Download New Chitin Production Process from Crab Shell using Sub

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