institute of Technology Madras, Chennai 600 036, India $This work constitutes a part of Indian Patent Application *Author for Correspondence E-mail for Correspondence: [email protected] Mailing address of all the authors: Department of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India. Abstract This report describes a facile process for separating chitin and simultaneous synthesis of carbon nanodots (CNDs) from shellfish waste using 333.3 mol/m3 (2 weight %) aqueous urea solution under hydrothermal condition (at 150 °C for 1 h). In this process, urea functions as a denaturant base and precursor for the hydrolysis of proteins that are linked with chitin by the glycosidic ester bond. The hydrolysed proteins in turn were used as a nitrogen-rich carbon source for synthesis of CNDs in the same pot. Additionally, this report describes a method for recovery of minerals from crab shell waste by gradient separation. The recovery of calcium carbonate in addition to chitin results in the reduction of acid consumption in the demineralization of crab shell and reduction in the evolution of carbon dioxide, a greenhouse gas. TEM analysis on CNDs demonstrates the formation of quasi-spherical nanodots of size 7 to15 nm. Solid-state NMR, FT-IR, ICP-OES and solid-state UV-Visible absorption spectroscopic analyses, P-XRD, SEM, and TGA studies as well as CHNS elemental analysis, confirm that better quality chitin is separated by this method in comparison to the chemical method that is widely used.

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Keywords: prawn shell waste, crab shell waste, deproteinization, demineralization, urea, hydrothermal treatment, calcium carbonate. Introduction Shellfish (shrimp, prawn, crab, lobster shells) waste from the seafood industries offer an opportunity to transform waste, which contains useful biomolecules such as chitin, protein and calcium carbonate into wealth.1,2 The crustacean shells are abundant and renewable natural resources and therefore the separation of chitin and chemicals from them acquire enormous significance. Globally, about 6 to 8 million tons of shell waste are produced annually. This causes surface pollution, especially in coastal areas. The estimated global annual production of chitin from all natural sources is about 100 billion tonnes.2 More significantly, chitin contains a nitrogen atom per repeat unit and can be an excellent source for the production of a series of nitrogen-containing organic chemicals in a sustainable manner.3,4 Few important chemicals such as N-acetyl glucosamine5, amino acids6, Ncontaining amino sugar alcohol and acetyl monoethanolamine have been synthesized from chitin.4,7,8 Chitin is used in cosmetics9, water treatment, textile and paper industries etc. It plays an important role in biomedical fields such as hemostasis, antimicrobial agents, tissue engineering and drug delivery.10–13 The primary crustacean sources for chitin extraction are prawn (shrimp) and crab shells. Although the components present in these sources are similar, the compositions are different. For example, in prawn shell chitin, protein and minerals are present virtually in equal ratio (~30 %), whereas in the case of crab shell, the major component is calcium carbonate (~ 70 %) with chitin and protein being present in smaller quantity and in equal ratio (~15 %). Currently, large-scale production of chitin from the shell waste is done by the chemical method, independent of the composition of crustacean shells and it consists of two harsh chemical conditions in sequence: demineralization and deproteinization using a mineral acid and concentrated sodium hydroxide, respectively.14,15 This process enables the separation of chitin alone while eliminating calcium carbonate as calcium salt, an effluent. For example, to separate 100 g of chitin from crab shell waste, ~ 1 L (972 mL) of 35% by weight of hydrochloric acid is required. In addition, it releases 220 g (112 L at STP) of carbon dioxide into the atmosphere16 which was trapped by crustaceans during the mineralization process.17 In fact, mineralization is sequestration of carbon dioxide by aquatic animals carried out for the formation of a reinforced exoskeleton.17 The chemical process of separation of chitin is


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destructive, wasteful, expensive and hazardous to the environment and is known to produce a very large quantity of contaminated water. There have been several alternative reports on deproteinization of chitin. Biotechnological method is thought to be a green alternative method for chitin extraction, which may enable the recovery of protein as amino acids.18–23 But the lack of efficiency in deproteinization required subsequent chemical treatments.24,25 Effective elimination of protein is important for biomedical application as the fraction of people allergic to crustacean proteins is large.25 It has been reported that the limit of residual protein content for pharmaceutical grade chitin/chitosan is 0.3 %.26 Recently, treatments using ionic liquids,27–29 deep eutectic solvents,30 subcritical water31 and hot glycerol pre-treatment32 method were reported for isolation of chitin. However, the protein present in the shells, which are important source of carbon and nitrogen, has been given less consideration in these methods. In so far as mineral recovery from the crustacean shells is considered calcium carbonate is separated by burning organic matter present in shells of the chicken egg, cockle and oyster, at high temperature, since they possess less organic content (~5 %).33–36 Although crab shell can be considered as a source for mining calcium carbonate in addition to chitin, the burning of ~30 % organic matter present and recovering calcium carbonate (70 %) would be energy intensive and could produce undesirable air pollutants. On the other hand, if the proportion of the organic content in crab shells can be reduced then recovery of calcium carbonate could be carried out as in the case of oyster shells. The facile and sustainable separation of chitin, protein and calcium carbonate from the shells of shellfish is a challenging task. The best method known to date for commercial production of chitin is the chemical method and it is not sustainable due to the use of mineral acid, caustic alkali, large quantity of water, the discharge of effluents such as calcium chloride and amino acids that are not viable for treatment. A simpler method of deproteinization and demineralization or one of the two with effective use of the organic content present in the shells is the need of the hour. We report here a simpler method for separation of chitin. The use of dilute ammonia, in contrast to the use of strong aqueous and hot caustic alkaline treatment for at least a day under reflux in the commercial process for deproteinization, makes our process novel and simpler. At the end of the process, ammonia can be driven out of the system by using compressed air while the use of sodium hydroxide would necessitate neutralization followed by removal of salts formed as effluent. The amino acids formed upon deproteinization with aqueous ammonia is used as a carbon source to prepare carbon nanodots, simultaneously and in a single-pot, thus effectively utilizing the protein present in 3 ACS Paragon Plus Environment


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the crustacean shells. In the second step of the treatment (following hot aqueous ammonia) citric acid is used for demineralization. The calcium citrate formed in this process can be used directly in contrast to the chemical process where calcium chloride is let out as effluent. A novel and simple method of separating calcium carbonate (without converting it to calcium chloride) from crab shell waste involving mechanical grinding followed by separation by density is also demonstrated. Since crab shell can contain nearly 70 % minerals the new process reduces the use of citric acid in the subsequent demineralization step. The method, for the first time, enables the recovery of calcium carbonate present in the shell refinery under milder conditions.

The current technology (chemical method) converts the same to carbon

dioxide and thereby generating more of the greenhouse gas. The simplicity of the process and the non-use of corrosive and hazardous chemicals could enable industrial scale production of chitin as well as calcium carbonate.

Materials Crab shell waste (CSW) and prawn shell waste (PSW) were collected from north Chennai harbour region and used after removal of tissue materials. Analytical grade urea and citric acid were purchased from Fisher Scientific Chemicals, Chennai. Ninhydrin was purchased from Sigma-Aldrich, Chennai. Sodium hydroxide, hydrochloric acid, nitric acid and sodium hypochlorite (NaOCl) were purchased from RANKEM, Chennai and were used as received.

Analytical studies Muffle furnace (Sigma Scientific, India) was used for heating the reaction mixture. The temperature fluctuation observed was ± 10 °C. An in house fabricated closed SS reactor equipped with a Teflon vessel was used for hydrothermal treatment. The weight averagemolecular weight was measured by dynamic light scattering (Horiba Scientific Model SZ100, Japan) from solution of chitin in 1 % (w/w) LiCl in dimethylacetamide of different concentrations of chitin (1.25 mg/mL, 0.75 mg/mL, 0.6 mg/mL, 0.3 mg/mL). Solid-state NMR (13C) spectra were acquired using Bruker Avance spectrometer operating at 100 MHz for 13C (probe diameter 4 mm; spinning rate 10,000 kHz, repetition time = 5 sec, contact time = 2000 microsecond). Fourier transform infrared spectra (FT-IR) was acquired using JASCO 4100 FTIR spectrometer (JASCO, Japan). The solid samples required for the analysis was prepared in the pellet form by mixing 3-5 mg of the dry sample with 100 mg of dry potassium 4 ACS Paragon Plus Environment

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bromide (KBr). The quantum yield for emission from carbon nanodots was measured with spectrofluorometer fluroMax 4 (Horiba Scientific) using quinine sulfate as reference. The thermogravimetric studies were carried out with TA Instruments Q500 Hi-Res TGA. The samples were heated at 10 °C min-1 under flowing N2 atmosphere. Solid and liquid phase UV-Visible spectroscopy analysis was carried out using JASCO-650. Photoluminescence analysis was done using JASCO-FP6300. X-ray diffraction patterns of all the materials were recorded with a Bruker D8 Advance diffractometer equipped with copper (Cu) anode (Cu Kα source of wavelength1.5406 Å).

Particle size and zeta potential were analysed using

MALVERN Zeta Sizer (ZS-90). High-Resolution Scanning electron microscopy (HR-SEM) images were acquired using Quanta 200 Scanning electron microscope, FEI, USA. HighResolution Transmission Electron Microscopy (HR-TEM) was performed at 200 kV using JEOL 3010, JAPAN. Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis was carried out using PerkinElmer Optima 5300 DV calibrated with Certipur multielement calibration standards supplied by Merck. Sulphur content was analysed using LECO TruSpec Micro Analyzer. Tensile tests were performed using Zwick/Roel 0.5 kN at a strain rate of 1mm/min. The test specimen required for the same was prepared as follows: 1.5 g chitin prepared by the current method was dissolved using 8 % LiCl in N,N dimethyl acetamide solution. The viscous solution was poured in a Petridish. The formation of a gel was observed. This gel was rinsed thoroughly with methanol (thrice) followed by double distilled water (thrice). The resulting gel was dried at 50 °C for three days. The dried samples was cut into the shape of a tensile testing specimen (dog bone shape) with a width of 10 mm. The thickness of the films were ~ 0.2 mm. The average of results from four different testing is reported. Three point bending test was carried out with AFM (Park Systems NX10), using NCHR tip (four sided pyramid), spring constant 40 N/m. Experimental Methods Isolation of raw prawn shell for extraction (RAW-P) 1000 g wet PSW (contains ~50 % moisture) was ground well in a domestic blender with tap water for 10 minutes. This operation enabled the separation of tissue material from the head and tail part of the prawn shell. It was then rinsed with tap water and filtered using a nylon/cotton cloth filter (size > 300 µm). The filtrate was heated, which resulted in the precipitation of protein (can be used directly as an animal feed or may be hydrolysed to prepare amino acids). The residue, denoted as RAW-P, was used for further directly or after drying at 70 °C for 12 h. Yield: 89.3 g (dry weight). It may be noted that this process can 5 ACS Paragon Plus Environment


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also be carried out without drying operation. The drying operation enabled the calculation of the exact quantity of solids. Isolation of chitin (CU-P) from RAW-P: deproteinization by hydrothermal treatment with urea followed by demineralization with citric acid 5 g of dry RAW-P and 0.82 g of urea was added to 40 mL of water in a hydrothermal reactor. The mixture, in a Teflon lined reactor, was heated to 150 °C for 1 h in muffle furnace whose temperature varied by ±10 °C during the process. It was then allowed to cool. The pH of the mixture was 9.7 while the initial pH was 9.0 (before the reaction). The solid and supernatant were separated by filtration. The liquid phase was found to contain carbon nanodots it was diluted with water and used for characterization. The wet solid was demineralized with 1.92 g citric acid followed by decolorization with 0.2 % aqueous NaOCl solution, at room temperature for 20 minutes. It was then rinsed with water and dried. The product isolated by this method was denoted as CU-P. The yield reported is the average of three independent experiments. Isolation of Chitin [CHE-P] from prawn shell by the chemical method (Control Experiment) To compare the properties of the chitin extracted using the methods described here, chitin was also extracted by the chemical method as reported in the literature.14 RAW-P was demineralized with 0.25 M HCl in the proportion of 40 mL/g at RT for 15 minutes followed by deproteinization with 1 M NaOH, in the ratio of 15 mL/g at 70 °C for 24 h and decolorized using NaOCl solution (0.2 %) at RT.37 Yield: 31 ± 2 % (average of three experiments). The chitin isolated by chemical method was denoted as CHE-P. Gradient separation of ground crab shell waste (CSW) for extraction of chitin and minerals 50 g of wet CSW (contains ~42 % moisture) was subjected to grinding in a domestic grinder for 2 minutes. The ground mixture was placed in a transparent plastic container containing 250 mL of water. The soft organic matter of lower density floats in water (segments I&II). This was separated by decantation over nylon filter and denoted as SOM. Yield: 8.60 g (dry weight 29.99 %). Some of the solids settle down as coarse material and this was denoted as Segment III (S-III). It was treated in two ways for isolation of minerals (with or without chitin) by pyrolysis. Yield: 18.36 g (dry weight 63.90 %). Total recovery of the solid was 94 6 ACS Paragon Plus Environment

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% from the initial dry crab shell; ~ 6.0 % was lost as soluble organic matter during grinding operation. Note that this is not extensive grinding, which may result in the mineral loss as well. Isolation of chitin (CU-C) from SOM: deproteinization by hydrothermal treatment with urea followed by demineralization with citric acid 8.60 g of dry SOM (contains % 53.3 of calcium carbonate) was treated with 2 % aqueous urea solution (8 mL/g) 150 °C for 1 h in a hydrothermal reactor of a capacity of 100 mL. After the reaction, the mixture was allowed to cool. The solids and liquids were separated by filtration. The liquid phase was found to contain carbon nanodots. To the wet solid, citric acid (6.4 g) was added to the same pot and stirred with occasional ultra-sonication for 30 minutes, at room temperature. Chitin was separated by filtration, rinsed with distilled water and decolorized using 0.2 % (v/v) aqueous NaOCl solution at room temperature for 20 minutes. The product isolated after rinsing with water and drying was denoted as CU-C. Yield: 1.81 g (21.0 %); the filtrate on setting aside resulted in the formation of calcium citrate. The same experiment was done on CSW powder to isolate without gradient separation chitin, yield was 13.2 %, initial and final pH was found to be 8.9 and 9.6 respectively. Pyrolysis and recovery of minerals S-III obtained from gradient separation of ground CSW was treated at two different temperature in order to recover minerals with or without chitin. The degradation temperature for chitin is ~ 250 °C under nitrogen atmosphere. 10 g of dry S-III was heated at 220 °C in the air atmosphere to remove protein alone resulting in the recovery of chitin and minerals. Yield: 9.16 g (91.6 %) Similarly, 10 g S-III was heated at 450 °C for 1 h to remove the organic contents and recover pure minerals. Yield: 7.81 g (78.1 %). Isolation of Chitin [CHE-C] from crab Shell by the chemical method (Control Experiment) 50 g of dry crab shell powder was demineralized with 500 mL of 10 % acetic acid (until no effervescence could be observed) and rinsed with water. The residue was treated with 1 M aqueous NaOH solution (15 mL/g) at 70 °C for 24 h. The product was isolated by rinsing with water and drying. Yield: 6.25 ± 0.13 g (12.5 ± 2.0 %) Deproteinization of membrane layer [M-CHE-DP] using sodium hydroxide 7 ACS Paragon Plus Environment


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The membrane layer from 50 g of fresh wet crab shell was manually peeled off. It was dried at 90 °C for 2 h. The yield of membrane layer was 0.63 g. It was treated with 1 M sodium hydroxide solution at 70 °C for 24 h and rinsed with distilled water up to neutral condition (pH~7). This did not require demineralization, as only traces of minerals were present. Yield: 0.49 g (2.3 %) Ninhydrin Test 50 mg of solid sample was added to a test tube followed by the addition of 2 mL of water and 1 mL of 1 % ninhydrin solution in methanol. It was heated to boiling. The change of color (from colorless) to purple indicates the presence of amino acids (indirectly protein). Xanthoproteic test 20 mg of sample was treated with 2 mL of concentrated nitric acid followed by the addition of 2 mL of 40 % aqueous NaOH. The appearance of yellow to orange color indicated the presence of aromatic amino acid(s). Results and Discussion Isolation of chitin from prawn shells The deproteinization is the crucial step in isolation of chitin because the interactions between protein and chitin are intimate and complex. To perform efficient deproteinization, the understanding about the interactions between protein and chitin such as quinone tanning and covalent bonding through ester linkage are important as reported in the literature. It has been reported that to make the shell surface less intractable to the environment, the protein present in the external surface of crustaceans are hardened by a process called ‘quinone tanning’.38,39 The literature also reports on covalent interaction between chitin and protein through an ester linkage.15,40 The possible interactions between chitin and protein in a crustacean shell are listed below. 1. Proteins physically associated with chitin by van der Waals (hydrophobic) and electrovalent (H-bonding) interactions- secondary interactions.15 2. Protein present in the crystalline unit of chitin15,24 3. Proteins that are linked by covalent bonds (ester linkage-primary interactions)15,40 4. Proteins hardened by quinone tanning 40–43


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The separation of chitin and protein can be done by a systematic approach. The proteins that are connected through secondary interactions can be separated using a denaturant such as urea. However, the proteins linked with chitin by covalent bonding (ester linkage)

15,40

require at least mild alkaline condition for hydrolysis.15 In the present method, urea was used to remove protein from prawn shell waste (PSW) under the hydrothermal condition at 150 °C as detailed in the experimental section. At this temperature, urea decomposes into ammonia and carbon dioxide44, which generates an in situ alkaline medium that was sufficient to remove the protein by hydrolysis. The initial pH of the reaction mixture was 9.0 (it may be due to dissolution of magnesium carbonate and the formation of ammonia from biochemical breakdown of the waste during drying) and after the reaction, it was found to be 9.7, which might be due to decomposition of urea (without catalyst or additives) resulting in the formation of ammonia or due to the formation of basic amino acids. The role of urea in dissolution of chitin/cellulose under special conditions is well known (it helps in penetration of alkali hydrate into cellulose by preventing hydrophobic association in alkali-swollen cellulose domains).45 Subsequent to the deproteinization the demineralization was carried out with citric acid as represented in Scheme 1. Chitin isolated in this manner contained a small quantity of protein bonded to chitin possibly through quinone tanning. This required treatment with dilute aqueous sodium hypochlorite, which also accomplished the task of decolourization. The details of the characterization of the chitin thus separated are discussed later. The deproteinization with urea under hydrothermal condition resulted in the simultaneous formation of CNDs.

Carbon nanodots (CNDs) are luminescent nanomaterials and are

potential green alternatives for inorganic semiconductor nanocrystals such as CdSe, Pbs, etc.46 They are

well known for their fascinating features of photoluminescence, water

solubility (dispersability), photostability, chemical inertness and excellent biocompatibility and this has led to their use in lighting and bioimaging.47 The absorption and emission spectra of CNDs were recorded (shown in Figure 1) to illustrate their photoluminescence (PL) property. The absorption spectrum of CNDs (Figure 1a) shows two peaks at 275 and 335 for π-π* and n-π* transition, which is similar to the results reported in the literature.48 The emission spectra of CNDs is shown in Figure 1b. The excitation wavelength was chosen from 280 to 580 with 10 nm increment. The maximum emission intensity was observed when the excitation was at 355 nm. The minimum emission intensity was observed at 415 nm. The normalized PL spectra are shown in Figure 1c. The analysis clearly demonstrates the 9 ACS Paragon Plus Environment


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dependence of the emission wavelength and intensity on λex, an important feature of CNDs.49 The CNDs exhibited unusual emission around 600 nm (yellow), which has been attributed in the literature to the presence of high abundance of nitrogen-containing functional groups on CND surface.50 The quantum yield for fluorescence was 5.84 % (with quinine sulfate as reference, excitation wavelength 350 nm). The TEM image of CNDs is shown in Figure 1d, which suggests the formation of quasi-spherical CNDs of different size in the range of 7-15 nm. The average size of CNDs as measured by dynamic light scattering was found to be 38 nm. The zeta potential of freshly prepared CND was -29.7± 1.3 mV. This decreased gradually with time and so was particle aggregation.

This suggests that the difference

between the size of the particles measured by TEM and dynamic light scattering (carried out few weeks later) might be due to aggregation of the particles with time. Further it may be noted that the particles were not agitated/stirred during the size measurement by dynamic light scattering. It should be emphasized here that the synthesis of (CNDs) from the crustacean shells such as shrimp and prawn shell proteins is reported in the literature.48,51 but these are limited to the synthesis of CNDs only.48 Moreover, in these processes large quantity of partially treated waste will be discarded again and this could not be a complete solution for effective transformation of a waste material. The widespread applicability of a synthetic methodology is decided by the step economy of the protocol as it controls the manpower, time, electricity, raw material and cost input, amount of waste and by-products.52 To accomplish this, simultaneous and single-pot procedures have been given paramount importance. Therefore, the concurrent synthesis of CNDs from protein and the separation of chitin in a single pot as demonstrated here would be more beneficial as it reduces the consumption of energy, time, water and more importantly the protein fraction is effectively utilized, which is otherwise being destroyed as effluent in the chemical method.53


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Scheme 1. Pictorial representation of the process used for deproteinization of shell material

Figure 1. a) Absorption spectrum of CNDs (insert: picture of CNDs under sunlight and UV light 235 nm), b) PL spectra of CNDs excited at different wavelength (280-580 nm), c) Normalized PL spectra and d) TEM image of CNDs (as synthesised).



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Gradient separation of ground crab shells for recovery of minerals without dissolution The separation of calcium carbonate in addition to chitin from crab shell waste was motivated due to the abundance of this mineral in its exoskeleton. The understanding of the inner structural organization of crustacean exoskeleton aided this execution. The components in the crustacean shell, which is a biological nanocomposite, are well-ordered in a hierarchical manner.54,55 The α-chitin crystalline nanofibers (~3 nm) are wrapped by protein and assembled into bundles of chitin-protein nanofibers of thickness ~50-300 nm.54,55 These nanofibers, in turn, form a flexible planar interwoven network and is further reinforced by minerals such as crystalline calcium carbonate, magnesium carbonate, and calcium phosphate. The planes are stacked in twisted plywood pattern (Bouligand) with a gradual change in orientation by rotation about their normal axis. This structural arrangement is replicated in the exoskeleton as three layers with distinct boundaries namely: epicuticle, exocuticle, and endocuticle with varying composition and density as shown in Figure 2 (Supporting Information Table S1). The fourth and innermost layer is a membranous layer54 as shown in the insert to Figure 2.


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Figure 2. SEM image of the crab shell (Portunus armatus) used. The insert picture is that of membranous layer. Among these layers, endocuticle (~380 µm thickness) and membranous layer (~28 µm thickness) are important from the viewpoint of separation of chitin. The membranous layer consists of chitin and protein and especially chitin (74 % by weight in the case of crab shell38) and is, therefore, the least dense. The endocuticle is the thickest layer. It is homogeneous and is mainly composed of amorphous calcium carbonate rather than calcite.56 The exocuticle (the layer above endocuticle; about 95 µm in thickness) and the epicuticle (thinnest and topmost layer of the shell is about 9 µm in thickness) consist of crystalline calcium carbonate and hardened by quinone tanning. These two layers are denser when compared to the endocuticle. The overall composition of the crab and prawn shells (average) used in this work is presented in Table 1. Table 1. Composition of blue crab shell and prawn shell used in this study (determined by TGA)



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Blue swimming crab

Prawn shell

shell

(Fenneropenaeus

(Portunus armatus)

indicus)

(dry weight)

(dry weight)

Organic contents % (Chitin, protein and others mass loss in the region 120 to 600 °C)

25.0 ± 3.3

54.3 ± 4.2

Calcium as CaCO3 %

65.2 ± 1.2

32.3 ± 2.4

9.7 ± 4.1

32.0 ± 0.5

Mg % (by ICP-OES)

1.86 ± 0.2

0.29 ± 0.1

Phosphate % (by ICP-OES)

1.4 ± 0.2

0.96 ± 0.03

Constituent (w/w)

(mass loss for CO2 in the region 600 to 800 °C) Ash at 800 °C % (excluding CaO)

Note:

#

Dry crab and prawn shells were stored in the freezer in a zip lock bag and TGA

analysis was done within 24 hours. The sample was heated in thermogravimetric analyzer under nitrogen atmosphere and at a heating rate of 10 °C/min. The result is the average of two measurements. The % composition is dependent on the gender, season and part of the animal etc.,57

Mechanical grinding of crab shells followed by separation by density The gradient separation in ground crab shell was intended to separate the chitin rich membraneous layer from mineral rich layers of endo, exo and epicuticle and to reduce mineral acid consumption in the demineralization step.

This is a very important and

significant novel step introduced in this work as crab shell is rich in minerals (the composition of minerals was as high as 73 % in samples used here). It may be noted that a mineral content of 88.6 % has been reported in crab finger (Cancer pagurus).57 In the current manufacturing process of isolating chitin from crab shells using hydrochloric acid, to extract 14 ACS Paragon Plus Environment

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12.5 g of chitin from 100 g of crab shell, (73 g of CaCO3 by weight; 5.84 times the mass of chitin) is converted into CaCl2 an effluent. If the mineral components of the shells were reduced through suitable separation, it would not only reduce the consumption of acid but would also reduce the quantity of carbon dioxide evolved during demineralization. Since the exoskeleton is a composite and known to exhibit hierarchical organization, as discussed above, it may be possible to separate the components, by gradient separation using the difference in physical properties. The initial attempt at separating chitin from the crab shells was to subject it to mechanical grinding followed by separation by the difference in density that arises from nature of components and composition. The response to the stress of the layers in the crab shell was expected to be different within a layer and across layers. Initially, the membranous layer was separated by manual pealing process and deproteinized using NaOH to isolate chitin. Subsequently, the shells were ground and suspended in water. The ground crab shell particles formed three distinct segments (represented in Scheme 1) in the column of water (I, II and III; due to different densities) as shown in Figure 3. The segment III (S-III) rich in high-density mineral was observed to settle down. The soft matter consisting of chitin, protein and the smaller quantity of minerals remained suspended at different column heights and could be separated with ease. The S-III showed clear and obvious pigmentation and this arises mostly from the epi- and exo-cuticle part of the shells. The first grinding operation resulted in 34.8 ±5.2 % (by dry weight) of soft matter in segments I&II and 65.2 ±5.3 % (by dry weight) of sediments as S-III. The composition of organic contents (chitin, protein, etc.,) and calcium carbonate in the different segments are presented in Table 2. The TGA and DTG of CSW and the different segments (SOM and SIII) obtained by the mechanical grinding from CSW are shown in ESI, Figure S1.



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Figure 3. Graphical representation of gradient separation of ground crab shell (Portunus armatus) and the differential TGA analysis of the three segments.

It is worth noticing that S-III contained the larger proportion of minerals and least organic content. Thus, S-III was used for isolating calcium carbonate with or without chitin. It was heated at two different temperatures, in an air atmosphere, based on the degradation temperature of chitin. Treating at 220 °C enabled the recovery of minerals along with chitin. The chitin obtained in this manner can be used as nanofibrous filler. Treating at 450 °C enabled the recovery of minerals devoid of organic contents.

The differential

thermogravimetric analyses of S-III before and after heating are shown in Figure 4 (the corresponding thermogravimetric analyses are shown in ESI, Figure S2). It shows distinctly that upon heating at 220 °C, protein peak vanishes and at 450 °C all the residual organic matters (chitin, protein, etc.) associated with S-III are lost by decomposition and the resultant calcium carbonate is ready to be used directly in various fields such as in pharmaceutical, paper and cement manufacturing industries.

It may be noted that this process of heat

treatment of S-III could result in the formation of undesirable gases and loss of chitin (a small fraction of that present in the shells).The structure of the recovered inorganic material was identified to be that of calcite from the P-XRD pattern (ESI, Figure S3). However, with 16 ACS Paragon Plus Environment

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proper optimization of the grinding process, it should be possible to minimize the organic content of S-III further and increase the organic content of S-I&II.

Table 2. Dry weight and percentage composition of material with different densities obtained on grinding of wet crab shells followed by separation by density using a column of water. Density Gradient in Water

SegmentI&II (floating; SOM) Segment-III (sediment) CSW

Dry weight %

% Organic matter (w/w)*

% CaCO3 a (w/w)

% Ash @ 800 °C (excludin g CaO)

34.80 ±5.2

29.53 ± 3.8

54 ± 4.8

12.56 ±2.4 20.66± 3.2

65.16 ±5.3

17.54±3.2

78.05± 6

8.7±3.2

12.05 ±2.5

91.6

100

25.01±3.3

65.2 ±1.2

9.7±4.1

14.28 ±2.1

79.26

Chitin Yield % ( by the current method)

CaCO3 and chitin Recovery by heating at @ 220 °C ---

a

1000 g of shell material. * Weight loss between 120-600 °C, Weight loss between 600-800 °C determined by TGA analysis under nitrogen atmosphere.

The prawn shells are relatively soft, flexible and contain a larger proportion of chitin and protein and smaller proportion of mineral. Owing to its lesser mineral content and smaller crystal size of calcium carbonate, it was not ground unlike crab shells. The advantages of the present method are: deproteinization was done under milder chemical condition using urea a sustainable chemical unlike the conventional method; the product of hydrolysis of protein is effectively used as a sustainable nitrogen-rich carbon source for the scalable synthesis of CNDs; in the case of crab shells, a large fraction of minerals is recovered by grinding followed by mild treatment and lesser quantity of mineral acid is used for separation of chitin.



Figure 4.

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Differential thermogravimetric analysis of S-III (the sediment obtained from

ground crab shell followed by separation via density gradient) before and after thermal treatment. The differential thermogravimetric analysis of dried CSW waste is also presented for comparison. Assessment of the quality of chitin The purity of the chitin obtained from the processes described here substantiate its efficacy, which was ascertained by diffused reflectance UV-visible spectroscopy (DRS), solid state NMR spectroscopy (SS-NMR), powder X-ray diffraction (P-XRD), thermogravimetric analysis (TGA), and fourier transform infrared spectroscopy (FT-IR). To analyze traces of residual protein qualitative analytical tests such as ninhydrin and xanthproteic tests were done while Lowry method using BSA standard enabled the quantitative estimation of residual proteins. The DRS spectra of RAW-P, chitin isolated by chemical method (CHE-P) and chitin isolated by current method (CU-P) are presented in Figure 5. The raw material (RAW-P) shows a broad absorption peak at ~ 280 nm, which is a characteristic peak for protein, known to be caused by the aromatic amino acids present in the protein.14 The disappearnce of the protein peak in CU-P evidently demonstrates that the chitin is free from protein (by the limits of detection of DRS used). However, the maximum residual protein content of chitin as assessed by Lowry test in three independent measurements was 0.22 % for chitin prepared 18 ACS Paragon Plus Environment

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from prawn shell (CU-P) and 0.16 % for chitin prepared from crab shells (CU-C). In contrast, the maximum protein content of chitin obtained by the chemical method, posthypochlorite bleaching, for prawn shell and crab shell were 0.41 % and 0.48 %, respectively. The average protein content before hypochlorite bleaching was 3.83 % for prawn shell while it was a maximum value of 0.22 % after bleaching. The difference of 3.61% of protein might arise from quinone tanning although hard experimental evidence is required to substantiate this hypothesis.

Figure 5. Diffuse reflectance UV-visible spectra: prawn shell (RAW-P); chitin isolated from raw prawn shell by the chemical method (CHE-P) and by the current method (CU-P). The solid-state

13

C NMR spectrum of chitin isolated form prawn and crab shell waste are

shown in Figure 6. For comparison, the SS-NMR spectrum of the raw materials are also presented. The chitin samples isolated show prominent and well-resolved peaks for chitin: 173 ppm (-CO- in -NH-CO-CH3), 103 (C1), 83 (C4), 75 (C5), 73 (C3), 61 (C6), 55 (C2) and 23 (-CH3) ppm, respectively.58,59 The spectrum for chitin isolated from crab and prawn shells were similar. Surprisingly no peak for carbonate was observed. The relative intensity at 173 ppm with respect to 23 ppm was greater in the case of CSW, which may be due to the carbonate peak arising from calcium carbonate.60 The degree of acetylation is one of the characteristic property of chitin isolated from crustacean shells. The degree of acetylation 19 ACS Paragon Plus Environment


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was found to be 93.6 % and 93.7 % for chitin isolated from prawn shell (CU-P) and crab shell (CU-C), respectively. The molecular weight (Mw) of chitin isolated from prawn shell by current method was 6.3x105 where as that for chitin isolated by the chemical method was found to be 4.8x105.61,62

s

Figure 6. Solid state NMR spectra of raw materials from prawn shell (RAW-P) and crab shell (CSW) and isolated chitin samples respectively CU-P and CU-C. The powder X-ray diffraction pattern of chitin obtained from prawn and crab shell by the process described here (CU-P and CU-C) are compared with their corresponding starting materials (RAW-P and CSW respectively) in Figure 7. RAW-P exhibits diffraction peaks for chitin (CH) along with calcite (Ca) at 2θ values 9.2 {CH (020)},13.3 {CH (021)}, 19.8 {CH (110)}, 23.4 {CH (130)}, 26.6 {CH (013)}, 29.7 {Ca (104)}, 36.4 {Ca(110)}, 39.8 {Ca (113)}, 43.4 {Ca (202)}, 47.8 {Ca (018)}, and 48.9 {Ca (116)}.63 The X-ray diffraction pattern of the chitin isolated by the process described here reveals the presence of the prominent crystalline peaks at 2θ values attributed to chitin [2θ =9.44° (020), 2θ =219.34° (110)].64 The elimination of other peaks arising from calcite suggests that it is removed completely. Thus, P-XRD results are in good agreement with the findings from SSNMR. The 20 ACS Paragon Plus Environment

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d-spacing values and crystallinity index obtained by P-XRD are shown in Table.3. Chitin separated by the present method CU-P and CU-C exhibits greater crystallinity index than that by the chemical method.65 From this, it can be hypothesized that native character of chitin is significantly unaltered by the current extraction method. Table 3. Crystallinity index of RAW-P and chitin separated (CU-P, CU-C and CHE-P)

Figure 7. P-XRD pattern of prawn shell waste (RAW-P), chitin separated from prawn shell (CU-P), crab shell waste powder (CSW) and chitin separated from crab shell (CU-C). For



Page 22 of 35

reference, chitin isolated form prawn shell by the chemical method is also given (CHE-P). The TGA and DTGA analysis of prawn shell waste (RAW-P) before and after treatment is shown in Figure 8. For the purpose of comparison, the degradation pattern of chitin obtained by the process described in this work (CU-P) with that obtained by the chemical method (CHE-P) is also shown. It is important to take into account, that the chitin isolated by the current method does not exhibit weight loss between 150-300 °C that is normally observed due to the presence of protein.66 Similarly, the TGA and DTG of chitin (CU-C) isolated by the current method from CSW and chitin isolated by chemical method are shown in ESI, Figure S4 (for comparison MEM-DP is also presented in this figure). It illustrates that the chitin isolated by current method is free from protein. Additionally, the absence of weight loss between 650-800 °C demonstrates the removal calcium carbonate by the limits of TGA. ICP-OES analysis on the isolated chitin for residual calcium and magnesium ions revealed the presence of traces of mineral ions (Ca2+ = 0.18 % and Mg2+ =0.05 % in CU-C and Ca2+ = 0.13 % and Mg2+ =0.02 % in CU-P).

Figure 8. TGA (left) and DTG (right) analysis of prawn shell before (RAW-P) and after treatment (CU-P and CHE-P) The FT-IR spectral data (ESI, Figure S5) also clearly demonstrated that the product isolated was α-chitin. From the IR spectral data, the purity of isolated samples can be evaluated by the splitting of the amide-I band at 1656 cm−1. This is attributed to two types of hydrogen bonding: one of them is between the N-acetyl carbonyl group with NH of the N-acetyl carbonyl of the adjacent chain and (CO⋯HN) and the other one is between carbonyl and


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primary hydroxyl group of the same chain (CO⋯HOCH2). This kind of splitting was not observed in the case of RAW-P and CSW and this may be due to amide peaks of protein overlapping with amide I bands of chitin.67 The other peaks observed at 874 and 725 cm-1 can be assigned to out of plane bending (ʋ2) and in-plane bending (ʋ4) of carbonate group from calcium carbonate, respectively. In CSW, splitting of peaks were observed at 1415 and 1487 cm-1 for asymmetric stretching (ʋ3) of carbonate ions confirming the existence of amorphous calcium carbonate (also confirmed by HRTEM; data not shown here).56,68 These peaks are reduced remarkably in the chitin isolated as compared to those of raw materials. The results from the spectroscopic analyses suggest the absence of calcium carbonate and protein in the chitin isolated by the limits of these techniques. Although thermal and spectral analysis were in good agreement and revealed the superior quality of chitin, qualitative tests were also done to analyse the traces of residual impurities for protein, peptides, and amino acids. Ninhydrin test was performed to find the existence of amine functional groups in raw and treated samples. The absence of color change in the chitin samples isolated by the method reported here confirmed the absence of amino acids/proteins (ESI, Figure S6). This test is beneficial to analyze the surface impurites. But to examine the protein present in the chitin fibrils, xanthoprotic test was performed. This test was accomplished by dissolution of chitin using nitric acid, which enables the collapse of the crystalline structure of chitin and exposes the residual protein. The aromatic amino acids are tranformed into their nitro derivatives upon treatment with nitric acid and the colour of sample changes from colorless to yellow. Upon addition of sodium hydroxide the appearnce of orange color confirms the presence of protein. The treated samples were not showing colour change indicating the removal of protein (ESI, Figure S7). In order to substantiate the above investigations, residual sulphur content using CHNS analyzer was evaluated and this revealed no sulfur contet in the chitin samples (CU-C and CU-P) isolated. Chitin is well known for its high tensile strength and low elongation at break.69 The chitin separated by the present method was solution cast into a film and was examined for tensile properties. The tensile strength, elongation at break (%) and the tensile modulus were 4.9±0.6 MPa, 73±6 MPa, and 7.8±0.9 %, respectively. The tensile strength is at least one order of magnitude below the values reported for wet spun fibers of chitin (153 MPa, 7.3 GPa, and 8 %, respectively).69-73 This could be due to the formation of films that are devoid of molecular orientation as crystalline regions in the chitin are fibers are dissolved by the solvent and the property of the film would depend on the crystallinity of the film as well as 23 ACS Paragon Plus Environment


Page 24 of 35

the possible presence of defects. The reduction in mechanical property may not arise due to degradation in molar mass as a consequence of the method of separating chitin since the molecular weight of chitin separated was reasonably high (6.3x105). It may be noted that we have not examined the mechanical properties of the wet spun fibers prepared in the form of membrane as reported earlier (by vacuum filtering a well-dispersed colloidal suspension of 0.07 w % chitin nanofibers over a DVPP filter membrane followed by drying in a semiautomated paper making equipment) and hence this data cannot be compared with the literature data74. The flexural modulus of chitin separated was evaluated using AFM. For this purpose, a dispersion of the chitin in water was coated on to a TEM grid and dried at room temperature.

A region of the grid was chosen such that the chitin fiber(s) could be

subjected to bending stress with the AFM tip. A plot of the bending stress versus elongation enabled the assessment of the flexural modulus, which was found to be 11.91 ± 0.6 GPa. The data was validated by testing poly(propylene) film of known bending modulus (2 GPa) under the same conditions. It may be noted that these measurements are not precise and probably pertain to a bundle of fibers and need to be validated using well accepted standard procedures. The SEM images of the external surface of prawn shell used in this work, its cross section and chitin isolated by current method are shown in Figure 9. The external surface of the prawn shell exhibits homogenous spots and pore canals as shown in Figure 9a. The cross section of prawn shell, which illustrates the presence of layered structure and Bouligand arrangement of the components as in the case of crab shell are shown in Figure 9b. The nanofibrous structure of chitin isoated by the current method shown in Figure 9c demonstrates that it is likely to be free from other components such as protein and minerals. The SEM analysis thus supports the spectroscopic and thermogravimetric studies.


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Figure 9. SEM images of a) prawn shell external surface, b) Bouligand arragment in prawn shell cross section and c) chitin fibers isolated by the current method (CU-P). The advantages of the method reported here over the coventional chemical method that is practiced indstrially in isolating chitin are summarized in Table 3. It is clear from this comparison that the present method holds promise for large scale industrial production of chitin in a sustainable manner. Table 3. Comparison of the present method with the conventional chemical method of isolating chitin from crustacean shell waste. S. No. 1.

Current method

Traditional chemical method

Harmless to the environment

Hazardous process involving strong mineral acid and caustic alkali

2.

Protein by-product is recovered as more

By-product (calcium chloride, carbon

value added CNDs

dioxide and amino acid salts are not recovered) have to be treated

3.

Minerals recovered as calcium citrate

Minerals are not recovered

4.

Shorter duration

Longer duration

5.

Requires 1.8 L of water for processing per

Consumes 55 L of water for processing per

kg of prawn shell ( excluding rinsing)

kg of prawn shell ( excluding rinsing)

Lower residual protein in chitin isolated

Greater residual protein in chitin isolated

(range: 0 to 0.22 % with prawn shells and 0

(minimum of 0.41 % with prawn shells

to 0.16 % with crab shells)

and 0.48 % with crab shells)

6.

Conclusions A simple and sustainable chemical approach for industrial-scale production of chitin from the seafood industry waste and more specifically crab shell and prawn shell waste are described. The advantages of this method are: deproteinization was done with 2 % aqueous urea solution instead of hot aqueous caustic soda treatment; demineralization with citric acid, which enables the recovery of calcium mineral as calcium citrate. The hydrolyzed proteins were used as a nitrogen-rich carbon source for the scalable synthesis of CNDs in the same 25 ACS Paragon Plus Environment


Page 26 of 35

pot. The recovery of calcium carbonate from crab shell waste will serve as an alternate source for mining calcium carbonate in a renewable approach instead of eliminating it as an effluent (CaCl2). Besides, the carbon dioxide trapped by the crustaceans by mineralization would not be released back to the environment during isolation of chitin with calcium carbonate being useful material for pharmaceutical industries. The by-product of demineralization, calcium citrate, can be effectively recovered by crystallization. The recovered minerals and mineral salts can be used as human and animal nutrients. Since the process utilizes sustainable and non-hazardous chemicals, much effort would not be required in processing the wastewater. This procedure is green, effective, simpler, relatively faster, and the quality of chitin obtained is better than the chemical method. Hence, this method could be an efficient, greener and sustainable method for industrial-scale production of high-quality chitin by addressing one of the significant environmental problems of the coastal areas where tons of seafood waste is either dumped back in the sea or buried in landfills or treated by harsh chemical method to separate chitin.

ASSOCIATED CONTENT Electronic Supplementary Information Thermal and Differential thermal analysis of CSW and the different segments (SOM and SIII) obtained by the mechanical grinding from CSW; Thermogravimetric analysis of S III (the sediment obtained from ground crab shell followed by separation via density gradient) before and after thermal treatment. The differential thermogravimetric analysis of dried CSW waste is also presented for comparison; P-XRD of the mineral obtained from crab shell by calcining the inorganic sediment (S-III; separated by grinding followed by suspension in water); TGA and DTG analysis of CSW, chitin (CU-C) and CHE-C; FT-IR spectra of raw material from crab shell waste (CSW), chitin separated from crab shell (CU-C), prawn shell waste (RAW-P) and chitin separated from prawn shell (CU-P). For reference, the same for chitin isolated form prawn shell by the chemical method is also given (CHE-P); Photographs


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of ninhydrin test for the presence of amino acid residues. CSW; chitin (CU-C) from crab shell waste using the present method and control (c); Photographs from Xanthoproteic test for the presence of amino acid residues: (a) CSW (b) chitin isolated from CSW using the present method (CU-C) and (c) chitin isolated from CSW by chemical method (CHE-C).

AUTHOR INFORMATION Corresponding Author Dhamodharan Raghavachari. E-mail: [email protected] Notes The authors declare no competing financial interests. Acknowledgements The authors thank IIT Madras for financial support and for the research facilities. They also thank Prof. E. Prasad (Department of Chemistry for zeta potential measurement), Prof. H. S. N. Murthy, Department of Aerospace Engineering for tensile testing), Prof. Sudakar Chandran (Department of Physics for AFM). R. Devi acknowledges CSIR for the award of the research fellowship and Vivek Anand, E. Ramachandran for valuable technical discussions. The work presented here is a part of Indian patent application (201641030691) filed by the authors.

References (1)

Kim, S. K. Chitin, Chitosan, Oligosaccharides and Their Derivatives: Biological

Activities and Applications; CRC Press, 2011. (2)

Yan, N.; Chen, X. Don’t Waste Seafood Waste. Nature 2015, 524, 155–157.

(3)

Chen, X.; Gao, Y.; Wang, L.; Chen, H.; Yan, N. Effect of Treatment Methods on 27 ACS Paragon Plus Environment


Page 28 of 35

Chitin Structure and Its Transformation into Nitrogen-Containing Chemicals.

Chempluschem 2015, 80, 1565–1572. (4)

Chen, X.; Yan, N. Novel Catalytic Systems to Convert Chitin and Lignin into Valuable Chemicals. Catal. Surv. from Asia 2014, 18, 164–176.

(5)

Yabushita, M.; Kobayashi, H.; Kuroki, K.; Ito, S.; Fukuoka, A. Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group. ChemSusChem 2015,

8, 3760–3763. (6)

Ohmi, Y.; Nishimura, S.; Ebitani, K. Synthesis of α-Amino Acids from GlucosamineHCl and Its Derivatives by Aerobic Oxidation in Water Catalyzed by Au Nanoparticles on Basic Supports. ChemSusChem 2013, 6, 2259–2262.

(7)

Chen, X.; Liu, Y.; Kerton, F. M.; Yan, N. Conversion of Chitin and N-Acetyl- D Glucosamine into a N-Containing Furan Derivative in Ionic Liquids. RSC Adv. 2015,

5, 20073–20080. (8)

Lv, Y. M.; Laborda, P.; Huang, K.; Cai, Z. P.; Wang, M.; Lu, A. M.; Doherty, C.; Liu, L.; Flitsch, S. L.; Voglmeir, J. Highly Efficient and Selective Biocatalytic Production of Glucosamine from Chitin. Green Chem. 2017, 19, 527–535.

(9)

King, C. A.; Shamshina, J. L.; Zavgorodnya, O.; Cutfield, T.; Block, L. E.; Rogers, R. D. Porous Chitin Microbeads for More Sustainable Cosmetics. ACS Sustain. Chem.

Eng. 2017, 5, 11660–11667. (10)

Riccardo A. A. Muzzarelli. Chitin and Its Derivatives: New Trends of Applied Research. Carbohydr. Polym. 1983, 3, 53–75.

(11)

Silva, S. S.; Mano, J. F.; Reis, R. L. Ionic Liquids in the Processing and Chemical Modification of Chitin and Chitosan for Biomedical Applications. Green Chem. 2017,

19, 1208–1220. (12)

He, M.; Wang, X.; Wang, Z.; Chen, L.; Lu, Y.; Zhang, X.; Li, M.; Liu, Z.; Zhang, Y.; Xia, H.; et al. Biocompatible and Biodegradable Bioplastics Constructed from Chitin via a “green” Pathway for Bone Repair. ACS Sustain. Chem. Eng. 2017, 5, 9126–9135.

(13)

Li, M.-C.; Wu, Q.; Song, K.; Cheng, H. N.; Suzuki, S.; Lei, T. Chitin Nanofibers as Reinforcing and Antimicrobial Agents in Carboxymethyl Cellulose Films: Influence of 28 ACS Paragon Plus Environment

Page 29 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Partial Deacetylation. ACS Sustain. Chem. Eng. 2016, 4, 4385–4395. (14)

Percot, A.; Viton, C.; Domard, A. Optimization of Chitin Extraction from Shrimp Shells. Biomacromolecules 2002, 4, 12–18.

(15)

Percot, A.; Viton, C.; Domard, A. Characterization of Shrimp Shell Deproteinization.

Biomacromolecules 2003, 4, 1380–1385. (16)

No, H. K.; Hur, E. Y. Control of Foam Formation by Antifoam during Demineralization of Crustacean Shell in Preparation of Chitin. J. Agric. Food Chem. 1998, 46, 3844–3846.

(17)

Wilson, R. W.; Wilson, J. M.; Grosell, M. Intestinal Bicarbonate Secretion by Marine Teleost Fish - Why and How? Biochim. Biophys. Acta-Biomembranes 2002, 1566, 182–193.

(18)

Liu, P.; Liu, S.; Guo, N.; Mao, X.; Lin, H.; Xue, C.; Wei, D. Cofermentation of

Bacillus Licheniformis and Gluconobacter Oxydans for Chitin Extraction from Shrimp Waste. Biochem. Eng. J. 2014, 91, 10–15. (19)

Cira, L. A.; Huerta, S.; Hall, G. M.; Shirai, K. Pilot Scale Lactic Acid Fermentation of Shrimp Wastes for Chitin Recovery. Process Biochem. 2002, 37, 1359–1366.

(20)

Arbia W, Arbia L, Adour L, A. A. Chitin Extraction from Crustacean Shells Using Biological Methods - A Review. Food Technol. Biotechnol. 2013, 51, 12–25.

(21)

Zhang, H.; Jin, Y.; Deng, Y.; Wang, D.; Zhao, Y. Production of Chitin from Shrimp Shell Powders Using Serratia Marcescens B742 and Lactobacillus Plantarum ATCC 8014 Successive Two-Step Fermentation. Carbohydr. Res. 2012, 362, 13–20.

(22)

Younes, I.; Ghorbel-Bellaaj, O.; Nasri, R.; Chaabouni, M.; Rinaudo, M.; Nasri, M. Chitin and Chitosan Preparation from Shrimp Shells Using Optimized Enzymatic Deproteinization. Process Biochem. 2012, 47, 2032–2039.

(23)

Manni, L.; Ghorbel-Bellaaj, O.; Jellouli, K.; Younes, I.; Nasri, M. Extraction and Characterization of Chitin, Chitosan, and Protein Hydrolysates Prepared from Shrimp Waste by Treatment with Crude Protease from Bacillus Cereus SV1. Appl. Biochem.

Biotechnol. 2010, 162, 345–357.



(24)

Page 30 of 35

Synowiecki, J.; Al-Khateeb, N. A. A. Q. The Recovery of Protein Hydrolysate during Enzymatic Isolation of Chitin from Shrimp Crangon Crangon Processing Discards.

Food Chem. 2000, 68, 147–152. (25)

Younes, I.; Rinaudo, M. Chitin and Chitosan Preparation from Marine Sources. Structure, Properties and Applications. Mar. Drugs 2015, 13, 1133–1174.

(26)

Venugopal., V. Marine Products for Healthcare; CRC Press, 2009.

(27)

Setoguchi, T.; Kato, T.; Yamamoto, K.; Kadokawa, J. ichi. Facile Production of Chitin from Crab Shells Using Ionic Liquid and Citric Acid. Int. J. Biol. Macromol. 2012, 50, 861–864.

(28)

Shamshina, J. L.; Barber, P. S.; Gurau, G.; Griggs, C. S.; Rogers, R. D. Pulping of Crustacean Waste Using Ionic Liquids: To Extract or Not to Extract. ACS Sustain.

Chem. Eng. 2016, 4, 6072–6081. (29)

Qin, Y.; Lu, X.; Sun, N.; Rogers, R. D. Dissolution or Extraction of Crustacean Shells Using Ionic Liquids to Obtain High Molecular Weight Purified Chitin and Direct Production of Chitin Films and Fibers. Green Chem. 2010, 12, 968–971.

(30)

Zhu, P.; Gu, Z.; Hong, S.; Lian, H. One-Pot Production of Chitin with High Purity from Lobster Shells Using Choline Chloride–malonic Acid Deep Eutectic Solvent.

Carbohydr. Polym. 2017, 177, 217–223. (31)

Espíndola-Cortés, A.; Moreno-Tovar, R.; Bucio, L.; Gimeno, M.; Ruvalcaba-Sil, J. L.; Shirai, K. Hydroxyapatite Crystallization in Shrimp Cephalothorax Wastes during Subcritical Water Treatment for Chitin Extraction. Carbohydr. Polym. 2017, 172, 332– 341.

(32)

Devi, R.; Dhamodharan, R. Pretreatment in Hot Glycerol for Facile and Green Separation of Chitin from Prawn Shell Waste. ACS Sustain. Chem. Eng. 2018, 6, 846– 853.

(33)

Cree, D.; Rutter, A. Sustainable Bio-Inspired Limestone Eggshell Powder for Potential Industrialized Applications. ACS Sustain. Chem. Eng. 2015, 3, 941–949.

(34)

Murakami et al. Physicochemical Study of Calcium Carbonate₃ from Egg Shells.

Ciência e Tecnol. Aliment. 2007, 27, 658–662. 30 ACS Paragon Plus Environment

Page 31 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(35)

Hamester, M. R. R.; Balzer, P. S.; Becker, D. Characterization of Calcium Carbonate Obtained from Oyster and Mussel Shells and Incorporation in Polypropylene. Mater.

Res. 2012, 15, 204–208. (36)

Islam, K. N.; Bakar, M. Z. B. A.; Noordin, M. M.; Hussein, M. Z. Bin; Rahman, N. S. B. A.; Ali, M. E. Characterisation of Calcium Carbonate and Its Polymorphs from Cockle Shells (Anadara Granosa). Powder Technol. 2011, 213, 188–191.

(37)

Hayes, M.; Carney, B.; Slater, J.; Brȕck, W. Mining Marine Shellfish Wastes for Bioactive Molecules: Chitin and Chitosan - Part B: Applications. Biotechnol. J. 2008,

3, 878–889. (38)

Roer, Robert, and R. D. The Structure and Calcificationof Crustacean Cuticle. Am.

Zool. 1984, 24, 893–909. (39)

Synowiecki, J.; Al-Khateeb, N. A. Production, Properties, and Some New Applications of Chitin and Its Derivatives. Crit. Rev. Food Sci. Nutr. 2003, 43, 145–171.

(40)

Tharanathan, R. N.; Kittur, F. S. Chitin — The Undisputed Biomolecule of Great Potential. Crit. Rev. Food Sci. Nutr. 2003, 43, 61–87.

(41)

Stevenson, J. R. Scelerotin in the Crayfish Cuticle Department of Biological Sciences , Kent State University , Kent , Ohio 44240 ,. Comp. Biochem. Physiol., 1969, 30, 503– 508.

(42)

Brown, C. H. K. A Review of the Methods Available for the Determination of the Types of Forces Stabilizing Structural Proteins in Animals. Q. J. Microsc. Sci. 1950,

91, 331–339. (43)

Andersen, S. O. Insect Cuticular Sclerotization : A Review. Insect Biochem. Mol. Biol. 2010, 40, 166–178.

(44)

Shaw, W. H. R.; Bordeaux, J. J. The Decomposition of Urea in Aqueous Media. J. Am.

Chem. Soc. 1955, 77, 4729–4733. (45)

Isobe, N.; Noguchi, K.; Nishiyama, Y.; Kimura, S.; Wada, M.; Kuga, S. Role of Urea in Alkaline Dissolution of Cellulose. Cellulose 2013, 20, 97–103.

(46)

Kwon, W.; Do, S.; Lee, J.; Hwang, S.; Kim, J. K.; Rhee, S. W. Freestanding 31 ACS Paragon Plus Environment


Page 32 of 35

Luminescent Films of Nitrogen-Rich Carbon Nanodots toward Large-Scale PhosphorBased White-Light-Emitting Devices. Chem. Mater. 2013, 25, 1893–1899. (47)

Strauss, V.; Margraf, J. T.; Dolle, C.; Butz, B.; Nacken, T. J.; Walter, J.; Bauer, W.; Peukert, W.; Spiecker, E.; Clark, T.; et al. Carbon Nanodots: Toward a Comprehensive Understanding of Their Photoluminescence. J. Am. Chem. Soc. 2014, 136, 17308– 17316.

(48)

Zhang, H.; Kang, S.; Wang, G.; Zhang, Y.; Zhao, H. Fluorescence Determination of Nitrite in Water Using Prawn-Shell Derived Nitrogen-Doped Carbon Nanodots as Fluorophores. ACS Sensors 2016, 1, 875–881.

(49)

Baker, Sheila N.; Gary A. Baker. Luminescent carbon nanodots: emergent nanolights." Angewandte Chemie International Edition 2010, 49, 6726-6744.

(50)

Jiang, K.; Sun, S.; Zhang, L.; Wang, Y.; Cai, C.; Lin, H. Bright-Yellow-Emissive NDoped Carbon Dots: Preparation, Cellular Imaging, and Bifunctional Sensing. ACS

Appl. Mater. Interfaces 2015, 7, 23231–23238. (51)

Zhang, X.; Liu, R.; Zang, Y.; Liu, G.; Liu, S.; Wang, G.; Zhang, Y.; Zhang, H.; Zhao, H..Shrimp-Shell Derived Carbon Nanodots as Precursors to Fabricate Fe,N-Doped Porous Graphitic Carbon Electrocatalysts for Efficient Oxygen Reduction in Zinc–air Batteries. Inorg. Chem. Front. 2016, 3, 910–918.

(52) Newhouse, T.; Baran, P. S.; Hoffmann, R. W. The Economies of Synthesis. Chem.

Soc. Rev. 2009, 38, 3010. (53)

Sholl, D. S.; Lively, R. P. Seven Chemical Separations to Change the World. Nature 2016, 532, 435–437.

(54)

Raabe, D.; Sachs, C.; Romano, P. The Crustacean Exoskeleton as an Example of a Structurally and Mechanically Graded Biological Nanocomposite Material. Acta

Mater. 2005, 53, 4281–4292. (55)

Chen, P.-Y.; Lin, A. Y.-M.; McKittrick, J.; Meyers, M. A. Structure and Mechanical Properties of Crab Exoskeletons. Acta Biomater. 2008, 4, 587–596.

(56)

Addadi, L.; Raz, S.; Weiner, S. Taking Advantage of Disorder: Amorphous Calcium Carbonate and Its Roles in Biomineralization. Adv. Mater. 2003, 15, 959–970. 32 ACS Paragon Plus Environment

Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(57)

Boßelmann, F.; Romano, P.; Fabritius, H.; Raabe, D.; Epple, M. The Composition of the Exoskeleton of Two Crustacea: The American Lobster Homarus Americanus and the Edible Crab Cancer Pagurus. Thermochim. Acta 2007, 463, 65–68.

(58)

Goodrich, J. D.; Winter, W. T. α-Chitin Nanocrystals Prepared from Shrimp Shells and Their Specific Surface Area Measurement. Biomacromolecules 2006, 8, 252–257.

(59)

King, C.; Stein, R. S.; Shamshina, J. L.; Rogers, R. D. Measuring the Purity of Chitin with a Clean, Quantitative Solid-State NMR Method. ACS Sustain. Chem. Eng. 2017, 5, 8011–8016.

(60)

Nebel, H.; Neumann, M.; Mayer, C.; Epple, M. On the Structure of Amorphous Calcium Carbonate--a Detailed Study by Solid-State NMR Spectroscopy. Inorg.

Chem. 2008, 47, 7874–7879. (61)

Ono, Y.; Ishida, T.; Soeta, H.; Saito, T.; Isogai, A. Reliable Dn/dc Values of Cellulose, Chitin, and Cellulose Triacetate Dissolved in LiCl/N,N-Dimethylacetamide for Molecular Mass Analysis. Biomacromolecules 2016, 17, 192–199.

(62)

Chang, C.; Chen, S.; Zhang, L. Novel Hydrogels Prepared via Direct Dissolution of Chitin at Low Temperature: Structure and Biocompatibility. J. Mater. Chem. 2011, 21, 3865–3871.

(63)

Heredia, A.; Aguilar-Franco, M.; Magaña, C.; Flores, C.; Piña, C.; Velázquez, R.; Schäffer, T. E.; Bucio, L.; Basiuk, V. A. Structure and Interactions of Calcite Spherulites with α-Chitin in the Brown Shrimp (Penaeus Aztecus) Shell. Mater. Sci.

Eng. C 2007, 27, 8–13. (64)

Cárdenas, G.; Cabrera, G.; Taboada, E.; Miranda, S. P. Chitin Characterization by SEM, FTIR, XRD, and 13C Cross Polarization/mass Angle Spinning NMR. J. Appl.

Polym. Sci. 2004, 93, 1876–1885. (65)

Focher, B.; Beltrame, P. L.; Naggi, A.; Torri, G. Alkaline N-Deacetylation of Chitin Enhanced by Flash Treatments. Reaction Kinetics and Structure Modifications.

Carbohydr. Polym. 1990, 12, 405–418. (66)

Romano, P.; Fabritius, H.; Raabe, D. The Exoskeleton of the Lobster Homarus

Americanus as an Example of a Smart Anisotropic Biological Material. Acta Biomater. 33 ACS Paragon Plus Environment


Page 34 of 35

2007, 3, 301–309. (67)

Brugnerotto, J.; Lizardi, J.; Goycoolea, F. M.; Argüelles-Monal, W.; Desbrières, J.; Rinaudo, M. An Infrared Investigation in Relation with Chitin and Chitosan Characterization. Polymer 2001, 42 , 3569–3580.

(68)

Chen, S.-F.; Cölfen, H.; Antonietti, M.; Yu, S.-H. Ethanol Assisted Synthesis of Pure and Stable Amorphous Calcium Carbonate Nanoparticles. Chem. Commun. 2013, 49 , 9564.

(69)

Torres-Rendon, J.G.; Schacher, F.H.; Ifuku, S.; Walther, A. Mechanical Performance of Macrofibers of Cellulose and Chitin Nanofibrils Aligned by Wet-Stretching: A Critical Comparison. Biomacromolecules 2014, 15, 2709–2717.

(70)

Fan, Y. M.; Saito, T.; Isogai, A.

Individual chitin nano-whiskers prepared from

partially deacetylated-chitin by fibril surface cationization. Carbohydrate Polymers 2010, 79, 1046-1051. (71)

Ifuku, S.; Saimoto, H. Chitin Nanofibers: Preparations, Modifications and Applications. Nanoscale 2012, 4, 3308-3318.

(72)

Mushi, N.E..; Butchosa, N.; Zhou, Q.; Berglund, L.A. Nanopaper membranes from chitin-protein composite nanofibers-structure and mechanical properties.

J. Appl.

Polym. Sci. 2014, 131, 40121. (73)

Mushi, N.E.; Butchosa, N.; Salajkova, M.; Zhou, Q.;Berglund, L.A.; Nanstructured membranes based on native chitin nanofibers prepared by mild process. Carbohydrate

Polymers 2014, 112, 255-263. (74)

Sehaqui, H.; Liu, A.; Zhou, Q.; Berglund, L. A. Fast Preparation Procedure for Large, Flat

Cellulose

and

Cellulose/Inorganic

Nanopaper

Structures.

Biomacromolecules 2010, 11, 2195–2198.


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For Table of Contents Use Only Synopsis A facile process for separation of chitin and simultaneous synthesis of carbon nanodots (CNDs) from shellfish waste using urea under hydrothermal condition is demonstrated.


Sustainable Process for Separating Chitin and Simultaneous

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