Pretreatment in Hot Glycerol for Facile and Green Separation of Chitin

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Pre-treatment in Hot Glycerol for Facile and Green Separation of Chitin from Prawn Shell Waste Devi Ramamoorthy, and Dhamodharan Raghavachari ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03195 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

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ACS Sustainable Chemistry & Engineering

Pre-treatment in Hot Glycerol for Facile and Green Separation of Chitin from Prawn Shell Waste Ramamoorthy Devi and Raghavachari Dhamodharan* Department of Chemistry IIT Madras, Chennai 600 036. India E-mail: [email protected] (Author for correspondence)

*Author for correspondence

Abstract A rapid and efficient process for the separation of chitin from waste prawn shells using hot glycerol pre-treatment is reported. The pre-treatment of waste prawn shell in hot glycerol enables the removal of protein possibly through dehydration and temperature induced fragmentation into low molecular weight water-soluble fragments, which are subsequently removed from the shell matrix by dissolution in water. In contrast, in the industrial process of preparing chitin from crustacean shells, the deproteinization is carried out with hot aqueous sodium hydroxide. The novel pre-treatment present here should be applicable to all crustacean shell waste, in principle. Chitin was isolated by two different methods after the pre-treatment in glycerol. In one of the methods, the pre-treated shells were treated directly with citric acid to remove protein and minerals (mostly as calcium citrate). In the second method, the pre-treated shells were ground and rinsed with water to remove protein fragments and part of the minerals (mostly as calcium carbonate). In the subsequent step, the residual minerals were demineralized with citric acid. The later method offers the additional advantage of removing significant quantity of minerals without dissolution in the first step and also reduces the consumption of citric acid used in the demineralization step resulting in reduction in the emission of carbon dioxide. In addition, the glycerol can be used again for three successive cycles of treatment and beyond that can be recovered with charcoal treatment (90 % recovery) and used again. The present method offers 1 ACS Paragon Plus Environment


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distinct advantages over the chemical method, such as lower residual protein (0.24 %), higher crystallinity index (80.9 %) of chitin in addition to the separation of nanofibers of chitin. The recovery of by-products, glycerol and simplicity of the present method guarantee that it could be a greener alternative to the chemical method, predominant in the current industrial scale production of chitin. Keywords: prawn shell waste, separation of chitin, deproteinization, demineralization, hot glycerol treatment, plasticization of shell, fragmentation of protein, biopolymers.

Introduction Crustacean (prawn, shrimp, crab, lobster) shell waste from the seafood industry serves as a useful source of molecules such as chitin, protein, calcium carbonate and carotenoids.1 A major share of the waste is returned to the sea and dumped in landfills. The rest cause surface pollution, especially in coastal areas and offer an opportunity to turn an environmental problem into a source of wealth especially if sustainable chemical approach can be followed for the separation of the molecules present in the shells. The elegant and green separation of chitin, protein and calcium carbonate from the waste shells is an important and challenging problem of economic and environmental relevance in today’s world2 in the context of the current practice of separating chitin alone by the chemical method. Chitin is the second most abundant biopolymer,1,3 which is synthesized by biological organisms to the extent of 100 billion tons, globally, every year. It is more prominently found in exoskeleton of crustaceans, cuticles of insects and fungal cell wall. About 6 to 8 million tons of crustacean shell waste is discarded by the seafood industry and this could serve as an inexpensive source of chitin.1 The exoskeleton of crustaceans is a composite with varying proportion of chitin (15-40 %), calcium carbonate (2050 %) and protein (20-40 %)1,4 that are bonded to each other in a complicated manner.5 The current industrial practice of extracting chitin from the shell waste is the chemical method.6,7 This employs two harsh conditions in sequence: demineralization using a mineral acid and deproteinization with boiling and concentrated sodium hydroxide or vice versa. This process enables the fractionation of chitin alone. This method results in the following disadvantages: release of carbon dioxide, a greenhouse gas; necessity to treat vast quantity of waste water 2 ACS Paragon Plus Environment

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containing calcium chloride along with smaller quantity of protein after demineralization; loss of valuable protein as amino acid salts in the effluent; substantial depolymerization and deacetylation8,9 effects associated with the use of strong mineral acid and hot caustic alkali on the people, infrastructure and environment. The process is destructive, wasteful, expensive and hazardous to the environment and is known to consume a very large quantity of water (the production of 1 kg of chitin from shrimp shells requires more than one ton of water10). Thus chitin can be expensive (50-200 $)1, especially if protein-free material is to be separated, although the raw material is a waste. The production of chitin is important from the utility point of view as it offers unique properties such as biocompatibility, biodegradability, antimicrobial activity, low immunogenicity, and accelerates wound healing.9,11 Because of its exceptional qualities it has found use in many applications including biomedical, cosmetics, food, agriculture, textile, paper, and water treatment.12–16 Chitin consists of seven weight percentage of biologically fixed nitrogen, which can be used to prepare value-added nitrogen containing chemicals similar to what is done using the chemical refinery.17, 18 There have been several reports on the extraction of chitin by biotechnological methods.8,19–23 These are time intensive, expensive and leave a small quantity of unextracted protein in chitin, that can cause allergy in some people,8 and will have be removed by subsequent alkaline hydrolysis. Thus the chemical method of extraction is the preferred industrial practice for the large-scale production of chitin because of its simplicity and relatively lesser residual impurities (proteins and minerals) when compared to the biotechnological methods. On the other hand, ionic liquid (IL) based extraction process24,25 are considered as a green alternative based on three arguments: vapor pressure, flammability, and toxicity. However, the wastewater treatment, biodegradability, and toxicity of degradation products have to be investigated in detail.26 Furthermore, the prior preparation of expensive ILs and recovery of ILs as well as by-products add few more processing complications. In the above content, the isolation of chitin by a sustainable and greener process acquires significance. If this can enable the recovery of protein and minerals while retaining the quality it would promote effective utilization of natural resources. The by-products could be used as animal feed or in agriculture eventually supporting the eco system. A sustainable separation method that enables the isolation of the useful chemicals from the shell refinery, which avoids 3 ACS Paragon Plus Environment


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corrosive or hazardous reagents and minimizes waste, is the need of the hour to establish sustainable as well as profitable shell refinery. We report here a very simple and sustainable method of separating chitin from prawn shell waste. The method involves pre-treatment of prawn shell waste in hot glycerol as a substitute for deproteinization step with, refluxing aqueous sodium hydroxide used in the chemical treatment. The pre-treated shells were treated directly with citric acid to remove protein and minerals (as calcium citrate) in a single step. In another method (two step process), the pre-treated shells were ground and rinsed with water to remove protein fragments and part of the minerals (as calcium carbonate). Subsequently, the residual minerals were demineralized with citric acid. The current processes are much faster, and uses chemicals from sustainable sources such as glycerol for pre-treatment and citric acid for demineralization.27,28 Glycerol is a by-product from the biodiesel industry, abundant and inexpensive.27 The methodology and the possible mechanism of the process are described in this paper. Materials Prawn shell waste (PSW) was collected from the harbour region of North Chennai (Sunday Fish Market), India. Analytical grade glycerol and citric acid were purchased from Fisher Scientific Chemicals, Chennai. Sodium hydroxide, hydrochloric acid and sodium hypochlorite were purchased from Rankem, Chennai and were used as received. Analytical studies Solid state NMR (13C; CPMAS) 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 micro sec, number of scans =1024). 1H and

13

C NMR spectra in

solution were also recorded using the above spectrometer operating at 400 MHz for proton in D2O. 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 bromide (KBr). The thermogravimetric analysis were carried out with TA Instruments Q500 Hi-Res TGA. The samples were heated at 10 °C min-1 under flowing N2 atmosphere. X-ray diffraction patterns of all the materials were recorded with a Bruker D8 Advance diffractometer equipped with copper 4 ACS Paragon Plus Environment

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(Cu) anode (Cu Kα source of wavelength1.5406 Å).

Scanning electron microscopy was

performed using Quanta 400 Scanning electron microscope (FEI, USA). Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis was carried out using Perkin-Elmer Optima 5300 DV calibrated with Merck standards.

Experimental Methods Separation of chitin from prawn shell waste 300 g of fresh, wet PSW was ground in a domestic blender for two minutes. It was filtered using metal-fibre filter (size 300 µm). The residue (material that was retained) was rinsed with tap water and dried using filter paper. Yield: 19.7 ± 0.3 % (average of three experiments)). The filtrate was heated upon which the precipitation of organic matter (37.8 g) consisting largely of protein takes place. This can be directly used as animal feed or may be hydrolysed to prepare amino acids. The residue was dried at 80 ° C overnight. Yield of RAW-P: 18.9 g (6.3 % by dry weight in this experiment). The average yield of RAW-P for three different experiments was 7.1 ± 0.8 %. Pre-treatment using glycerol RAW-P (18.9 g) was heated by suspending in 200 g of glycerol maintained at 200 °C for 4 minutes. The solid and glycerol were separated from the hot mixture by filtration. The residual glycerol was reused for the succeeding treatment processes.

The heat-treated shells were

allowed to cool. Deproteinization and demineralization after pre-treatment The pre-treated shells were ground with citric acid (8.5 g; 0.044 moles) for 10 minutes in a wet pan mill. This was followed by the addition of 300 ml of water, soaking for 20 minutes, and filtration. The solid obtained was rinsed again with 600 ml of water. The filtrate upon maintenance at room temperature for about 10 h (overnight) resulted in the formation of crystals of calcium citrate that could be filtered, enabling the recovery of calcium. The residual water was slightly acidic (pH ~ 5). The chitin isolated was decolorized using 0.12 % of sodium hypochlorite solution at room temperature for 20 minutes, rinsed with distilled water and dried at 5 ACS Paragon Plus Environment


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80 °C. This chitin was denoted as HTC-P. Yield: 6.13 g (32.4 % by weight of RAW-P); Residual mineral content was Ca2+ = 0.23 % and Mg2+ = 0.02 %. Yield of calcium citrate: 6.88 g (0.014 moles; 70 % recovery). Grinding and Rinsing after Pre-treatment (Two-step process) RAW-P (5 g) was suspended in hot glycerol (85 g) maintained at 200 °C. After 4 minutes (the colour of the fibrous shell material turns brown), the mixture was drained to recover excess glycerol (47 g) using a SS filter. The pre-treated shells were was ground with 50 ml of water in a domestic blender and filtered (pH of filtrate ~ 8-9). It was rinsed with 100 ml of water and the fibrous material thus obtained (G-PRS) was dried at 80 °C. Yield: 2.11 g (42.2 % by weight of RAW-P). Demineralization of G-PRS 5 g of dry G-PRS (15 weight % of CaCO3 by TGA and 12.5 weight % by ICP-OES) was treated with a slight excess of citric acid [0.96 g; 0.0049 moles] in 50 ml of water for 20 minutes at room temperature. The resultant solid was separated by filtration followed by rinsing with 250 ml of water and dried at 80 °C. Yield: 3.32 g (66.4 % by weight of G-PRS; and 29.6 % by weight of RAW-P). Yield of Calcium Citrate: 0.94 g (90 % recovery). Plasticization using Glycerol To demonstrate the plasticization effect of glycerol on prawn shells, 2 g of RAW-P was ground with 2 ml of an aqueous mixture of glycerol and water in the ratio of 1:1 (v/v). The glycerol treated RAW-P was denoted as G-RAW-P and used for PXRD analysis. Recovery of Glycerol After pre-treatment, used glycerol was recovered by filtration while hot and reused without further modification for two subsequent treatments. Further, it was treated with charcoal to absorb soluble organic matter. The overall recovery of glycerol was 90.4 % and the glycerol was denoted as HTC-3. Isolation of Chitin [CHE-P] from Prawn Shell by the Chemical Method (Control Experiment) 6 ACS Paragon Plus Environment

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To compare the yield and properties of chitin extracted using the methods described here, control experiment was performed by the chemical method of separating chitin as reported in literature.6 100 g of wet PSW was ground mechanically, rinsed with water and filtered. The residue (RAWP, 7.8 g, dry weight) was demineralized with 0.25 M HCl (in the proportion of 40 mL/g) at ambient temperature for 15 minutes followed by deproteinization with 1 M NaOH (in the ratio of 15 mL/g) at 70 °C for 24 h. Yield: 2.59 g by dry weight, 33.2 % by weight of RAW-P). The chitin isolated by chemical method was denoted as CHE-P. Deproteinization of prawn shell [CHE-DP] using sodium hydroxide (control experiment) 5 g of RAW-P powder was treated with 75 ml of 1 M sodium hydroxide solution for 24 h at 70 °C, rinsed with 1500 ml of distilled water to neutral pH, and dried. Yield: 2.94 g (58.80 % sodium hydroxide insoluble). Demineralization of prawn shell using citric acid (control experiment) 5 g of RAW-P powder was treated with 2.55 g (0.0133 moles) of citric acid dissolved in 50 mL of water. After 20 min, the fibrous and entangled solid was rinsed with distilled water to neutral pH, and dried. Yield: 2.633 g (52.65 % citric acid insoluble). Tests for residual protein i)

Biuret Test

20 mg of the solid treated sample was mixed with 2 ml of 1 % sodium hydroxide solution followed by the addition of 1 ml of 1 % copper (II) sulfate solution. The inference is change in colour of copper sulphate solution to purplish violet. ii)

Ninhydrin Test

20 mg of solid sample was added to a test tube followed by the addition of 2 ml of water and 1 ml of 0.1 % ninhydrin solution in methanol. It was heated to boiling. The change of color (from colorless) to purple indicated the presence of protein or peptide residue with terminal amine group. iii)

Lowry Test

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50 mg of sample was treated with 1 M aqueous sodium hydroxide solution for 24 h at 70 °C. The supernatant was made up to 10 ml and analyzed as per the standard protocol.29 The protein extracted from RAW-P using 2 M sodium hydroxide solution (boiled for 20 minutes) was used as a standard, after dialysis, for quantitative purpose. iv)

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 Deproteinization and Demineralization of Prawn Shells The thermogravimetric analysis of dry raw prawn shell (RAW-P) shown in Figure 1, revealed that the order of thermal stability of its main components was: calcium carbonate > chitin > protein. In view of the fact that the onset temperature for chitin degradation is ~250 °C, heat treatment below this temperature is likely to enable thermal degradation of protein, which may result in partial fragmentation of the protein backbone.30 In fact it has been reported that chitin remains structurally intact when it is heated from room temperature to 250 °C.31 Therefore the ground prawn shells were treated with hot glycerol with the objective of separating the protein from the composite matrix. Consequent to heat treatment and after draining the glycerol, the demineralization was also carried out by grinding with citric acid followed by water rinse. The TGA and differential thermal analysis of the material thus obtained (HTC-P) is also shown in Figure 1. By comparison of the data presented in Figure 1 it appears that that the new method results in the separation of chitin from the shell matrix as evident from the absence of peaks in the region 200-250 °C (protein) and 600-700 °C (assigned to calcium carbonate).


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Figure 1. Thermogravimetric (a) and differential thermal (b) analysis of dry RAW-P and chitin (HTC-P). With the view to understand the impact of glycerol treatment on removing protein from prawn shell, the treated shell material was ground and rinsed with water, dried (G-PRS) and analysed by TGA for residual protein content. This revealed that proteins are removed by hot glycerol treatment (Figure 2) as can be inferred from the absence of weight loss observed in the region 200-250 °C, which is typically observed for proteins in the untreated shell material as shown in Figure 1. For the purpose of comparison the TGA of RAW-P deproteinized by the conventional hot aqueous NaOH treatment (1 M NaOH for 24 h at 70 °C)5 is also shown in Figure 2. Thus, it is clear that the treatment of RAW-P in hot glycerol is successful in removing protein vis-à-vis that carried out by the conventional chemical process with 1 M sodium hydroxide. Surprisingly, it was observed from the TGA that the hot glycerol treatment resulted in the elimination of significant quantity of calcium carbonate (within the analytical limits of TGA) in addition to proteins as evident from the absence of weight loss in the region 600-900 °C, which arises due to the decomposition of calcium carbonate into calcium oxide and carbon dioxide. The percent residue at 900 °C, which can also be used to indirectly assess the mineral content as it arises largely from calcium oxide and chitin present in the shells. For the NaOH treated RAW-P [CHE-DP] the residue at 900 °C was 38.2 % while that for the glycerol pre-treated sample (GPRS) was 7.2 %.

This demonstrates that glycerol pre-treatment is not only effective in

deproteinization but also depletes mineral content to a greater extent. The calcium carbonate content of RAW-P as assessed by TGA was 27.5 ± 2. 6 % (average of four measurements) while 9 ACS Paragon Plus Environment


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that by ICP-OES was 28.9 %.

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Therefore the pre-treatment in glycerol in addition to

deproteinization enables the removal of about 50 % calcium carbonate from RAW-P. The elimination of significant quantity of calcium carbonate by treatment with hot glycerol may be understood from the inner structural organization of crustacean shells where the protein wraps around the crystalline chitin nanofibril and acts as a bridge between chitin and minerals.5 The heat treatment can be expected to result in dehydration followed by partial fragmentation of the protein that in turn should result in the formation of a more brittle envelop which could break up upon the application of mechanical stress. As the connection between chitin and protein is broken down, the minerals associated with proteins through strong ionic interaction could have been removed along with the protein. The removal of minerals may also be attributed to the loss of smaller size particles of calcite (0.5-200 µm) as well as loss of amorphous calcium carbonate that has been reported to be present in the case of shrimp shells.32,33 The residual minerals present in G-PRS were demineralized with 20 weight % of citric acid that was used in the one step process to separate chitin (Supporting Information Figure S1).34

Figure 2.

Thermogravimetric and differential thermogravimetric analysis of RAW-P

deproteinized with (a) hot aqueous [CHE-DP] and (b) hot glycerol [G-PRS]. The advantages of the present method in contrast to the chemical method of separating chitin from the shells are: i) shorter processing time (~ 30 minutes including demineralization); ii) water required in the current method for the two step process (hot glycerol pre-treatment, 10 ACS Paragon Plus Environment

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grinding, rinsing with water (G-PRS) and demineralization with citric acid) is one-third that of the chemical method; iii) the facile recovery of calcium carbonate from the filtrate of G-PRS as sediment; iv) the recovery of calcium as calcium citrate crystals, at room temperature (therefore the effluent treatment is minimized while in the conventional chemical treatment method, calcium chloride effluent may have to be treated); v) the evolution of CO2, a greenhouse gas, is significantly reduced (if the two step process is performed as there is a reduction of 80 % of citric acid used when compared to the one step process); vi) the greater degree of crystallinity and deproteinization of chitin than chemical method. Yield of chitin from dry RAW-P was 32.40 % and 33.05 % for HTC-P and CHE-P respectively, which is comparable to the yield of chitin obtained from shrimp shells.4 It may be noted that pre-treatment using glycerol does not require water and the optional intermediate step can be carried out with seawater. Hence, the present method appears to be promising for large-scale production from the viewpoints of greener process of separation and lesser time involved. The relative ease of separation of chitin from protein and other constituents was made possible by pre-treatment of the shells in glycerol and the picturization of the same is represented in Scheme 1. One of the roles of glycerol is to function as a plasticizing medium (see supporting information Figure S2 for the PXRD pattern of glycerol treated prawn shell) and enable the penetration of heat into the organic matrix.35,36 thereby providing uniform distribution of heat. Subsequently, dehydration of the swollen/ plasticized matrix may occur followed by partial breakdown of the protein chains resulting in the formation of brittle matrix. It may be noted that any high boiling liquid, can replace glycerol, although glycerol with its well-established nature of inhibiting protein aggregation, hydrophilic nature, high boiling point, eco-friendliness, and availability is more suitable. The present process is represented by the flow chart shown in Scheme 2. It may also be noted that the current work does not discuss the mechanism of protein degradation in hot glycerol and rather focuses on the development of a method to remove protein from the chitinous matrix. It may very well involve temperature induced random scission of peptide linkages as is normally observed during the breakage of proteins in thermogravimetric analysis.30 In fact, control experiments involving the hot glycerol treatment of bovine serum albumin (BSA) followed by the qualitative analysis with ninhydrin indicated the presence of amino acids (Supporting Information Figure S3).



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Scheme 1. Pictorial representation of pre-treatment in hot glycerol for isolation of chitin

Scheme 2. Process flow chart representing the present method of separating chitin from prawn shell waste. Recovery of glycerol The glycerol after continuous usage turned brown in colour after three uses. Upon treatment with activated charcoal, it is decolourized and could be reused (Figure S4). NMR spectra of fresh and recovered glycerol are also shown in Figure S5-8. 12 ACS Paragon Plus Environment

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Assessment of the Quality of Chitin The purity of the chitin obtained from the processes described above was ascertained by powder x-ray diffraction (PXRD), thermogravimetric analysis (TGA), Fourier-Transform infrared spectroscopy (FT-IR), solid state NMR spectroscopy and qualitative analytical tests such as biuret, ninhydrin and Lowry methods. The PXRD data of chitin separated from prawn shells is shown in Figure 3. This revealed the prominent crystalline peaks associated with chitin37 at 2θ values of 9.57° (020), 12.95 (021), 19.61° (110), 21.15 (120), 23.43 (130), 26.58 (013) attributed to chitin.38,39 The other peaks associated with calcite are not observed suggesting that is essentially pure by the limits of PXRD. For the purpose of comparison the PXRD pattern of the prawn shell along with the peak assignments are also shown in the Figure 3. The PXRD pattern of the chitin obtained by the method described here are in good agreement with that of chitin obtained by the chemical treatment (CHE-P) and also suggest that the chitin obtained is free from minerals. Further more, the crystallanity index of the chitin as assessed following the procedure reported in the literature40 are found to be 76.7 % for CHE-P and 80.9 % for chitin from prawn shell waste. The intensity ratio of (020) with respect to (110) is 0.90 for chitin obtained by the present method, while the ratio for chitin obtained by the chemical method (CHE-P) is 0.79. This suggests that the chitin obtained by present method does not undergo significant deaetylation while the chitin obtained from the chemical method could be undergoing deacetylation during deproteinization.37 Since, the process described here, does not involve any such harsh chemical environment, it may be inferred that the chitin remains intact after the separation processes.



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Figure 3. PXRD pattern of chitin isolated by a) current method (HTC-P), b) chemical method (CHE-P). For comparison c) the PXRD pattern of RAW-P. CH-Chitin, Ca-Calcite. The

13

C SS-NMR is a one of the imprtant tools to assess the quality of chitin.41 The SS-NMR

spectrum of prawn shell and chitin separated from prawn shell by the present method are presented in Figure 4. The spectrum of prawn shell reveals all the peaks characteristic of chitin, namely, 173 ppm carbonyl carbon in N-acetylamine, 103 (C1), 82.5 (C4), 76 (C5), 73 (C3), 61 (C6), 55 (C2) and 22 (C8) and in addition reveals peaks for proteins in the region of 20-40 ppm (highlighted) as well as by the broadening of the carbonyl carbon in the region 160 to 170 ppm that arises typically from amide carbonyl. The peaks characteristic of proteins were not detected in the chitin isolated (HTC-P) thus demostrating its purity by the limits of SS-NMR.38,41


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. Figure 4. Solid state 13C NMR spectra of a) chitin (HTC-P) isolated by current method and b) RAW-P The FT-IR spectrum of prawn shells and that of the chitin isolated by the present as well as the chemical methods are presented in supporting information (Figure S9). The spectra of chitin isolated illustrated the presence of characteristic peak for α-chitin at 1660 cm−1 (splitting of amide-I band is one of the indication for removal of protein. This may be attributed to two types of hydrogen bonding; one of them is between the N-acetyl carbonyl group with NH of the Nacetyl carbonyl of the adjacent chain and the other one is between carbonyl and primary hydroxyl group of the same chain CO⋯HN and CO⋯HOCH2).37,42 Such a splitting of amide-I band was not observed in RAW-P due to amide peaks of protein overlapping with the chitin amide I bands, and this suggested that the chitin isolated is free from protein (by the limits of FTIR) and supports the result obtained from SS-NMR. The other peaks characteristic of carbonate group [observed at 1440, 874 and 725 cm-1 and assigned to (ʋ3) asymmetric stretch, (ʋ2) asymmetric stretch and (ʋ4) symmetric stretch of carbonate group, respectively] were not observed in the chitin samples suggesting the absence of calcium carbonate (by the limits of FTIR). The DTG and TGA analysis of prawn shell waste (RAW-P) before and after treatment and that of the intermediate product G-PRS are presented in supporting information (Figures S10 and S11). For the purpose of comparison, the thermal degradation pattern of chitin obtained by the 15 ACS Paragon Plus Environment


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process described in this work (HTC-P) and that obtained by the chemical method (CHE-P) are shown in Figure S10. It is important to take into account, that the chitin isolated by the current method does not exhibit weight loss between 150-330 °C, in contrast to the chitin isolated by chemical method, where a small hump was observed (highlighted in insert Figure S11). This may indicate the presence of residual protein, which are present in the crystalline matrix and might be protected from alkaline hydrolysis.6 The current method perhaps enables the penetration of heat energy into the crystalline matrix by convection and probably results in breakdown of the backbone of the protein, which is then removed upon grinding. The TGA data suggests that protein is not present along with chitin obtained by the current method by the limits of detection of the method. Additionally, the absence of weight loss between 600-900 °C demonstrates the complete removal of calcium carbonate by the limits of TGA. The thermal analysis suggests that demineralization using citric acid was as efficient as that with a mineral acid such as hydrochloric acid. The intermediate product G-PRS shows less intense weight loss for minerals (highlighted in the Figure S11) due to their removal upon grinding. The product isolated from this (G-PRS) resulted in less recovery of calcium citrate (shown in Figure S12). Thus, the removal of minerals without dissolution can be substantiated by this observation. In order to analyze the presence of trace quantity of residual protein, qualitative and quantitavie chemical tests for peptides and amino acids were also carried out with the chitin isolated. The biuret test reveals the presence of peptide bonds up to a chain length at least three amino acids with a sensitivity range of 5–160 mg/mL of proteins (Figure S13). This test was carried out to compare the residual protein content in raw and treated samples. The absence of color change in the chitin samples isolated by the method reported here (Figure S13) confirmed the absence of peptides. To further confirm this implication, ninhydrin test was also performed, which reveals the presence of amine functional group from residual amino acid, if it was present. The lack of colour change in the chitin (HTC-P) samples that were isolated by the method described here suggests the absence of residual amino acids (Figure S13). The quantitaive analysis for degree of deproteinization was determined by the Lowry test (shown in Figure 5). The chitin isolated by the present method (HTC-P) showed a residual protein content of 0.24 % while chitin separated by the chemical method (CHE-P) showed a residual protein content of 0.41 % (R2 value was 0.998 against protein isolated from prawn shell waste).


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Figure 5. Lowry test for protein residues in demineralized and isolated samples. A) RAW-P, B) CHE-P, C) HTC-P and D) Control. The morphology of the chitin obtained by the present method was assessed by HRSEM (Figure 6), which clearly demonstrated the formation of nanofibrous structure. In contrast, the chitin separated from the chemical method showed the presence of thicker bundles of fibers. Thus the width of the chitin fibres separated by the present method are observed to be in the range of 20100 nm, while it was 5-10 µm for the chitin isolated by chemical method. The length of the chitin nanofibers was in the range of several hundred nanometres in nanofibers in the present method could be due to partial protonation of free surface amine groups during the demineralization with citric acid, that in turn could result in fiber-fiber repulsion leading to dispersion. In fact, the chitin obtained by the present method was a fine powder in comparison to flakes of chitin obtained by the chemical method where the pH of the medium is alkaline in the final step and would not facilitate the formation of quaternary ammonium salt of free surface amine groups. If the chitin separated by the present method was rinsed with water, repeatedly, to neutral pH and dried, the reorganization of fibrils was observed along with the formation of hard lumps. This was also observed in the case of prawn shell waste demineralized with citric acid, rinsed with water to neutral pH and dried. The isolation of nanofibers of chitin directly from the waste may eventually facilitate the further modifications.43-45



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Figure 6. SEM images of a) RAW-P (broken surface), b) G-PRS and c) chitin (HTC-P) (scale=1µm) Conclusion In conclusion, we have developed a greener protocol for large-scale production of chitin in shorter duration from prawn shell waste using glycerol and citric acid that are obtained from sustainable resources. The pre-treatment of the prawn shells with hot glycerol followed by grinding with citric acid enables the removal of protein and minerals in a single step. The plasticization of the prawn shell waste by glycerol enables uniform heat transfer and possibly is the driving force for thermal depolymerization of protein, which takes place in the absence of acid or base catalysts. Importantly, this method enables the removal of significant quantity of minerals in its native form, if the two step process is performed. This in turn results in the use of 80 % less quantity of citric acid required for demineralization in comparison to the single step process thereby reducing the volume of carbon dioxide evolved. In addition, the glycerol used in the pre-treatment was effectively reused and recycled. The spectroscopic and thermal analysis showed that the quality of chitin was superior while the quantitative protein analysis by Lowry method demonstrated the greater efficiency of deproteinization than the conventional chemical method. The current method is simple, scalable, and sustainable. Hence, it might be ideal for sustainable production of chitin in industrial scale.


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ASSOCIATED CONTENT Supporting Information Photographs of RAW-P after pre-treatment in glycerol, G-PRS and chitin (HTC-P); PXRD pattern of RAW-P and glycerol treated RAW-P; Qualitative ninhydrin test for amino acids; Photographs of glycerol after pre-treatment of RAW-P and after treatment with activated charcoal followed by filtration; 1H NMR spectra of recovered glycerol in D2O; 1H NMR spectra of glycerol in D2O;

13

C NMR spectra of recovered glycerol in D2O;

13

C NMR spectra of

glycerol in D2O; FT-IR spectra of chitin isolated by, the present method (HTC-P), the chemical method (CHE-P) and RAW-P; Thermogravimetric analysis of chitin HTC-P: separated by the method reported here from RAW-P; G-PRS: separated from hot glycerol treated RAW-P followed by grinding and rinsing with water; CHE-P: separated by the conventional chemical method; Differential thermogravimetric analysis of chitin separated from prawn shell. Hot glycerol treatment followed by grinding and rinsing with water (G-PRS); chemical method (CHE-P); and hot glycerol treatment followed by grinding in the presence of citric acid followed by rinsing with water (HTC-P); Photographs of crystals of calcium citrate obtained by demineralization of RAW-P (I) and G-PRS (II) using citric acid; Photographs of samples of RAW-P, CHE-P and HTC-P after different tests. Biuret test (a,b,c), ninhydrin test (d,e,f) and xanthoprotic test (g,h,i). AUTHOR INFORMATION Corresponding Author Dhamodharan Raghavachari. E-mail: [email protected] 19 ACS Paragon Plus Environment


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Notes The authors declare no competing financial interests. Acknowledgements R. Devi thanks CSIR, Government of India for fellowship. The authors thank IIT Madras for financial support and for the research facilities. This work was made possible due to the special support of IIT Madras. The work presented here is a part of Indian patent application (201641030691) filed by the authors. References (1)

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For Table of Contents Use Synopsis Protein and minerals in prawn shell waste are removed by hot glycerol pre-treatment, a novel substitute for hot alkaline hydrolysis.


Pretreatment in Hot Glycerol for Facile and Green Separation of Chitin

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