Accepted Manuscript

Title: Hydroxyapatite crystallization in shrimp cephalothorax

wastes during subcritical water treatment for chitin extraction

Authors: Angélica Espı́ndola-Cortés, Rosario Moreno-Tovar,

Lauro Bucio, Miquel Gimeno, José Luis Ruvalcaba-Sil, Keiko
Shirai

PII: S0144-8617(17)30572-6
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2017.05.055
Reference: CARP 12341

To appear in:

Received date: 10-3-2017

Revised date: 27-4-2017
Accepted date: 18-5-2017

Please cite this article as: Espı́ndola-Cortés, Angélica., Moreno-Tovar,

Rosario., Bucio, Lauro., Gimeno, Miquel., Ruvalcaba-Sil, José Luis., &
Shirai, Keiko., Hydroxyapatite crystallization in shrimp cephalothorax wastes
during subcritical water treatment for chitin extraction.Carbohydrate Polymers
http://dx.doi.org/10.1016/j.carbpol.2017.05.055

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 Hydroxyapatite crystallization in shrimp cephalothorax wastes during

subcritical water treatment for chitin extraction

Angélica Espíndola-Cortés1, Rosario Moreno-Tovar1, Lauro Bucio2, Miquel Gimeno3, José

Luis Ruvalcaba-Sil4, and Keiko Shirai1*

1
Universidad Autonoma Metropolitana-Iztapalapa, Biotechnology Department, Laboratory of

Biopolymers and Pilot Plant of Bioprocessing of Agro-Industrial and Food By-Products, Av. San

Rafael Atlixco, No. 186, 09340, Mexico City, Mexico.

Universidad Nacional Autónoma de México, Circuito de la Investigación Científica s/n, Ciudad

Universitaria, Coyoacán, C.P. 04510 Ciudad de México, México. 2Laboratorio de Cristalofísica y

Materiales Naturales, Instituto de Física. 3Depto. Alimentos y Biotecnología, Facultad de

Química. 4Instituto de Física.

Corresponding author (KS) E-mail: smk@xanum.uam.mx

Chemical compounds studied in this article

Water (PubChem CID: 962); Chitin (PubChem CID: 6857375); Calcium carbonate (calcite)

(PubChem CID: 516889); Aragonite (Ca(CO3) (PubChem CID: 6335838); Hydroxyapatite

(Ca5(PO4)3OH) (PubChem CID: 14781); Quartz (SiO2) (PubChem CID: 24261); Lanthanum

hexaboride (PubChem CID: 71308229).

1
Highlights

 Taguchi model was used to optimize conditions of water under subcritical treatment

 Subcritical water was used for recovery of chitin from shrimp wastes

 Deproteinization was achieved along with the crystallization of minerals

 The crystalline phases identified were α-chitin, calcite, hydroxyapatite, quartz

 Subcritical water treatment produces calcite and hydroxyapatite crystallization

Abstract

The extraction of calcareous chitin from shrimp cephalothorax was successfully achieved using a

subcritical water treatment to attain a deproteinization up to 96%. The treatments also increased

the crystalline domain size in the -chitin fibers. An experimental design of Taguchi allowed the

optimization of experiments. The macroelements identified in all samples were Ca, P, S, K, Cl

and Al, whereas Cr, Mn, Fe, Ni, Cu, Zn, Br and Sr were also detected as microelements. The

assigned crystalline phases by XRD were α-chitin, calcite, HAP and traces of quartz. The

presence of these phases was corroborated by ATR-FTIR and SEM-EDS analyses. The highest

content of α-chitin (82.2 wt%) was obtained for the 0.17 chitin:dH2O (wt/wt) ratio for 30 min

treatment at 260 °C. Noteworthy, this treatment promotes the crystallization of both minerals as

microcrystals of calcite and nanocrystals of hydroxyapatite with needle and flake shapes as well

as intermediate morphologies.

Keywords: Subcritical water; Chitin; Calcite; Hydroxyapatite; shrimp wastes.

2
1. Introduction

The chitin, polysaccharide composed of β-(1-4)-N-acetyl-D-glucosamine units (>50%) and

glucosamine units, is a linear semi-crystalline biopolymer which displays α, β, and γ polymorphic

forms. This biopolymer is regarded as biocompatible and biodegradable, among many other

properties and present in the exoskeletons of insects, crustaceans and arachnids (Kandra, Challa,

& Kalangi Padma Jyothi, 2012). Commercial chitin is usually extracted from crustacean wastes,

mostly by thermochemical methods (Percot Viton, & Domard, 2003). Therein, acid and alkali

treatments are carried out in order to remove minerals and protein. Complete deproteinization has

been a major issue on chitin purification owing to restricted hydrolysis of the fraction embedded

in the complex chitin-protein matrix network. The residual protein might cause allergic reactions

in sensitive consumers; therefore, several efforts toward complete protein removal without

diminishing the biopolymer characteritics have been investigated including biological methods

with enzymes or microorganisms. Generally, the biotechnological approaches proved

advantageous for this purpose (Cira, Huerta, Hall, & Shirai, 2002; Pacheco, Garnica-Gonzalez,

Gimeno, Bárzana, Trombotto, David, & Shirai, 2011; Flores-Albino, Arias, Gómez, Castillo,

Gimeno, & Shirai, 2012) compared to other chemical or physicochemical routes (Aye, &

Stevens, 2004; Percot et al., 2003). However, their main drawback is the long fermentation times,

usually more than 2 days (Kaur & Dhillon, 2015). Alternatively, hydrothermal treatments to

biomass using water under subcritical conditions (Tc of 374 °C and Pc of 22.1 MPa) allow the

recovery of added-value products in short reaction times with low environmental impact. The use

of water treatments under subcritical conditions has been applied for the recovery of compounds

3
such as amino acids (Quitain, Sato, Daimon, H., & Fujie, 2001; Kang, Daimon, Noda, Hu, &

Fuji, 2001) and fatty acids (Yoshida, Terashima, & Takahashi 1999), as well as the modification

of chitin morphology for enhanced enzymatic digestion (Osada, Miura, Nakagawa, Kaihara,

Nikaido, & Totani, 2012) or protein removal from the crab shells (Osada, Miura, Nakagawa,

Kaihara, Nikaido, & Totani, 2015). The reaction conditions, such as temperature, residence time,

particle size, water content and reactor configurations generally need careful handling in order to

avoid side reactions, such as hydrolysis, depolymerization, dehydration, decarboxylation,

alteration or degradation of compounds (Iryani, Kumagai, Nonaka, Nagashima, Sasaki, &

Hirajima 2014; Lavoie, Capek-Menard, Gauvin, & Chornet, 2010). Subcritical water processes

have taken advantatge of these side reactions at industrial scale, for instance polymer

decomposition in recycling technologies (Goto, 2016).

The exoskeleton of crustacean also contains calcium carbonate, which can be converted to

calcium phosphate composites (Raya, Mayasari, Yahya, Syahrul, & Latunra, 2015). Among

them, the hydroxyapatite (HAP) has been pointed out as an interesting material owing to its

biocompatibility and its osteogenic potential. Several methods to produce HAP have been studied

and most of them employed solution-based reactions with Ca2+ and PO43- reagents but also using

biowastes, including those from crustaceans (Sadat-Shojai, Khorasani, & Jamshidi 2013). In this

regard, Raya et al. (2015) reported the synthesis of HAP from crab shells, where the organic

matter was removed by calcination and the ash used as the Ca2+ source, which was then reacted

with (NH4)2PO4 at high temperatures (400-1000°C). Other high-temperature methods, including

the hydrothermal process, employs chemicals in aqueous solution at elevated temperature (>100

ºC) (Sadat-Shojai et al., 2012).

4
In spite of the efforts on the application of water under subcritical condition for chitin extraction,

there is no information, to the best of our knowledge, of the effect of high temperature on the

mineral fraction of the shrimp cephalothorax waste (CW) and deproteination for the biopolymer

extraction. The present work is first to report subcritical water treatment of CW for chitin

recovery in short reaction times. The optimization of the process conditions was achieved using

the Taguchi model with an orthogonal matrix L9 (33).

2. Materials and methods

2.1 Preparation of samples

CW was obtained from the central seafood market (Mexico City) and minced through a 1/8-inch

sieve in a Torrey (Mexico) meat mincer, then dried in an oven at 50 °C and stored at room

temperature packed in a thermosealed plastic bag prior to use.

2.2 The Taguchi model

Taguchi orthogonal arrays, factors and levels are shown in Table 1. Variables for an orthogonal

arrangement L9 (33) of Taguchi model were CW to distilled water (CW/dH2O wt/wt) ratio (R),

reaction time (t) and reaction temperature (T). Statistical analyses with the signal to noise (S/N)

indicator was related to the multivariate statistic distance (MSD) and calculated according to

equation 1 (Taguchi, 1990). S/N indicator was used to identify control factors that reduce the

variability of the process of water under subcritical condition. The S/N indicator in this study was

targeted as “larger is better” for soluble protein in the liquid fraction as the dependent variable,

and “smaller is better” for Ca, P, K and S contents in the solid fraction.

5
  10 log10 MSD 
S
(1)
N

where MSD was S (smaller is better) and L (larger is better) according to equations 2 and 3,

respectively.

1 n 1 
S: MSD    2  (2)
 n i 1Y i 

1 n 
L: MSD    Y i2  (3)
 n i 1 

where: Yn is the response variable; n is the number of trials for experiment i, i is the experiment

number

Statistical analysis of the experimental data was performed with analysis of variance (ANOVA)

for the determination of the significance of control factors on soluble protein, Ca, P, K and S

contents. Data was also analyzed by the test Tukey-Kramer multiple comparison of means (p ≤

0.05).

2.3 Experimental methodology

Experiments were conducted in a 100 mL home-made stainless steel 316 cylindrical reactor

equipped with a magnetic stirrer, an external ceramic heating jacket, manometer (Swagelok,

USA), a high-pressure valve (Swagelok, USA), a safety disc release valve and two independent

thermocouples, one measuring the temperature inside the reactor and the other at the ceramic

jacket, which were connected to a temperature control device. Reactor was loaded to its

maximum capacity with the each described R variable of the experimental design and heated at

6
each T and t of the model. Reactor contents were magnetically stirred throughout the experiments

by an external stirring plate. After each experiment, the reactor was cooled to room temperature

in a cool chamber at 5 °C before to open at atmospheric pressure. Products were recovered and

filtered through a 40 μm membrane. Solid fraction was dried at 40 °C for 24 h and the liquid

fraction was centrifuged at 10,000 rpm for 15 min at 4 °C. Control samples were treated with

identical CW and C1, C2 and C3 ratios but not subjected to subcritical water conditions. Each

control sample was magnetically stirred in flasks for 30 min (Thermo Scientific model

SP131015Q level 5), then filtered to separate liquid and solid fractions prior to measurements.

Control samples are identified as C1, C2 and C3 according to the levels R1, R2 and R3,

respectively.

2.4 Soluble protein, pH and X-ray fluorescence (XRF) determinations

Soluble protein was determined following the method described by Lowry-Peterson (1977). All

absorbance measurements were conducted on a Genesys 6 spectrophotometer (Thermo

Spectronic, USA). pH measurements were conducted in a HANNA pH 210 potentiometer.

Elemental analysis by XRF was determined in situ by a Non-Destructive X-Ray Analysis System

as reported by Ruvalcaba-Sil, Ramírez Miranda, Aguilar Melo, & Picazo (2010), which detects

elements of atomic number 13 and above. Si detector at 45 ° from the excitation direction of the

X-rays was used with available X-ray tubes of Mo, Rh, and anodes with Be window and N filter.

The system was also equipped with two lasers manually centered using a camera in order to

establish the region of analysis. Maximum power of the X-ray tubes was 75 W (50 kV, 1.5 mA).

Analyses conditions were 0.125 mA, 35 kV in an active area of 0.03 cm2 at 2 cm distance. Data

were collected from channel 20 to 270 and acquisition time of 120 s for each spectrum using

7
DppMCA software Amptek (Redus, Huber, & Sperry, 2009) and processed by PyMCA software

(Solé, Papillon, Cotte, Walter, & Susini, 2007). Quantitative analysis calculations were

performed using AXL software with standard reference materials (SRM) from the National

Institute of Standards and Technology (NIST SRM 1573A).

2.5 Characterization of the solid fractions

Water and ash contents were determined following the AOAC (1990) method. Total nitrogen

content in samples was determined by Kjeldahl (Buchi, Switzerland). Corrected protein contents

were calculated by the subtraction of the chitin nitrogen to the total nitrogen content and

multiplied by 6.25. FT-IR spectra were obtained in a Perkin Elmer (UK) FTIR-ATR Spectrum

100 (32 scans). All samples were previously milled to a particle size of 140 μm prior to analyses.

Scanning electron microscopy (SEM) images were acquired in a JEOL JSM-7800F (Japan)

equipped with microanalyses by characteristic X-ray energy dispersion system (EDS) between 3

and 5 kV for SEM and between 12 and 20 kV for EDS. Samples (177 µm) were covered with

carbon and gold on aluminum plates prior to analyses. Powder X-Ray diffraction (PXRD)

analyses were carried out in a Rigaku Ultima V diffractometer at 40 vkV and 30 mA (CuKα λ =

1.5406 Å). Data were collected between 2θ = 5 - 80° using a D/teX-ULTRA solid state detector

at 10°/min. Crystalline phases were assigned according to the chemical analyses data from EDS

and the powder diffraction file of the International Centre of Diffraction Data (ICDD, 2007). For

the quantitative analyses, crystallographic data for the identified phases were obtained from the

Inorganic Crystal Structure Database (ICSD, 2013), Cambridge Structural Database (CSD, 2015)

and American Mineralogist Crystal Structure Database (AMCSD) (Sikorski et al., 2009).

Crystalline phases were identified as α-chitin (Sikorski, Hori, & Wada, 2009); calcite, CaCO3

8
(ICSD-16710); hydroxyapatite, Ca5(PO4)3OH (ICSD-22059) (Kay, Young, & Posner, 1964) and

quartz, SiO2 (ICSD-27826). Cell parameters, crystal symmetry and atomic coordinates were

introduced in the Rietveld GSAS II program (Toby & Von Dreele, 2013). Refined parameters

were scale factor, peak shape parameters, preferred orientation parameters and mineral cell

parameters at the end of the refinement. Peak shape parameters were kept constant and

determined by analysis of a standard sample of lanthanum hexaboride, LaB6 (NIST SRM 660)

which allowed modeling the shape of the peak by refining the microstructural parameters.

Atomic coordinates remained fixed during refinement and background was modeled using a set

of points to which a polynomial Chevyschev function was fitted with 12 terms. α-Chitin

microstructure was modeled considering a uniaxial symmetry for the crystalline domain size,

thereby equatorial size D and the axial size L parameters were refined. Axial size is the size of

the crystal domain along the c-axis -the direction of the fiber- which is the direction of the chitin

polymer chains; this parameter was not refined, since its size is of the order of μm and does not

contribute to the peak width of the reflections; then, for chitin only the equatorial size D was

refined. α-chitin shows orthorhombic structure with space group P212121. Structurally, the α-

chitin chains are aligned in a corrugated and anti-parallel fashion on the bc plane. With the

antiparallel position, strong hydrogen bonds are established and, therefore, a more stable structure

is reached (Sikorski et al., 2009). For HAP microstructure, which crystallizes in the hexagonal

system described by the space group P63/m, was modeled with an axial component L (in the

direction of the hexagonal c-axis) and an equatorial D for the crystalline domain size. For calcite

(and quartz when present), crystal size effects were not considered to describe the width of the

reflections.

9
3. Results and discussion

3.1 The Taguchi model analyses

The results obtained from the experimental design displayed the main effect of the control factors

on the response variables. The S/N analysis was carried out and function “larger is better” was

chosen for soluble protein and pH in the liquid fraction owing to the expected removal of protein

from the chitinous matrix as reported by Osada et al. (2015). Furthermore, the function “smaller

is better” was used for Ca, P, K and S contents in the solid fraction because demineralization was

expected with the process (Fig. 1). The S/N indicators presented in Figure 1 indicate that R, ratio

of CW/dH2O, shows the greatest and significant effect over all the response variables. R2

displayed the maximum values for soluble protein, Ca and P contents. The temperature of the

process also infers significantly in the soluble protein and mineral contents, while the time of

reaction presented the lowest meaning among the control factors. For soluble protein, the

experiment R2t1T2 (0.09gCW/g dH2O, 5 min and 260°C) displayed the maximum protein yield

(140.73 ±18.03 mg/g sample) with p ≤ 0.05 (Figure 1a). In a related work by Quitain et al. (2001)

using hydrothermal treatment for the production of valuable material from shrimp waste, the flow

of protein from solid to the liquid fraction in shrimp cephalothoraxes using 1:125 (g

exoskeleton/g water) ratio was also ascribed to the subcritical water conditions (250 °C, 4MPa

for 60 min). Another researchs by Yoshida et al. (1999) and Kang et al. (2001) described that

subcritical water reaches its maximum ion product (Kw 1 x 10-11) between 250 and 268 °C and

between 9 and 4 MPa pressure. According to these authors, the proteins in the solid fraction are

hydrolyzed at the maximum Kw thereby migrating to the liquid phase. Figures 1 b-e represent the

10
results of Taguchi model for Ca, P, S and K, respectively, and the data of pressure achieved

during treatments are summarized in Table 1. As observed, Ca is minimized for R2t2T3 (7.26 ±

0.54 % Ca) but contrarily, it increases for R1t3T3 (10.97 ± 0.52 % Ca). Additionally, Figure 1 c

points out that P is minimized with R2 and T1. Generally, levels R1 and T3 displayed the highest

percentages of this element, while the multiple comparisons of means displayed the lowest value

for R2 (2.2 ± 0.39 % P). On the K and S contents (Fig.1d and e), these are minimized in R1 but

maximized for R3 and interestingly, changes in pH among treatments were not significant, with

the lowest value found for R1t1T1 (8.46 ± 0.28), while R2t2T3 displayed the highest value (8.89 ±

0.03) (Table 1).

11
3.2 X-Ray Fluorescence (XRF) analyses

The XRF results for the samples of solid fractions are shown in Table 2 where Ca, P, S, K, Cl and

Al are major elements, and Cr, Mn, Fe, Ni, Cu, Zn, Br and Sr appear as trace elements (see

supplementary data 1 for microelement composition of treated samples and controls). Both

groups have been reported as macroelements and microelements essential for a nutritional diet in

shrimps and crustaceans, which are acquired from food intake as well as their ability to absorb

minerals from the water (Tacon, 1989). The mineralized tissues of crustaceans are the result of

interaction among these inorganic ions contained in biological fluids, such as Ca2+, Mg2+, Na+,

K+, phosphate, bicarbonate, Cl- and sulphates within the formation of ion-substituted calcium

phosphates at nanoscale which are responsible for the hardening process of exoskeletons (Tas

2014). Moreover, the concentration of Ca and P increases in the pre-moulting period. The

alkaline-earth metal is related to calcification of the skeleton and the latter is probably needed for

chitin synthesis (Weaver et al., 2012). In the study of the application of hydrothermal treatment

for HAP synthesis carried out by Sadat-Shojai et al. (2012), no control of the Ca/P ratio was

needed during reaction to achieve the CaP desired phase, but other parameters such as pH, flow

rate of Ca2+ and HPO42- solutions, temperature and time. In the present work, the Ca/P ratios

varied from 2.06 to 3.86 (average of 2.84), which were fairly different to the theoretical Ca/P

ratio in HAP (1.67). The positive differences on the Ca/P ratio have been attributed to the

substitution of carbonate for phosphate in the crystal lattice (Sadat-Shojai et al., 2012). In this

regard, P data in Table 2 displayed no significant differences among treatments, whereas three

groups were detected for Ca with significant differences, R1t3T3 with the highest and in the group
12
with the lowest mean are R2t1T2, R2t2T3, R2t3T1 and R3t3T2.

13
3.3 Attenuated Total Reflectance-Fourier transform infrared spectroscopy (ATR-FTIR),

Scanning electron microscopy with X-ray energy dispersion system (SEM-EDS), Powder X-

Ray diffraction (PXRD) analyses of samples

The FTIR spectrum shown in Fig. 2 presents the characteristic bands for chitin in agreement to

the literature (Cárdenas, Cabrera, Taboada, & Miranda, 2004). The FTIR spectra of the treated

samples displays two bands at 1660 and 1620 cm-1, which are assigned to the carbonyl group

(amide I), and that at 1560 cm-1 to NH (amide II). However, these bands assigned to the

acetamido group of chitin are defined in lesser extent than that for α-chitin, whereas for C2 only

one band is observed at 1660 cm-1 instead of the doublet, which might be adscribed to the

presence of proteins (Barth, 2007) (Fig. 2). This was corroborated with the treated samples, in

which the spectra evidence the protein removal. The chitin and calcite bands are observed before

and after the treatments, although those for CO32- increase and new bands assigned to PO4-3 are

observed. Additionally, the band at 1409 cm-1 is assigned to the vibration of C=O of carbonate

(Mikkelsen, Engelsen, Hansen, Larsen, & Skibsted 1997; Miller & Wilkins, 1952) and signals at

874 and 725 cm-1 to calcite form, which presence was also corroborated by PXRD analyses

(Fowler, 1974; Mikkelsen et al., 1997; Rahman & Halfar, 2014). The signals at 1032, 1021, 603

and 565 cm-1 could be assigned to PO4-3 moiety for HAP (Ca5(PO4)3(OH)), which was also

corroborated by X-ray analysis (Fowler, 1974; Miller & Wilkins, 1952).

The results of Rietveld fitting are shown in Table 3 summarizes the crystallographic data of the

identified phases, equatorial D, axial L, crystalline domain sizes for α-chitin and HAP, as well as

the RF, the discrepancy factor as a useful indicator of refinement (see supplementary data 2 for X-
14
ray diffraction patterns of control and treated samples adjusted by the Rietveld method). The α-

chitin and the polymorphs of calcium carbonate, calcite, aragonite and amorphous CaCO3 are the

most studied structures in crustaceans by XRD (Mikkelsen et al., 1997). According to Heredia

et al. (2007), calcite interacts into the α-chitin matrix in specific sites related to nucleation and

spheroidal crystal growth resulting in the formation of spherulites, which is conspicuous with the

appearance of white spots. The results in Table 3 indicate that the wt% of chitin for control C1, C2

and C3 samples are higher than that for the samples subjected to subcritical water treatments.

These results can be explained because calcite and HAP were not detected and because they are

found as amorphous calcium carbonate and calcium phosphates in the control samples (Becker et

al., 2005). The crystalline SiO2 quartz in all samples is less than 1.5 wt% compared to the

biopolymer, calcite and HAP phase; its presence is attributed to the crustacean food intake,

consistent mainly by seaweed (Boßelmann et al., 2007; Neues et al., 2011). It was also noticed by

EDS and SEM that silicon is not homogeneously distributed in the samples; however, it was

locally identified in sample C2 (Figure 3 and Table 4), whereas calcite was detected in all samples

being the lowest content for R1t2T2 and the highest for R2t3T1. Generally, the calcite content for

samples C1, C2, and C3 is notably lower than those measured for the treated samples. The

presence calcium carbonate either crystalline or amorphous has been described in the exoskeleton

of crustaceans, precipitated in the organic matrix of protein and chitin. The adopted crystal or

amorphous arrangement depends on environmental factors and the biomineralization process that

is closely related to the crustacean molting cycle (Luquet & Marin, 2004; Neues et al., 2011).

The presence of calcite was corroborated by FTIR and SEM-EDS. The SEM micrographs

and results of the elemental analysis are shown in Fig. 3 and in Table 4. Fig. 3a corresponds

15
to sample C2 where amorphous minerals are observed and Fig. 3b and 3d show amorphous

ore clusters. The structural morphology observed in the form of spherulites (Heredia et al.,

2007) in Fig. 3d was identified as amorphous calcium carbonate by elemental analysis-EDS

(Table 4), which shows the presence of C, O, Ca as major elements, and Na, Mg, Cl and Cu

below 1 wt%. Interestingly, a needle-shaped morphology is observed in the mineralized

sample of Fig. 3c, which might be ascribed to the presence of aragonite, according to the

description of this polymorphic form of calcium carbonate in marine shells by Hou et al.,

(2016). Fig. 3 shows the of SEM images of the samples R1t2T2 (3e); R3t3T2 (3f); R2t3T1

(3g-k) and R2t1T2 (3l-p). The micrographs of Fig. 3h show minerals on the surface and each

specimen were analyzed at higher magnification in Fig. 3i, 3j, and 3k where aggregates of

crystallized minerals with different crystalline forms are observed. Elemental analysis by

EDS for the regions indicated in Fig. 3i1 (5000x) and 3i2 (22000x) revealed the presence of

C, O and Ca, which are related to several phases of calcium carbonate. Figures 3l-p

correspond to the sample R2t1T2. According to the elemental analysis by EDS of the

mineral aggregates in 3n, their composition matches with crystalline CaCO3. The

crystallization of these minerals is attributed to the treatment with subcritical water because

high pressures and temperatures favor the crystallization of these minerals (Koga et al.,

1998; Fratzl et al., 2010; Sadat-Shojai et al., 2012; Gal et al., 2013; Zhang et al., 2012).

It is worth to remark that HAP was identified in all samples treated with water in subcritical

conditions with percentages between 3.4 and 25.7 wt%, corresponding to R3t3T2 and

R2t3T1, respectively). Contrarily, HAP is not observed in samples C1, C2, and C3 but only a

small percentage of calcite (3-4 wt%) (Table 3 and supplementary data 2). This analytical

evidence suggests the formation of HAP under the subcritical water process. Additionally,

16
the subcritical water treatment increased the content of calcite between 10 and 22.1 wt%,

for R1t1T1 and R2t3T1, respectively.

According to the XRD results for the treated samples, the identified HAP mineral appears

as nanocrystals with an axial size between 1 and 15.8 nm (average of 7.9 nm) and an

equatorial size between 1.2 and 14.1 (average of 3.8 nm). The nanosized crystals generally

tend to be needles although platelet-like morphologies are also observed which depends on

the size relation between the equatorial domain D and the axial L (Table 3). These small

domain crystalline sizes (D and L) indicate that a fraction of HAP is amorphous. The

production of HAP from marine shells by hydrothermal treatment has been reported

employing calcium phosphate solution (Hou et al., 2016). In this work, the FTIR and

SEM-EDS analyses sustain that the phosphorus present in control samples is part of HAP

in the amorphous phase, as the bands assigned to PO43- were identified by FTIR (Fig. 2)

and therefore the addition of an external source of phosphorous is unnecessary.

On the other hand, a flake-type structural morphology was observed on the surface of the

sample showed in Fig. 3f among the amorphous part of the material. Chemical analysis by

EDS revealed the presence of C, O, Ca and P, which could be interpreted as the presence of

HAP. This suggests that the HAP identified in the treated samples, which was obtained in

situ without added phosphorus, might be the result of a crystallization phenomenon of P,

present in samples C1, C2, C3 (Table 2) under the subcritical water treatment. The variation

of the diameter of the α-chitin fiber, the equatorial crystalline domain size D (Table 3,

supplementary data 2) might be ascribed to a rearrangement of these fibers in the structure

17
due to protein removal (see supplementary data 3 for SEM micrographs of subcritical water

treated samples R2t1T2, R2t3T1 and R3t3T2). Therefore, the chains of chitin and the fibers are

rearranged when removal of the protein, thus increasing the equatorial domain size D from

an average of 2.3 to 6.3 nm. It is worth to mention that the crystallization of α-chitin occurs

with an increase in the size of the equatorial crystalline domain D and the Bragg reflection

caused by the (110) planes, which run lengthwise through the α-chitin fiber (Fig. 4), is

particularly affected by the rearrangement because this is the most intense reflection of α-

chitin and keeping in mind that the peak width is inversely related to the equatorial

crystallite domain size. As expected, the arrangements of chitin chains characteristic of an

amorphous state prevail although it must be affected by several conditions and,

arrangements of chitin chains characteristic of an amorphous state will partially prevail (a

possible model is shown in Fig. 4). Regarding the micrographs, the samples subjected to

the treatments R1t2T2, R2t1T2, R2t3T1 and R3t3T2 show chitin fibers and amorphous minerals

on the surface, which is indicated in Fig. 3 as arrows pointing upwards and downwards,

respectively.

On the other hand, the chemical composition analysis displayed 73.21% of initial moisture

in the CW sample with protein and ash contents of 51.45 ± 0.17% and 25.05 ± 0.08%,

respectively. After treatments, sample R2t1T2 displayed 2.03 ± 0.28% and 36.81 ± 0.80%

for protein and ash, respectively, that stands for a deproteinization of 96.06 ± 0.54%, which

resulted in the most efficient treatment for protein removal. These chitins contain relatively

high amount of ash notwithtanding it can be eliminated by mild acid treatment, while the

18
residual protein can be removed during conventional thermochemical deacetylation process

for chitosan production. The geometric shapes of crystalline phases found in R2t1T2 and

R2t3T1 treatment samples in which plates, flakes, and sheets prevailed, while that from

R3t3T2 thick braids of chitin fibers with oval voids with less compact structures were

observed (supplementary data 3). Furthermore, these micrographs also evidence the

removal of protein in these samples.

4. Conclusions

The Taguchi model was used to optimize the experimental conditions of subcritical water

treatment. Chemical, structural and morphological characterizations evidence the complete

protein removal from the chitin matrix network by the use of water under subcritical

conditions in short reaction times. Additionally, the presence of amorphous and crystalline

calcite and HAP was demonstrated, thus showing the crystallization produced during water

under subcritical process.

Acknowledgements

The authors would like to thank CONACyT for founding Project 237292 and scholarship

(AEC). Special thanks to Dr. Samuel Tehuacanero Coapa, M en C Manuel Aguilar Franco

and M en C. Mayra Dafne Manrique Ortega at the Instituto de Física (UNAM) for their

assistance in the acquisition of the SEM, XRF, and use of the software.

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FIGURE CAPTIONS
Figure 1. Effect of water under subcritical condition process of CW on soluble protein
content determined in the liquid fraction (a) and Ca (b), P (c), K (d) and S (e) in the solid
fraction by elemental analysis with XRF.

Figure 2. FTIR spectra of calcite, HAP, α-chitin obtained by a biological method and
experimental samples: R1t1T1 is 0.05gCW/gdH2O at 5 min 230 ºC; R1t2T2 is
0.05gCW/gdH2O at 15 min 260 ºC; R1t3T3 is 0.05gCW/gdH2O at 30 min 280 ºC; R2t1T2 is
0.09gCW/gdH2O at 5 min 260 ºC; R2t2T3 is 0.09gCW/gdH2O at 15 min 280 ºC; R2t3T1 is
0.09gCW/gdH2O at 30 min 230 ºC; R3t1T3 is 0.17gCW/gdH2O at 5 min 280 ºC; R3t2T1 is
0.17gCW/gdH2O at 15 min 230 ºC and R3t3T2 is 0.17gCW/gdH2O at 30 min 260 ºC.

Figure 3. SEM micrographs of C2 (a-d) and of subcritical water treated samples: R1t2T2 (e);
R3t3T2 (f); R2t3T1 (g-k) and R2t1T2 (l-p). SEM b1 and b2 were analyzed by EDS in the
indicated area (box). Arrows up indicate chitin fibers, arrows down amorphous minerals.

Figure 4. Crystal structure of α-chitin, the projection of the unit cell on the ab and the
planes (110) according to the most intense reflection experimentally observed for chitin
(top). Uniaxial model for microstructure used in the refinements (middle left). D-diameter
27
fiber (equatorial crystalline domain size) is shown longitudinally, with the chitin polymer
chains oriented along the c-axis and antiparallel arrangement along the b-axis: cross section
of the fiber where the packing of the chitin chains and the planes (110) with the highest
electronic density (bottom). Suggested model of the amorphous arrangement of chitin
chains that could contribute to the amorphous background represented in the diffractograms
(middle right).

FIGURE 1
45 -17,0
a
40
-17,5 b
S/N=-10log10(MSD)
S/N=-10log10(MSD)

-18,0
35

-18,5
30
-19,0

25
-19,5

20
-20,0

15 -20,5
0.05 0.09 0.17 5 15 30 230 280 0.05 0.09 0.17 5 15 30 230 280
260 260
R(CW/dH2O) Time (min) Temperature (°C) R(CW/dH2O) Time (min) Temperature (°C)

19
-6

-7 c 18 d
S/N=-10log10(MSD)
S/N=-10log10(MSD)

17
-8

-9 16

-10 15

-11 14

-12 13

-13 12
5 15 30 0.05 0.09 0.17 5 15 30 230 280
0.05 0.09 0.17 230 260 280 260
R(CW/dH2O) Time (min) Temperature (°C) R(CW/dH2O) Time (min) Temperature (°C)

28
 11

e
10
S/N=-10log10(MSD)

6
0.05 0.09 0.17 5 15 30 230 280
260
R(CW/dH2O) Time (min) Temperature (°C)

FIGURE 2

29
30
 Figure 3

aa b c d

e f g h i

i j k l

i1
i2

m n o p .

31
FIGURE 4

32
Table 1. Control factors, level, and Taguchi orthogonal arrays L9 (33)

Control Factors
Level Sample/dH2O Time Temperature
R (g/g) t (min) T (°C)*
1 0.05 5 230
2 0.09 15 260
3 0.17 30 280
Entry Independent Variables Pressure (MPa) pH
R1t1T1 1 1 1 2.78 ± 0.09 8.46±0.28

R1t2T2 1 2 2 10.21 ± 3.59 8.32±0.08

R1t3T3 1 3 3 20.18 ± 2.78 8.52±0.03

R2t1T2 2 1 2 5.36 ± 0.64 8.57±0.01

R2t2T3 2 2 3 8.94 ± 0.60 8.89±0.03

R2t3T1 2 3 1 3.94 ± 0.95 8.37±0.14

R3t1T3 3 1 3 10.39 ± 1.98 8.48±0.03

R3t2T1 3 2 1 3.96 ± 0.66 8.52±0.14

R3t3T2 3 3 2 5.00 ± 1.66 8.64±0.15

*Standard deviation of ± 4 °C for each level.

33
Table 2. Elemental analysis by XRF for solid fractions of CW control and treated samples of macroelements and the ratio of Ca/P.
Entry Macroelements (%)
Ca P S K Cl Al Ca/P
C1 9.04 ± 0.72 3.46 ± 0.33 0.36 ± 0.05 0.17 ± 0.01 0.38 ± 0.09 0.09 ± 0.02 2.61
C2 7.82 ± 1.29 2.83 ± 0.41 0.44 ± 0.09 0.20 ± 0.01 0.86 ± 0.27 0.08 ± 0.02 2.76
C3 8.60 ± 0.76 2.63 ± 0.50 0.49 ± 0.04 0.24 ± 0.00 1.22 ± 0.06 0.07 ± 0.00 3.27
R1t1T1 9.64 ± 2.22ab 3.08 ± 2.96 0.31 ± 0.08c 0.10 ± 0.02c 0.15± 0.01 d 0.08 ± 0.02 3.13
R1t2T2 9.66 ± 0.01 ab 3.75 ± 0.47 0.28 ± 0.00 d
0.11 ± 0.01 c
0.16 ± 0.05 d
0.07 ± 0.00 2.58
R1t3T3 10.97 ± 0.53a 4.83 ± 0.36 0.34 ± 0.00c 0.14 ± 0.01c 0.28 ± 0.06d 0.10 ± 0.01 2.27
R2t1T2 7.27 ± 0.54c 2.09 ± 0.48 0.41 ± 0.02 b
0.19 ± 0.00 b,c
0.84 ± 0.04 b
0.06 ± 0.00 3.60
R2t2T3 7.66 ± 0.04 c 2.55 ± 0.37 0.40 ± 0.00 b,c
0.17 ± 0.01 b,c
0.66 ± 0.13 c
0.07 ± 0.01 3.00
R2t3T1 7.58 ± 1.22 c 1.96 ± 0.15 0.37 ± 0.00 c
0.14 ± 0.01 c
0.47 ± 0.03 c
0.07 ± 0.00 3.85
R3t1T3 8.80 ± 0.11 ab
3.99 ± 0.47 0.49 ± 0.05ª ,b
0.19 ± 0.00 b,c
0.69 ± 0.08 c
0.07 ± 0.00 2.21
R3t2T1 9.05 ± 1.28ab 3.17 ± 0.80 0.40 ± 0.00 b,c
0.20 ± 0.01 b
0.70 ± 0.02 c
0.07 ± 0.01 2.85
R3t3T2 7.13 ± 0.86 c 3.46 ± 0.34 0.51 ± 0.06a 0.27 ± 0.05a 1.19 ± 0.23a 0.06 ± 0.00 2.06

a, b, c and d in a column means that groups are statistically different among inoculum level (Tukey-Kramer p ≤0.05).

34
 Table 3. Crystallographic data and results of the quantification of identified crystalline phases.

Identified phases
Name α-Chitina Calciteb HAPc Quartzd
Formula (C8H13O5N)n CaCO3 Ca5(PO4)3OH SiO2
Weight formula
812.78 100.088 502.32 60.084
(g/mol)
P212121 (19) R-3c (167) P63/m (176) P3221 (154)
Space group

Crystalline
system
orthorhombic Trigonal hexagonal trigonal
Z 1 6 2 3
Reticular a = 4.750
a = 4.989 a = 9.432 a = 4.91
parameters (Å) b = 18.890
c = 17.062 c = 6.881 c = 5.40
c = 10.333
Entry D D L
wt% RF** wt% RF* wt% RF** wt% RF**
(nm)* (nm)* (nm)*
C1 95.4 1.7 0.08 4.3 0.08 ND ND ND ND 0.3 0.11
C2 96.7 2.5 0.09 3.1 0.07 ND ND ND ND 0.2 0.11
C3 95.9 2.7 0.09 3.8 0.08 ND ND ND ND 0.2 0.16
2.3***
R1t1T1 72.0 7.0 0.03 10.0 0.04 18.0 3.3 6.3 0.03 ND ND
R1t2T2 78.8 7.7 0.05 12.3 0.06 8.9 4.1 8.9 0.07 ND ND
R1t3T3 68.6 7.9 0.04 16.9 0.03 14.5 2.4 1.0 0.04 ND ND
R2t1T2 76.7 5.5 0.08 16.3 0.06 6.7 3.1 11.6 0.09 0.3 0.12
R2t2T3 64.0 7.7 0.04 19.0 0.04 15.6 1.5 11.8 0.05 1.5 0.08
R2t3T1 52.2 2.3 0.04 22.1 0.03 25.7 1.2 1.0 0.04 ND ND
R3t1T3 76.0 6.7 0.05 16.1 0.04 8.0 2.4 15.8 0.06 ND ND
R3t2T1 67.9 4.9 0.06 20.6 0.05 11.2 1.8 13.7 0.07 0.4 0.10
R3t3T2 82.2 6.6 0.09 14.4 0.06 3.4 14.1 1.0 0.11 ND ND
6.3*** 3.8*** 7.9***
* In the monoaxial model used for crystallite size with the axis along [001] direction (c-axis), D and L are the equatorial and axial domain sizes respectively. ** Rietveld
discrepancy value given by where Fobs,hkl and Fcalc,hkl are the observed and computed structure factor amplitudes for the
specific crystalline phase. ***Average values, ND = not detected. a Sikorski et al. (2009); bICSD-16710; cICSD-22059, Kay et al. (1964); dICSD-27826

Table 4 Elemental composition analysis by EDS of the areas indicated in the images by SEM

35
 Content (% wt)
Element
C2 C2 C2 R2t3T1 R2t3T1 R2t1T2 R2t1T2 Standard
Fig. Fig. Fig. Fig. Fig. Fig. 3n Fig. 3p
3b 3c 3d 3i1 3i2
C 64.15 73.37 29.77 19.88 36.42 14.80 ± 1.29 15.47 ± 1.59 C Vit
± ± ± ± 1.85 ± 1.84
0.87 0.77 0.93
O 27.31 17.48 44.06 45.52 48.82 42.84 ± 1.398 21.78 ± 1.69 SiO2
± ± ± ± 2.51 ± 2.08
0.88 0.77 0.96
Na 0.74 0.92 0.61 ND ND ND ND Albite
± ± ±
0.11 0.10 0.16
Al ND 0.74 ND ND ND ND ND Al2O3
±
0.06
Si ND 1.41 ND ND ND ND ND SiO2
±
0.07
Mg 0.56 ND 0.55 ND ND 2.44 ± 0.34 2.09 ± 0.31 MgO
± ±
0.08 0.10
P 1.65 1.11 ND ND ND ND 13.54 ± 0.88 GaP
± ±
0.11 0.09
S ND 0.50 ND ND ND ND ND FeS2
±
0.07
Cl 0.50 0.37 0.26 1.33 ± ND ND ND NaCl
± ± ± 0.34
0.06 0.05 0.06
Ca 5.08 3.62 24.15 33.27 14.76 39.91 ± 1.62 47.13 ± 1.59 Wollastonite
± ± ± ± 1.74 ± 0.9
0.15 0.12 0.50
Cu ND 0.49 0.61 ND ND ND ND Cu
± ±
0.13 0.19

ND Not detected

36