WO2018222137A1

WO2018222137A1 - A method of processing shell waste

Info

Publication number: WO2018222137A1
Application number: PCT/SG2018/050264
Authority: WO
Inventors: Ning Yan; Huiying Yang; Xi Chen
Original assignee: National University Of Singapore
Priority date: 2017-05-30
Filing date: 2018-05-30
Publication date: 2018-12-06
Also published as: CN110944761A

Abstract

There is provided a method of processing shell waste for recovering components comprised in shell of shellfish, the method comprising: mixing the shell waste with a denaturing agent or water at a pre-determined temperature for a pre-determined period of time to form a first mixture; filtering the first mixture to obtain a filtrate and a residue, wherein a first component is recovered from the filtrate; contacting the residue obtained from the filtering with carbon dioxide to form a suspension comprising a second component; and collecting a third component as balance residue following the contacting, wherein the first component is one or more proteins, the second component is a calcium salt, and the third component is chitin.

Description

A method of processing shell waste

Technical Field

The present invention relates to a method of processing shell waste. In particular, the method may enable recovery of components comprised in the shell.

Background

Global fish production has grown at a very fast pace and accordingly, huge quantities of waste are also generated, especially the shells from shellfish including crustacean animals.

Although the share of crustaceans in fishery production is only at around 9%, the shell waste takes up a significant portion because these species are featured with low meat content but high shell yield. Currently, the shell waste is landfilled but this can cause serious environmental issues and human health risk in view of bacterial contamination and strong odour during decomposition.

Shells of shellfish comprise useful chemicals such as proteins, chitin, minerals (e.g. calcium carbonate), pigments (e.g. astaxanthin and carotenoids) and lipids. Current methods of extracting chitin from shell waste involve large amounts of corrosive acids, such as hydrochloric acid, and bases, such as sodium hydroxide, to peel-off proteins and calcium carbonate (CaC0₃) from chitin. Such reagents cause environmental concerns and also require costly corrosion-resistant equipment, special handling and wastewater treatment, therefore riding up the cost of extracting chitin.

Other methods which involve solvent extraction using an ionic liquid or biological fermentation have been proposed. Nevertheless, the solvent ionic liquids are expensive, sensitive to water (in air) and difficult to handle due to their high viscosity. Biological fermentation is environmentally benign and scalable, however, it has low efficiency in view of the long reaction time and the relatively low purity of chitin product.

There is therefore a need for an improved method of processing shell waste and for recovering major components comprised in the shell. Summary of the invention

The present invention seeks to address these problems, and/or to provide an improved method of processing shell waste for recovering components comprised in shell of shellfish. For example, the major components comprised in shell of shellfish may be proteins, calcium salt and/or chitin.

In general terms, the invention relates to a simple and environmentally friendly method of processing shell waste and recovering major components such as protein, calcium salt and chitin from the shell. The method is advantageous in that it utilises non- corrosive and low-cost reagents and enables high purity chitin to be recovered.

According to a first aspect, the present invention provides a method of processing shell waste for recovering components comprised in shell of shellfish, the method comprising: mixing the shell waste with a denaturing agent or water at a pre-determined temperature for a pre-determined period of time to form a first mixture;

filtering the first mixture to obtain a filtrate and a residue, wherein a first component is recovered from the filtrate;

contacting the residue obtained from the filtering with carbon dioxide to form a suspension comprising a second component; and

collecting a third component as balance residue following the contacting, wherein the first component is one or more proteins, the second component is a calcium salt, and the third component is chitin.

The shell waste may comprise shell from any shellfish. For example, the shell may be from, but not limited to, crab, clam, oyster, shrimp, lobster, mussel, abalone, scallop, crayfish, limpet, winkle, or a combination thereof. According to a particular aspect, the shell waste may comprise crushed shell.

The mixing may be with a denaturing agent or water. According to a particular aspect, the mixing of the shell waste may be with a denaturing agent. The denaturing agent may be any suitable denaturing agent. For example, the denaturing agent may be selected from the group consisting of: urea, guanidine hydrochloride, and a combination thereof. The mixing of the shell waste with the denaturing agent may be at a pre-determined temperature for a pre-determined period of time. The pre-determined temperature may be any suitable temperature for the purposes of the present invention. For example, the pre-determined temperature may be 0-50°C.

The pre-determined period of time when the mixing of the shell waste is with the denaturing agent may be any suitable period of time. For example, the pre-determined period of time may be 1-24 hours.

The denaturing agent used for the mixing may have a suitable concentration. For example, the concentration of the denaturing agent may be 1-8 mol/L.

According to a particular aspect, the mixing of the shell waste may be with water. The mixing of the shell waste with water may be at a pre-determined temperature for a predetermined period of time. The pre-determined temperature may be any suitable temperature for the purposes of the present invention. In particular, the pre-determined temperature may be≥140°C.

The pre-determined period of time when the mixing of the shell waste is with water may be any suitable period of time. For example, the pre-determined period of time may be ≤ 4 hours.

The contacting may be carried out under suitable conditions. According to a particular aspect, the contacting may be carried out in water in a chamber.

The carbon dioxide used for the contacting may be at a suitable pressure. In particular, the carbon dioxide may be at a pressure of 1-100 bar.

According to a particular aspect, the contacting may result in the formation of carbonic acid. In particular, the suspension formed during the contacting may be from a reaction of the carbonic acid with calcium salt comprised in the shell waste comprised in the residue.

The method may further comprise recovering the second component from the suspension. Brief Description of the Drawings

In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments, the description being with reference to the accompanying illustrative drawings. In the drawings:

Figure 1 shows a process flow diagram of production of chitin using the traditional chitin extraction method (prior art);

Figure 2 shows a process flow diagram of production of chitin according to one embodiment of the present invention, i.e. urea as the denaturing agent;

Figure 3 shows a process flow diagram of production of chitin according to one embodiment of the present invention;

Figure 4(a) shows the kinetic profile of mixing with urea solution for deproteinization at different concentrations wherein 0.4 mg of shrimp shell is mixed with 40 mL urea solution at 0-8 mol/L at room temperature for 5, 15, 30, 60, 120, 240, 360 and 720 minutes and Figure 4(b) shows the mixing for deproteinization for three cycles wherein 0.4 mg of shrimp shell is mixed with 40 mL pure water or 8 mol/L urea solution at room temperature, 6 hours per cycle;

Figure 5 shows deproteinization in urea solution at different concentrations;

Figure 6 shows deproteinization in 8 mol/L urea solution at different solid to solvent ratio;

Figure 7 shows deproteinization using different stirring rates; Figure 8 shows deproteinization at different temperatures;

Figure 9(a) shows deproteinization using water wherein 0.2 mg of shrimp shell is mixed with 3 mL water at room temperature, 140, 160, 180, 200, 220°C for 15 or 30 minutes and Figure 9(b) shows GPC profile of protein isolates from deproteinization process using water at room temperature, 140, 180, 220°C; Figure 10 shows the kinetic profile of demineralization by high pressure C0₂ wherein 0.1 mg of shrimp shell is mixed with 10 mL water at room temperature under high pressure C0₂ at 10 or 20 bar for 5, 15, 30, 60 and 120 minutes;

Figure 1 1 shows deproteinization of demineralized shrimp shell via hydrothermal method;

Figure 12(a) shows FTIR spectra of fractionated products from shrimp shell via different methods and Figure 12(b) shows XRD spectra of shrimp shell and chitin extracted via different methods - tra chitin: chitin extracted via traditional method; urea- C0₂-chitin: chitin extracted via deproteinization by 40 mL urea solution at 8 mol/L at room temperature after three cycles and demineralization by high pressure C0₂ at 10 bar for 1 hour at room temperature; HT-C0₂-chitin: chitin extracted using water at 220°C for 30 minutes; and

Figure 13 shows FESEM images of shrimp shell at different fractionation stages. Figure 13(a) shows shrimp shell's layer-by-layer structure; Figures 13(b), (c) and (d) show shrimp shell after deproteinization with pure water at room temperature, urea method and hydrothermal method, respectively; Figures 13(e) and (f) show chitin extracted via urea-C0₂ method and hydrothermal-C0₂ method, respectively.

Detailed Description

As explained above, there is a need for an improved method of processing shell waste for recovering components comprised in the shell of shellfish. Shell of shellfish are generally composed of about 30% chitin, 30% proteins and 40% CaC0₃. Current methods of extracting chitin include: the traditional industrial method utilising hydrochloric acid and sodium hydroxide for demineralization and deproteinization, respectively; the solvent extraction method in which chitin is extracted from the shells by dissolving the shells in ionic liquids and precipitation by addition of water; and the bioprocessing method in which the shells are fermented with microorganisms and the minerals and proteins are gradually dissolved respectively by lactic acid and proteases released by the microorganisms. The problem with the traditional industrial method is that strong acid and base waste is generated, and further generating large quantities of waste water for diluting the strong acid and bases after the reaction which is not suitable for the environment. The solvent extraction method is an expensive method since the ionic liquids used in the reaction are expensive and therefore need to be recycled. Further, as a result of the reaction, chitin dissolution accompanies CaC0₃ dissolution and therefore an additional mineralization step is required using citric acid to obtain the chitin. This makes recovery of the ionic liquids more difficult, leading to an increase in cost of using the method. In the case of the bioprocessing method, while the method utilised mild reagents and is environmentally friendly, the quality of the chitin obtained is relatively low due to the poor accessibility of proteases caused by inefficient demineralization.

The present invention relates to a method which is environmentally friendly, low cost and at the same time able to recover chitin with high purity. The method also enables other components of the shell, such as proteins and minerals, to be recovered. In particular, the method of the present invention utilises mild reagents under mild conditions, making the method safe and economical.

Generally, the method of the present invention involves deproteinization and demineralization. In particular, the deproteinization may comprise recovering the proteins in the shell waste by dissolving the shell waste either in a denaturing agent or in water under suitable conditions and recovering by filtration. The demineralization may comprise dissolving the shell waste under high pressure carbon dioxide in water without heating and precipitating the mixture to recover the CaC0₃ by simple filtration.

collecting a third component as balance residue following the contacting, wherein the first component is one or more proteins, the second component is a calcium salt, and the third component is chitin. For the purposes of the present invention, the shell waste may comprise the shell from any shellfish. For example, the shell waste may be from shell of the shellfish discarded following the use of the shellfish. The shellfish may be defined as any exoskeleton- bearing aquatic invertebrate. In particular, the shellfish may comprise crustaceans. For example, the shell waste may comprise shell from, but not limited to, crab, clam, oyster, shrimp, lobster, mussel, abalone, scallop, crayfish, limpet, winkle, or a combination thereof. According to a particular aspect, the shell waste may comprise crushed shell.

According to a particular aspect, the method may further comprise crushing the shell waste prior to the mixing. The crushing may be by any suitable means. For example, the crushing may be by utilising a crusher. The crusher may be a hammer mill, or the like.

Mixing

The mixing may be with a denaturing agent or water. According to a particular aspect, the mixing of the shell waste may be with a denaturing agent. The denaturing agent may be any suitable denaturing agent. For example, the denaturing agent may be selected from the group consisting of: urea, guanidine hydrochloride, and a combination thereof. In particular, the denaturing agent may be urea. According to another particular aspect, the mixing of the shell waste may be with water.

The mixing of the shell waste with the denaturing agent or water may be under suitable conditions to form a first mixture. For example, the mixing of the shell waste with the denaturing agent or water may be at a pre-determined temperature for a predetermined period of time. The pre-determined temperature and the pre-determined period of time may vary depending on whether the mixing of the shell waste is with a denaturing agent or water.

The pre-determined temperature may be any suitable temperature for the purposes of the present invention. According to a particular aspect, when the mixing of the shell waste is with a denaturing agent, the pre-determined temperature may be 0-50°C. In particular, the pre-determined temperature may be 5-45°C, 10-40°C, 15-35°C, 20- 30°C, 25-28°C. Even more in particular, the pre-determined temperature may be 25°C. According to a particular aspect, when the mixing of the shell waste is with water, the pre-determined temperature may be ≥ 25°C. In particular, the pre-determined temperature may be≥140°C. For example, the pre-determined temperature may be 140-250°C, 150-240°C, 160-230°C, 170-220°C, 180-210°C, 190-200°C. Even more in particular, the pre-determined temperature may be 180°C.

The pre-determined period of time may be any suitable period of time for the purposes of the present invention. According to a particular aspect, when the mixing of the shell waste is with a denaturing agent, the pre-determined period of time may be 1-24 hours. In particular, the pre-determined period of time may be 2-22 hours, 4-20 hours, 5-18 hours, 6-15 hours, 8-12 hours, 9-10 hours. Even more in particular, the pre-determined period of time may be 12 hours.

According to a particular aspect, when the mixing of the shell waste is with water, the pre-determined period of time may be ≤ 4 hours. In particular, the pre-determined period of time may be≤ 3 hours,≤ 1 hour,≤ 30 minutes, 5-30 minutes, 10-25 minutes, 15-20 minutes, 16-18 minutes. Even more in particular, the pre-determined period of time may be 15-60 minutes.

When the mixing is with water, following the mixing at the pre-determined temperature, the first mixture formed may be cooled to room temperature. The cooling of the first mixture may be by any suitable method. For example, the cooling of the first mixture may be by cooling water.

When a denaturing agent is used for the mixing, the denaturing agent may have a suitable concentration. For example, the denaturing agent may be in the form of a solution and the concentration of the solution of denaturing agent may be 1-8 mol/L. In particular, the concentration may be 1.5-7.5 mol/L, 2-7 mol/L, 2.5-6.5 mol/L, 3-6 mol/L, 3.5-5.5 mol/L, 4-5 mol/L. Even more in particular, the concentration may be 4 mol/L.

Filtering

Following the mixing of the shell waste with the denaturing agent or water under suitable conditions, the first mixture may be filtered to separate a solid phase (residue) from a liquid phase (filtrate). The liquid phase is separated from the solid phase in order to recover a first component from the shell waste. The first component may be one or more proteins comprised in the shell of the shellfish within the shell waste. In particular, the proteins may be comprised in the liquid phase while the chitin and the minerals may be comprised in the solid phase.

The filtering of the first mixture may be by any suitable means for the purposes of the present invention. For example, the filtering may comprise centrifugation. In particular, the filtering may be by using filter paper or filter cloth. The residue obtained from the filtering may be subjected to further steps as will be discussed below. The filtrate obtained from the filtering may comprise the proteins extracted from the shell waste and may be subjected to further separation techniques to obtain the proteins. For example, the filtrate may be subjected to separation techniques such as, but not limited to, membrane dialysis, distillation, precipitation, or a combination thereof.

According to a particular aspect, the first mixture may be centrifuged to separate the liquid phase and the solid phase prior to the filtering.

Contacting and collecting

The residue obtained from the filtering may then be subjected to contacting with carbon dioxide. The contacting may be carried out under suitable conditions. According to a particular aspect, the contacting may be carried out in a chamber. In particular, the chamber may comprise water and the contacting may be carried out in water. The water may be deionized water. Even more in particular, the residue may be loaded into a closed filter bag and the filter bag may then be immersed in water comprised in a chamber pressurised by carbon dioxide. The contacting may further comprise constantly stirring the water comprised in the chamber.

The carbon dioxide used for the contacting may be at a suitable pressure. In particular, the carbon dioxide may be at a pressure of 1-100 bar. For example, the carbon dioxide may be at a pressure of 5-95 bar, 10-90 bar, 15-85 bar, 20-80 bar, 25-75 bar, 30-70 bar, 35-65 bar, 40-60 bar, 45-55 bar, 48-50 bar. Even more in particular, the carbon dioxide may be at a pressure of about 10-20 bar.

According to a particular aspect, the contacting may result in the formation of carbonic acid. The carbonic acid may form due to the reaction between water and the carbon dioxide. The carbonic acid may then react with the calcium salt comprised in the shell waste comprised in the residue to form calcium bicarbonate. After a sufficient period of time, the carbon dioxide is released from the chamber. As the formation of calcium bicarbonate is a reversible reaction, the calcium salts may be re- precipitated in the water to form a suspension. The suspension may be collected to recover the calcium salts. In particular, calcium carbonate may be recovered from the suspension.

Accordingly, the method may further comprise recovering the second component from the suspension, wherein the second component is a calcium salt.

The collecting comprises collecting the balance of the shell waste remaining to recover the third component, wherein the third component is chitin. In particular, the balance of the shell waste may remain in the filter bag placed within the chamber during the contacting.

Whilst the foregoing description has described exemplary embodiments, it will be understood by those skilled in the technology concerned that many variations may be made without departing from the present invention.

Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration, and is not intended to be limiting.

EXAMPLE

Chemicals and materials

Shrimp shells used in the example were from a single species Crangon crangon purchased from Sheng Siong supermarket, Singapore. Crangon crangon (common name: grey shrimp) is commercially common species fished mainly in the North Sea. The collected shells (back and tail) were washed thoroughly with tap water, rinsed with deionized water and dried at room temperature. The dry shrimp shells were grinded into powders by a home-use blender. Commercial chitin (92% purity, high molecular weight) was purchased from Wako Pure Chemical Industry. Sodium hydroxide (NaOH) was from Schedelco. Hydrochloride acid (HCI) and sodium chloride were from VWR. Carbon dioxide (C0₂) was from Air Liquid Singapore Private Limited. Urea, 1 , 1 , 1 ,3,3,3- hexafluoro-2-propanol, lithium chloride (LiCI), dimethylacetamide (DMAc) and other chemicals were purchased from Sigma Aldrich and used as received. Shrimp shell composition

Moisture content in the shell was determined by the weight loss after oven dry at 105°C for 24 hours. The contents of the shell comprising chitin, minerals (dominantly CaC0₃) and proteins were quantified as follows. 1 g of dry shrimp shell powder was stirred in 20 mL of 5 wt% NaOH solution at 90°C for 2 hours. The residue was washed and dried to obtain the deproteinized sample. Next, the deproteinized sample was stirred in 10 mL of 5 wt% HCI solution at room temperature for 1 hour to remove the minerals. Finally, the unconverted solids were washed and dried as chitin. The weight losses in steps one and two were determined to be the amounts of protein and mineral, respectively, while the remaining weight was used to calculate the content of chitin in the shell. The percentages of water, proteins, minerals and chitin in the shell were determined to be 5.8%, 26.5%, 17.0% and 50.4%. The balance 0.3% comprised minor components such as pigments.

Shrimp shell fractionation

The method of the present invention was applied on the shrimp shell. In particular, the shell fractionation involved two main steps of deproteinization (mixing) to obtain the proteins and demineralization (contacting) to obtain the calcium salts from the shell.

For deproteinization, two methods were tested: a) mixing with concentrated urea solution; and b) mixing with water. For method b), heat treatment was further provided. In view of the mixing of the water and heat treatment provided, method b) is termed as providing the shell with hydrothermal (HT) treatment.

When urea was used as the deproteinization reagent, 0.4 g of shrimp shell powder was stirred in a glass beaker in an aqueous urea solution (0 to 8 M, 4-40 mL) at varying stirring speed (0-1000 rpm), duration (5-720 min) and temperatures (0 to 40°C). The proteins dissolved in the solution were quantified using a UV-vis spectrometer (Shimadzu UV-vis 3600). An external calibration curve was plotted by using the solutions of extracted shrimp proteins as standards, based on the absorbance at 280 nm.

In the HT method, the proteins were directly extracted using water at elevated temperature. In a typical experiment, 0.2 g of shrimp shell powder and 3 mL of water with a stirrer bar were loaded into an autoclave (inner volume: 20 mL) and mixed for a desired period of time. The temperature of the autoclave was pre-set at a desired value (140-220°C) prior to the mixing. Afterwards, the autoclave was taken out and cooled down to room temperature by cooling water. The product mixture was separated by a centrifuge (10000 rpm, 5 minutes). The liquid phase was diluted and subjected to total organic carbon (TOC) and inorganic carbon (IC) analysis using a TOC analyzer (Shimadzu TOC-L CSH). The solid residue was washed and lyophilized for further treatment.

The demineralization step was conducted in an autoclave in deionized water at room temperature in the presence of C0₂. 0.1 g of shrimp shell powder was loaded into a closed filter bag and then the whole bag was immersed into 10 mL water inside the autoclave under 10-20 bar C0₂ gas. A stirrer bar was put beneath the bag in the water, and the solution was constantly stirred for a desired of period. The gas was then released, during which the majority of Ca²⁺ leached into the solution re-precipitated. The bag was taken outside the autoclave, and washed gently with deionized water. The washing solution was combined with the suspension, diluted by 5% nitric acid to a volume of 50 mL for inductively coupled plasma optical emission spectrometry (ICP- OES) analysis to quantify the amount of Ca removed from the shell. Later, 3.5 wt% and 5 wt% of sodium chloride aqueous solution (mimic seawater) were used respectively to replace deionized water as dissolution medium, while the other experiment procedures were the same as described above.

Characterization

The products after different reaction stages, were characterized by a series of analytical techniques. Field Emission Scanning Electron Microscope (FESEM) was conducted on a JEOL JSM-7610F scanning electron microscope. Samples were treated via Pt sputtering for 120 seconds before observation. Powder X-ray diffraction (PXRD) patterns were obtained on a Rigaku MiniFlex 600 X-ray powder diffractometer equipped with a Cu sealed tube (λ = 1.541 A) operating at 40 kV, 15 mA with a scan rate of 0.05 ° s^~ The equation for the CI calculation is shown below:

C/[%] 110 x l00%

110 where l₁₁₀ is the maximum intensity of the diffraction for (1 10) plane at around 2Θ = 19° while \_am is the intensity of the amorphous diffraction at around 2Θ = 12.7°.

Fourier-transform infrared spectroscopy (FTIR) was conducted on a Thermo Scientific Nicolet iS50 FTIR spectrometer with both attenuated total reflection (ATR) mode and transmission mode. Gel permeation chromatography (GPC) analysis was carried out with a system equipped with a Waters 2410 refractive index detector, a Waters 515 high-performance liquid chromatography (HPLC) pump, and two Waters Styragel columns (HT3 and HT4) using ultrapure water as eluent at a flow rate of 0.5 mL/min at room temperature. Viscometry for viscosity average molecular weight (M_n) measurement was conducted on an Ubbelohde type viscometer (AVS-360). 5 wt% of Ν,Ν-dimethylacetamide (DMAc)/lithium chloride (LiCI) solution was freshly prepared as mixed solvent and different chitin samples were dissolved in DMAc/LiCI at concentration of 0.1 wt%. The intrinsic viscosity [η] and M_n can be calculated by following equations:

where [η] is the intrinsic viscosity; t and t₀ is the elution time for sample solution and the solvent, respectively; c is the concentration; K and a are empirical constants valid for a specific polymer-solvent system at a given temperature (i.e. 25°C).

Three process models for traditional method, urea-C0₂ method and HT-C0₂ method, respectively, were developed for different shrimp shell fractionation processes using Aspen Plus V9, on the basis of shrimp shell treatment rate of 100 kg/h as starting materials. The starting materials were standardized as consisting of 20 wt% chitin, 30 wt% calcium carbonate, 30 wt% protein and 20 wt% of moisture.

The traditional method utilized hot aqueous NaOH to remove protein in the shrimp shell in the deproteinization step, and dilute HCI at room temperature to dissolve minerals in the demineralization step. After these two reaction steps, chitin would be the remaining solid residue. These reaction conditions are consistent with the procedure of shrimp shell composition determination. The urea-C0₂ method relied on the use of urea at room temperature to solubilize protein by denaturing the protein in the shrimp shell in the deproteinization step, after which pressurized C0₂ was used to create the acidic conditions needed to dissolve minerals in the deproteinized products. In this design, all the product (CaC0₃) and reagents (water and C0₂) could be recovered when C0₂ was released after reaction and then CaC0₃ was re-precipitated. Chitin was then obtained from the shrimp shell after these treatment steps.

In the HT-C0₂ method, the demineralization step followed that of urea-C0₂ method. For deproteinization, this method applied a fast HT treatment. The operation conditions of the other two methods were consistent with the optimal experiment conditions (see Table 1).

Method Description De-proteinization De-mineralization

Chemical 5 wt% NaOH 5 wt% HCI Temperature 90°C 25°C

Traditional Pressure 1 atm 1 atm

Residence time 2 hours 1 hour

Ratio 1 g : 20 mL 1 g : 10 mL

Chemical 8M Urea Aqueous C0₂ Temperature 25°C 25°C

Urea Pressure 1 atm 10 atm

Residence time 12 hours 2 hours

Ratio 1 g : 100 mL 3 g : 500 mL

Chemical Hot water Aqueous C0₂ Temperature 180°C 25°C

Water Pressure 20 atm 10 atm

Residence time 1 hour 2 hours

Ratio 1 g : 10 mL 3 g : 500 mL

Table 1 : Summary of the reaction steps in each shell-to-chitin process. Ratio refers to the solid to chemical loading ratio.

The overall process flow diagrams (PFDs) of the traditional method, urea-C0₂ method and HT-C0₂ method are shown in Figures 1 to 3, respectively, which can be systematically described as follows: shrimp shell was first crushed to powder via a hammer mill crusher to obtain finer particles for downstream reaction. The shrimp shell powder then passed through the deproteinization reactor. After reaction, the solid was separated by filtration, washed with water in a multi-stage solids washer, and then separated again by another round of filtration for further reaction in the demineralization reactor, after which the solid residue was washed and dried, leading to chitin as the final product.

Results and discussion

Deproteinization by using urea solutions

Urea is a classic protein denaturant applied in protein unfolding/refolding experiment. Urea can expel water from the hydration shell of the protein to form preferential hydrogen bond with protein backbone and further intrude the hydrophobic core to promote protein unfolding, which augments the flexibility and stability of peptide chains, accounting for increased solubility. First, urea solutions with a concentration ranging from 0 M to 8 M were attempted for deproteinization. A kinetic study was conducted to examine the extent of deproteinization with the evolution of time at different urea concentrations. The results are shown in Figure 4a. Surprisingly, the control experiments using water solution led to 29.8% dissolution of proteins within 2 hours, which may be ascribed to the soluble protein exposed after the destruction of the lipid wall at the surface of shells and soluble hydrolysates from proteolysis by endogenous enzymes and bacteria. The increase in urea concentration resulted in an almost linear improvement of the protein removal efficiency (Figure 5). Besides, a fast removal rate was noticed at the initial stage of the first half an hour, and it slowed down afterwards with negligible protein removal after 6 hours. This may be because the protein dissolution reached equilibrium at 6 hours and the urea solution was not able to dissolve any more of the proteins. Therefore, the urea treatment was repeated for three cycles. In each cycle, fresh urea solution of 8 M concentration was used and the extraction time was set at 6 hours. After three cycles, the deproteinization efficiency ascended remarkably to 84% with 8 M urea solution at room temperature (Figure 4b).

The influences of the feedstock to solvent ratio, stirring speed and temperature were also investigated. The feedstock to solvent (g to ml_) ratios of 0.01 , 0.025 and 0.1 were used and there was negligible change of protein removal in the first cycle (Figure 6). Nevertheless, in the second and third cycles, higher protein removal rate was observed with lower feedstock to solvent ratio. For the stirring speed, three values of 0, 500 and 1000 rpm were attempted and the results are shown in Figure 7. The efficiency dropped significantly without stirring that only 9.4% of the proteins was removed even after 12 hours, which shows that stirring is important for deproteinization since it enhances the contacts between the extraction liquid and the solid. When the stirring speed exceeded 500 rpm, a satisfactory removal performance was achieved which was comparable with that at 1000 rpm.

In many extraction processes, temperature can be a great factor. Thus, the deproteinization was conducted at 0°C, room temperature and 40°C. The results are shown in Figure 8. Low temperature at 0°C slightly inhibited the removal efficiency which dropped to 38.1 % compared to the 45% at room temperature with the extraction time of 6 hours, whereas, the increase of temperature to 40°C did not lead to enhanced performance and the removal efficiency was similar with that at room temperature. Higher temperatures were not attempted since decomposition of urea may occur.

Deproteinization by high temperature water

Since it was observed that deproteinization happened in pure water solution, it was envisaged that pure water may be able to extract proteins from shrimp shells, which is even greener and cheaper. Deproteinization in water was conducted at a wide range of different temperatures. From Figure 9a, there was no obvious increase of removal efficiency when the temperature increased from room temperature to around 160°C. However, a sharp increase in removal efficiency was observed when the temperature was further increased to 180°C and above. Besides, the extraction rate was much faster that almost 100% removal was achieved within 30 minutes at 200°C.

This may be due to higher temperatures improving water to generate protons (H⁺) and anions (OH^") capable of performing acid- and base-catalyzed reactions to hydrolyze proteins and/or the abundant hydroxyl groups in chitin catalysed protein hydrolysis. Overall, according to the GPC result (Figure 9b), the protein hydrolysis favours high temperature. The proteins removed at room temperature and 140°C had shorter elution times while a shift toward longer elution time was observed at 180°C and 220°C, indicating the decrease of the molecular weight and size of the protein. There were two peaks in the spectra of HT-180 and HT-220 at about 1 1.3 and 13.2 min, and the peak at 11.3 min was relatively higher than that at 13.2 min in HT-180 spectrum, whereas the situation was reversed in HT-220 spectrum. This shows that there was a large portion of low-molecular-weight proteins in the solution at 220°C. Demineralization by pressurized CO?

The deproteinized samples were then used for demineralization. A green and efficient method to remove minerals in shrimp shells is by using high pressure C0₂ because it not only considerably increases the solubility of C0₂ in water but also remarkably accelerates the dissolution rate due to the lower pH of saturated carbonic acid solution. Kinetic study was conducted using deproteinized samples via both urea treatment and high temperature water treatment under 10 or 20 bar C0₂ gas (Figure 10). According to the data (Figure 1 1), the sample obtained via hydrothermal treatment was easier to be demineralized than that obtained by urea treatment. Almost 100% demineralization was achieved from HT-sample within 15 minutes and from urea treated sample within 30 minutes. It indicates that hydrothermal treatment impairs the binding between the minerals and chitin. On the other hand, the original shrimp shell powder was also used for demineralization and it took a longer reaction time of 1 hour to fully remove the minerals, which is reasonable considering that the untreated shrimp shell possesses more compact structure and some mineral particles inside the shell structure are less accessible. Besides, the demineralization performances are comparable when 10 and 20 bar C0₂ was used. Hence, 10 bar was sufficient for efficient demineralization and there was no need to further increase the pressure.

Characterization of the chitin product

The optimal conditions for deproteinization and demineralization were selected and the solid residue was collected for analysis. From FTIR analysis (Figure 12a), it was seen that shrimp proteins had two characteristic peaks at 1620 and 1656 cm^"1 and CaC0₃ had a major peak at 1395 cm^"1. The Urea-C0₂-sample and HT-C0₂-sample displayed similar FTIR spectra with that of chitin obtained from the identical shrimp shell by traditional method. They both possessed the characteristic peaks of chitin, and there is a very trace amount of proteins left. Besides, the residual CaC0₃ was determined by ICP-OES to be 0.43% and 0.65% in Urea-C0₂-sample and HT-C0₂-sample. Overall, the developed methods are capable to fractionate shrimp shell and obtained chitin products with a high purity.

XRD spectra profile of shrimp shell at different fractionation stages and their corresponding crystalline index (CI) via different methods were compared, as shown in Figure 12b. It was found that the CI values were comparable for crude shrimp shell, Urea-C0₂-sample and HT-C0₂-sample, while the CI value increased by 5% for the sample obtained via traditional method. As a result, the traditional method may promote the crystallization of chitin.

SEM was an efficient technique to observe the structures of shrimp shell at different fractionation stages. As shown in Figure 13, the pristine shrimp shell was a layer-by- layer structure (Figure 13a), in which each layer is made up of arranged bundles containing chitin fibres wrapped by protein and particles with size of 50 to 200 nm are embedded inside the bundles or dispersed between the layers. After deproteinization, chitin fibres could be identified (Figures 13b, c, d). It can be noted that after water extraction at room temperature, chitin fibres were still partially wrapped (Figure 13b) while chitin fibre structure could be more clearly identified in the samples treated via urea method (Figure 13c) and hydrothermal method (Figure 13d). As shown in Figures 13c and 13f, the chitin extracted via both methods possesses similar rigidity and crystallinity as the one via traditional method, which are in agreement with the XRD results.

The molecular weight of chitin samples obtained by different approaches were measured based on viscometry. Generally, commercial chitin possessed lower viscosity average molecular weight M_n (-260 k) than all the chitin samples extracted by the different methods, presumably due to the different shrimp shell sources. Comparing the chitin from the same shrimp shell sources obtained via different treatments, it was noted that chitin obtained from urea-C0₂ method had the highest M_n (-590 k), similar to the one from industrially-practiced method (-570 k), while chitin produced by hydrothermal method had a relatively lower M_n (-390 k). The results are reasonable because urea-C0₂ method is the mildest one and HT-C0₂ method is conducted under harsher conditions. As mentioned previously, high temperature water generates protons and -OH anions which may also slightly promote the depolymerization of chitin.

Conclusion

From the above, it can be seen that the method of the present invention is a simple, low-cost, efficient and environmentally friendly method and yet the quality of the chitin obtained from the method is comparable to that obtained by traditional methods.

Claims

1. A method of processing shell waste for recovering components comprised in shell of shellfish, the method comprising:

mixing the shell waste with a denaturing agent or water at a predetermined temperature for a pre-determined period of time to form a first mixture;

contacting the residue obtained from the filtering with carbon dioxide to form a suspension comprising a second component; and collecting a third component as balance residue following the contacting, wherein the first component is one or more proteins, the second component is a calcium salt, and the third component is chitin.

2. The method according to claim 1 , wherein the mixing comprises mixing the shell waste with a denaturing agent at a pre-determined temperature of 0-50°C.

3. The method according to claim 1 or 2, wherein the denaturing agent is selected from the group consisting of: urea, guanidine hydrochloride, and a combination thereof.

4. The method according to any preceding claim, wherein the denaturing agent is at a concentration of 1-8 mol/L.

5. The method according to any preceding claim, wherein the pre-determined period of time is 1-24 hours.

6. The method according to claim 1 , wherein the mixing comprises mixing the shell waste with water at a pre-determined temperature of≥140°C.

7. The method according to claim 6, wherein the pre-determined temperature is 140-250°C.

8. The method according to claim 6 or 7, wherein the pre-determined period of time is≤ 4 hours.

9. The method according to any preceding claim, wherein the contacting is carried out in water in a chamber.

10. The method according to any preceding claim, wherein the carbon dioxide is at a pressure of 1-100 bar.

1 1. The method according to any preceding claim, wherein the carbonic acid is formed during the contacting.

12. The method according to claim 11 , wherein the suspension is formed from a reaction of the carbonic acid with calcium salt comprised in the shell waste comprised in the residue during the contacting.

13. The method according to any preceding claim, further comprising recovering the second component from the suspension.

14. The method according to any preceding claim, wherein the shell waste comprises crushed shell.

15. The method according to any preceding claim, wherein the shell waste comprises shell from: crab, clam, oyster, shrimp, lobster, mussel, abalone, scallop, crayfish, limpet, winkle, or a combination thereof.