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Conversion of Crab Shell to Useful Resources Using Sub-critical Water Treatment Hidemi Nakamura, Hiroki Oozono, Norihisa Nakai and Hiroyuki Yoshida Department of Chemical Engineering, Osaka Prefecture University, Sakai, Japan Abstract: Some efficient methods for production of useful resources from crab shell using sub-critical water treatment were proposed. We found that the sub-critical water decomposed the protein and pigments, and removed them rapidly and completely under the conditions of reaction temperature of 533 - 593 K and reaction time of 1 - 20 min. The sub-critical water also extracts lipids completely from crab shell. However, most of calcium carbonate were not decomposed and removed. Since the residual calcium carbonate was removed by additional treatment of 2 N aqueous HCl solution for two days, this two stage method is useful for new chitin producing method compared to the conventional method with many processes, long time treatment and large cost. In the aqueous phase, various useful organic acids and amino acids were produced by the hydrolysis reaction of protein in the crab shell. On the contrary, by using the sub-critical aqueous acetic acid solution (stoichiometric molar ratio of acetic acid is 3:1 for calcium carbonate) as a reaction solvent, it was possible to produce 95 - 100% purity chitin with white color under the conditions of relatively low temperature of 453 - 493 K and very short reaction time of 1 - 10 minutes in one stage process. In addition, by the sub-critical water treatment in further high temperature of 593 - 623 K, a part of the chitin was decomposed. Decomposition products were oligosaccharides, glucosamine and erythrose, etc. It was clarified that this sub-critical water treatment method was useful as a saccharic manufacturing method of low molecular weight from the chitin. Furthermore, it was possible to produce the chitosan by the deacetylation of chitin using sub-critical aqueous NaOH solution in very short time compared with the conventional method. 74.0% of deacetylation degree was obtained under the conditions of 10 wt% aqueous NaOH solution, reaction temperature of 493 K and reaction time of 60 min. Keywords: Sub-critical Water, Chitin, Chitosan, Organic Acids, Amino Acids 2. EXPERIMENTAL Crab shell used in this study was the freeze-dried carapace supplied from Kani Doraku Co., Ltd., Japan. Before the experiment, the shell was crushed so that their size has about 3 - 5 mm. Chitin used in this study also was powder reagent obtained from Tokyo Chemical Industry Co., Ltd., Japan. A stainless tube (SUS 316, i.d.: 0.0064m × 0.15m) with Swedgelok caps, was used as a reactor. For the sub-CW hydrolysis reaction, 1.0 2.0 × 10-4 kg of crab shell and 2.0 × 10-3 kg of Milli-Q water, aqueous acetic acid solution or 5 40 wt% aqueous NaOH solution were charged to the reactor tube. The reactions were carried out in the temperature range of 453 - 623 K at predetermined time intervals from 1 to 60 min. After the reaction, the residual solid was separated from the aqueous solution and dried on a filter. The HPLC (Shimadzu, SCL-10A) with the conductivity detector was used to determine the concentrations of organic acids. The concentrations of amino acids were determined by HPLC system (Shimadzu, LC-10A) using the fluorescence detector. And also, the HPLC (Shimadzu LC-10A) was used to determine the concentrations of saccharide in the aqueous solution using the refractometer detector. Total 1. INTRODUCTION Large amounts of organic wastes including chitin such as crab or shrimp shells are discharged from fish market and food processing plant. Therefore, the constructions of effective treatment method or recycling technologies of the organic waste as a renewable resource are an urgent problem. As one of the new processing methods of organic waste, the sub-critical water (hereafter called sub-CW) treatment is expected. Yoshida et al. [1-9] have proposed the reaction using the sub-critical region of 473 - 647 K in order to provide much wider and milder reaction conditions and showed that sub-CW hydrolysis was an efficient method for the production of useful substances such as amino acid, organic acids, fatty acids, oil, heavy metals and so on, from fish meat, waste squid entrails, waste woods and bone meal. Those studies demonstrated the possibility of converting organic wastes to useful substances and resources using sub-CW process. In this work, a new, very fast and low initial and running costs production of useful resources from crab shell using sub-CW treatment were proposed. Corresponding author: H. Nakamura, [email protected] 1 Production of chitin and chitosan ●Sub-critical treatment method ●Conventional method Crab shell and shrimp shell etc. Crab shell and shrimp shell etc. Treatment in 2 mol/l aqueous HClsolution 2 days (one liquid exchange) Sub-critical water treatment about 40% weight loss Decalcification Treatment in 1 mol/laqueous NaOH solution 36 hours boiling (one liquid exchange) ・cost reduction few process short time treatment ・high purity ・production of useful by-products about 25% weight loss De-protein Ethanol reflux 6 hours heating (omissible) Delipidation and decolorization some weight loss Sufficient washing 333 K,3 days drying Isolation of chitin Isolation of chitin about 35% residual solid Deacetylation Deacetylation 30-60% melting NaOH aqueous solution 353-393 K, 30 min - 5 hours agitation Production of chitosan Production of chitosan Fig. 1. Chitin and Chitosan producing method organic carbon (TOC) of the reaction products was measured with the TOC analyzer (Shimadzu, TOC-500). Concentration of calcium ion dissolved in the aqueous phase was determined by ICP plasma emission spectrophotometer (Seiko Instruments INS., SPS7800). In the sub-critical aqueous NaOH solution treatment, the residual solid was washed and dissolve in 1% acetic acid solution. The molecular weight of obtained chitosan was estimated using size exclusion chromatography (Shimadzu LC-10AS). The degrees of deacetylation were also estimated using MBTH method [10]. process is also required. 3.2. New method proposed in this work On the contrary, sub-CW treatment method proposed in this work using the sub-CW and sub-critical aqueous acid solution is totally new method of chitin production with a view to obtaining cost reduction by few process and short time treatment, high purity chitin and production of useful by-products. 4. RESULTS AND DISCUSSION Figure 2 shows the effect of the temperature on the time course of decomposition of the crab shell in the sub-CW under the conditions of reaction temperature of 533 - 593K and reaction time of 1 - 20 min. The residual solid decreased 3. PROPOSED METHOD The conventional method and a new method proposed in this study to produce chitin and chitosan from crab shells is shown in Figure 1. 1 Ratio of residual solid [kg/kg-dry crab shell] 3.1. Conventional method In the conventional method, to begin with, the treatment of crab shell in 2 kmol/m3 aqueous HCl solution for two days is required for removal of calcium carbonate in the crab shell. Next, treatment in 1 kmol/m3 aqueous NaOH solution under boiling condition for 36 hours is carried out to remove the protein. Furthermore, by using ethanol, the delipidation and decolorization is necessary for the production of chitin. Thus, more than one week treatment to produce chitin using large amount of expensive reagents and large amount of water for washing are required. Therefore, large scale waste water treatment 0.8 0.6 0.4 533 K 563 K 593 K 0.2 0 0 5 10 15 20 Reaction time [min] 25 Fig. 2. Effect of reaction temperature on time course of decomposition of crab shell 1 0.02 Organic acid yields [kg/kg-dry crab shell] with reaction temperature and became to 75 % (533 K), 65 % (563 K) and 53 % (593 K) of initial weight of crab shell. The reaction was fast and almost finished within 10 min. In the conventional method of producing chitin, we confirmed that the crab shell used here contained protein (24 %), calcium carbonate (41 %), chitin (35 %), some pigments and lipids. We found that the sub-CW decomposed the protein and pigments, and removed them rapidly and completely under these conditions, because the decreased amounts of solid phase for each temperature is almost equal to that of the protein which is included for the crab shell. In the high temperature of 563 K or 593 K, some of chitin in the crab shell also may be decomposed with decomposition of protein. However, most of calcium carbonate were not decomposed and/or removed. Figures 3 and 4 present the effects of reaction time on yields of main amino acids and organic acids produced by the sub-CW hydrolysis of protein in the shell, respectively. In the aqueous-phase, various valuable amino acids such as alanine and arginine, and organic acids such as acetic acid and pyroglutamic acid were produced by the hydrolysis reaction. As shown in these figures, this reaction was very fast and it almost finished within 10 min. The yields of produced amino acids and organic acids are also dependent on reaction time and temperature. The residual calcium carbonate was removed completely by contacting of 2 N aqueous HCl solution in normal condition for two days. This two stage method was effective not only to produce chitin with the high-purity and other valuable substances such as amino acids and organic acids, but also to decrease drastically the initial and running costs, and treatment period of time in comparison with the conventional method with many complicated processes, long time treatment and large cost. On the contrary, by using the sub-critical aqueous acetic acid solution (3 times molar quantity for stoichiometric ratio of reaction of acetic acid and calcium carbonate), higher than 95 % purity chitin with white color, which was equivalent in purity to the commercial reagent produced from the crab shell with the conventional method, was produced under the conditions of relative low temperature of 453 493 K and very short reaction time of 1 minute, as shown in Figure 5. Furthermore, almost 100% purity chitin was obtained in longer additional sub-critical treatment of 2 - 10 minutes. The decalcification, delipidation, decolorization and de-protein of crab shell were done simply and simultaneously by using the acetic acid solution which was activated as a sub-CW condition. From these results, it has become apparent that the highly pure chitin can be produced without using strong acid, strong base under the process of being less than the conventional method, short time treatment and cost reduction. Figure 6 shows the result of decomposition for 0.01 0 0 Phosphoric acid Pyruvic acid Maric acid Succinic acid Lactic acid Formic acid Acetic acid Pyroglutamic acid Isobutyric acid 533K 5 10 Reaction time [min] 15 Fig. 3. Effect of reaction time on organic acids yield in aqueous solution. Amino acid yields [kg/kg-dry crab shell] 0.015 Proline Alanine Metohionine Histidine Arginine 533K 0.01 0.005 0 0 5 10 Reaction time [min] 15 Fig. 4. Effect of reaction time on amino acids yield in aqueous solution. Residual solid [kg/kg-dry crab shell] 1 acetic acid 1.74 kmol/m 3 0.8 1min 2min 5min 10min 0.6 commercial chitin 0.4 100% pure chitin 0.2 0 450 460 470 480 490 Reaction temperature [K] 500 Fig. 5. Effect of reaction temperature on ratio of residual solid by sub-critical aqueous acetic acid solution Ratio of residual solid [kg/kg-dry chitin] 1 0.8 493K 543K 573K 603K 613K 0.6 0.4 0.2 0 0 10 Reaction time [min] 20 Fig. 6. Effect of reaction temperature on ratio of residual chitin in sub-critical water treatment of commercial chitin. 1 Technol., 39 (2005), pp. 2357-2363. O. Tavakoli and H. Yoshida; J. Green Chemistry, 8 (2006), pp. 100 –106. 9. O. Tavakoli and H. Yoshida; Ind. Eng. Chem. Res., 45 (2006), pp. 5675-5680. 10. Mamual of chitin and chitosan, Gihodo Shuppan (1991), p.50 (in Japanese) commertial chitin by the sub-CW hydrolysis reaction. In the low temperature of 493 K and reaction time of 1 - 20 min, the chitin was also hardly decomposed. On the contrary, in high temperature of 603 - 613K, most of the chitin was decomposed. Decomposition products were oligosaccharides, glucosamine and erythrose, etc. As a typical example, Figure 7 shows the effects of reaction temperature on erythrose yields in the aqueous phase of the sub-CW treatment. It was clarified that this sub-CW treatment method was useful as a saccharic manufacturing method of low molecular weight from the chitin. Figure 8 shows depolymerization of chitosan in the sub-critical aqueous NaOH solution under the conditions of reaction temperature of 473 493K and reaction time of 1 - 4 min. Chitosan made from chitin in the sub-critical NaOH aqueous solution was depolymerized. Figure 9 shows degree of deacetylation of chitosan in 5 20 wt% aqueous NaOH solution at relatively long reaction time 40 - 60 minute and 493 K. 74.0% of deacetylation degree was obtained under the conditions of 10 wt% aqueous NaOH solution, reaction temperature of 493 K and reaction time of 60 min. Using sub-critical aqueous NaOH solution enables deacetylation of chitin in very short time and depolymerization of chitosan. 8. ACKNOWLEDGMENT A part of this research funds was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan in the form of the 21st century COE program (“Science and Engineering for Water-Assisted Evolution of Valuable Resources and Energy from Organic Wastes,” E-1). Erythrose yields [kg/kg-dry chitin] 0.0008 483K 503K 523K 543K 573K 603K 0.0006 0.0004 0.0002 0 0 10 Reaction time [min] 20 Fig. 7. Effect of reaction temperature on time courses of erythrose yields in aqueous solution 5. CONCLUSIONS The sub-CW treatment was carried out for the purpose of the construction of new chitin, chitosan and the other useful resources production process from the waste crab shell. Especially, it is possible to obtain the necessary products such as chitin, chitosan, amino acids, organic acids and saccarides by dealing with the crab shell at the decomposition condition of various modes. It became clear that the proposed process was technically and economical feasible with a view to obtaining cost reduction by few process and short time treatment, high purity chitin and production of useful by-products. 453K 473K 493K 473K (chitin from crub shell) Molecular weight of chitosan [g/mol] [×106] 4 3 2 NaOH 40wt% 1 0 0 REFERENCES 1. H. Yoshida, M. Terashima and Y. Takahashi; Biotechnol. Prog., 15 (1999), pp. 1090-1094. 2. H. Yoshida, M. Terashima and Y. Takahashi; J. Japan Soc. Waste Management Experts, 12 (2001), pp. 163–167. 3. H. Yoshida, Y. Takahashi and M. Terashima; J. Chem. Eng. Japan, 36 (2003), pp. 441-448. 4. H. Yoshida, and O. Tavakoli; J. Chem. Eng. Japan, 37 (2004), pp. 253-260. 5. H. Yoshida and Y. Katayama, Proc. of 10th Asian Pacific Confederation of Chemical Engineering Conference, 3P-03-025 (2004), pp. 1-9. 6. H. Yoshida and T. Nakahashi, Proc. of 10th Asian Pacific Confederation of Chemical Engineering Conference, 3P-03-026 (2004), pp. 1-8. 7. O. Tavakoli and H. Yoshida; Environ. Sci. 1 2 3 4 Reaction time [min] Fig. 8. Depolymerization of chitosan in sub-critical aqueous NaOH solution Relation of Deacetylation[%] 100 80 493K NaOH 20wt% 493K NaOH 10wt% 493K NaOH 5wt% 60 40 20 0 0 10 20 30 40 50 60 Reaction time [min] Fig. 9. Degree of deacetylation in sub-critical 5-20 wt% aqueous NaOH solution 4 1