Bioresource Technology 97 (2006) 488493

Characterization and adsorption properties of eggshells and eggshell membrane

W.T. Tsai
a

a,*

, J.M. Yang b, C.W. Lai a, Y.H. Cheng a, C.C. Lin a, C.W. Yeh

Department of Environmental Engineering and Science, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan, ROC b Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan, ROC Received 8 October 2004; received in revised form 28 February 2005; accepted 28 February 2005 Available online 17 May 2005

Abstract The objective of this work was to study the chemical and physical characterization of eggshell and eggshell membrane particles prepared from the hen eggshell waste. Under the characterization measurements investigated, it was found that the pore structures of the two biomaterials belong to a typical Type II, indicating that they should be basically characteristic of nonporous materials or materials with macropores or open voids. Further, the chemical composition of the resulting eggshell particle was strongly associated with the presence of carbonate minerals from the Fourier transform infrared (FTIR) spectra. In contrast to the resulting eggshell membrane particle, the presence of functional groups of amines and amides was observable because of its chemical composition of brous proteins. From the isotherm data of methylene blue at 25 C, the Freundlich model yielded a somewhat better t than the Langmuir model. The adsorption isotherms revealed the eggshell biosorbents could only uptake the basic dye of less than 1.0 mg/g in aqueous medium, which was attributed to their poor pore properties. 2005 Elsevier Ltd. All rights reserved.
Keywords: Eggshell; Eggshell membrane; Pore property; FTIR; Methylene blue adsorption

1. Introduction Hen eggshell typically consists of ceramic materials constituted by a three-layered structure, namely the cuticle on the outer surface, a spongy (calcareous) layer and an inner lamellar (or mammillary) layer (Tullett, 1987; Stadelman, 2000). The spongy and mammillary layers form a matrix composed of protein bers bonded to calcite (calcium carbonate) crystal. The two layers are also constructed in such a manner that there are numerous circular openings (pores). This structure permits gaseous exchange throughout the shell. The outer surface of the eggshell is covered with a mucin protein that acts as a soluble plug for the pores in the shell. The cuticle is also permeable to gas transmission. The chemical composi*

Corresponding author. Tel.: +886 6 2660393; fax: +886 6 2669090. E-mail address: wwtsai@mail.chna.edu.tw (W.T. Tsai).

tion (by weight) of by-product eggshell has been reported as follows: calcium carbonate (94%), magnesium carbonate (1%), calcium phosphate (1%) and organic matter (4%) (Stadelman, 2000). Notably, the by-product eggshell generated from food processing and manufacturing plants is inevitably composed of calcium carbonate (eggshell) and eggshell membrane (ESM). The ESM resides between the egg white (albumen) and the inner surface of the eggshell. There are two shell membranes around the egga thick outer membrane attached to the shell and a thin inner membrane (Parsons, 1982; Nakano et al., 2003). The total thickness of these two membranes has been found at approximately 100 lm. Each of these membranes is composed of protein bers that are arranged so as to form a semi-permeable membrane. Therefore, the ESM possesses an intricate lattice network of stable and water-insoluble bers and has high surface area

0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.02.050

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resulting in various applications such as adsorbent (Suyama et al., 1994; Ishikawa and Suyama, 1998; Allen et al., 2000; Gota and Suyama, 2000; Ishikawa et al., 2002a,b; Koumanova et al., 2002) and immobilization support (Xiao and Choi, 2002; Yang et al., 2003; Choi and Yiu, 2004). Notably, these researches have no data on the pore properties of ESM. The by-product eggshell represents approximately 11% of the total weight (%60 g) of egg (Stadelman, 2000). It was also reported that about 28% of all eggs produced were sent to commercial breaking operations for manufacturing egg products (Poland and Sheldon, 2001). In Taiwan, for example, the annual generation of eggshell waste from the food processors was estimated to be over 1.3 104 metric ton on the basis of 7.1 109 of pieces of hen eggs (COA, 2003). The byproduct eggshell from these breaking operations represents a signicant waste for the processor because they are traditionally useless after the production of eggs and egg derivatives. Most of the waste was commonly disposed of for landll without any pretreatment. It is obvious that these approaches are not desirable practices in view of the odor from biodegradation. Occasionally, few of the by-products were reused as a fertilizer or soil conditioner because of their high nutrition contents such as calcium, magnesium and phosphorus (Tacon, 1982). Based on the bioresource recovery and reuse, the utilization of this food processing by-product has slightly increased in recent years. Rivera et al. (1999) reported a novel procedure to porous hydroxyapatite from eggshells. Moreover, there have been studies aiming at the calcium supplement and other nutrition sources from the albumin, membrane and matrix of the eggshell, which was processed by crushing and milling to obtain ne particles (ours) for animal use (Christmas and Harms, 1976). Taking into account the sustainable utilization of eggshell and its intrinsic pore structure (Rauch, 1952), the characterization of the biomaterial is very scarce in the literature. Creger et al. (1976) observed the various stages of formation of the eggshell with scanning electron microscope. Garcia-Ruiz et al. (1995) aimed at the textural analysis of eggshell with a powder diractometer. They found that the mineral structure of eggshell could be properly described in terms of competitive crystal (calcite) growth. The mineral phase (calcite) present in the eggshell was demonstrated and observed by X-ray powder diractometry (Rivera et al., 1999). Thus, the main objectives of this work are to study the main physical and chemical characteristics of the hen eggshell and eggshell membrane particles. Further, the applicability of common isotherm models (i.e., Langmuir and Freundlich) has been evaluated based on the adsorption capacities from the removal of methylene blue from aqueous solution at 25 C.

2. Methods 2.1. Materials Raw membrane-bound eggshell was collected from a campus breakfast shop and immediately stored in the iced water. The hen eggshell was manually stripped from eggshell membrane after cleaning of the raw material. The stripped eggshell membrane was immersed in aqueous acetic acid (70%) for two days to dissolve residual eggshell, followed by rinsing with deionized water repeatedly to weak acidity and nally dried in the oven (50 C) for two days. The eggshell and eggshell membrane were further ground to prepare powder particles. 2.2. Physical characterization measurements 2.2.1. Surface area, pore volume and pore size distribution The pore structures of eggshell and eggshell membrane particles relating to surface area, pore volume and pore size distribution were obtained by measuring their nitrogen adsorptiondesorption isotherms at 196 C in an ASAP 2010 apparatus (Micromeritics Co., USA). BrunauerEmmettTeller (BET) surface area (SBET, m2 g1) and total pore volume (Vt, cm3 g1 at STP) were thus obtained by the N2 adsorption data. The pore size distribution was calculated based on dierential pore volume of BarrettJoynerHalenda (BJH) adsorptiondesorption (Lowell and Shields, 1991). All measurements were carried out in duplicate. 2.2.2. True density The true density (qs) of the sample was measured by a helium displacement method with an AccuPyc 1330 pycnometer (Micromeritics Co., USA). From the data of Vt and qs, the particle density (qp) and porosity (ep) can be thus obtained (Smith, 1981). 2.2.3. Scanning electron microscope (SEM) observation In order to elucidate the porous properties of the resulting eggshell particles from the measurement of nitrogen adsorptiondesorption isotherm, the porous texture was further examined by scanning electron microscope (SEM) observation. The SEM analysis was carried out on the JEOL JXA-840 operated at a 20 kV accelerating potential. Prior to the observation, the surface of the sample was coated with a thin, electric conductive gold lm. 2.3. Chemical characterization measurements 2.3.1. Elemental analysis Carbon/hydrogen/oxygen/nitrogen/sulfur (C/H/O/N/ S) contents were used as a means of examining the remaining residues in the preparation of the eggshell products. The elemental analysis of the sample

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(13 mg) was made using an elemental analyzer (model CHN-O-RAPID, Heraeus Co., Germany). For each analysis, the standard sample (i.e., sulfanilic acid) was rst analyzed for checking the experimental error within 1%. All measurements were carried out in duplicate. 2.3.2. Fourier transform infrared spectroscopy (FTIR) analysis FTIR has been used for the examination of functional groups on the surface of activated clays or carbon materials previously (Tsai et al., 2001, 2002). To obtain the observable absorption spectra, the dilution and homogenization of the dried, ne samples with KBr (spectroscopic grade), were carried out with additional grinding and mixing in an agate mortar. Discs (12.7 mm ID and %1 mm thick) were prepared in a manual hydraulic press (model 15.011, Perkin Elmer Co., USA) at about 10 tonnes for a pressing time of 3060 s. The spectrum was measured and recorded (5004000 cm1) on a spectrometer (model system 2000 FTIR, Perkin Elmer Co., USA) with a resolution of 2.0 cm1. 2.4. Adsorption of methylene blue The methylene blue adsorption equilibrium of adsorbent was often used as one of the important pore properties (Brady and McKay, 1996). The adsorption behaviors of eggshell and eggshell membrane particles were tentatively determined to evaluate its preferability for removal of methylene blue from aqueous solution. All the experiments of adsorption capacities of methylene blue were determined by using a batch method as described previously (Tsai et al., 2004). In each experiment, 0.1 g of sample was used after degassing at 105 C for 24 h in the oven. Adsorption was carried out by shaking (at %110 rpm) with xed value (i.e., 0.05 dm3) of methylene blue solutions at dierent initial concentration of 1, 2, 4, 6, 8, 10 mg dm3 in a thermostated shaker bath at 25 0.1 C for all experiments. The methylene blue solutions were contacted for 2 h, which had been shown previously to approach adsorption equilibrium. All adsorption experiments were

carried out in duplicate and the maximum deviation was found within 5%.

3. Results and discussion 3.1. Particle size of the eggshell samples The results of mean diameter, median diameter and mode diameter of eggshell and eggshell membrane particles are 77.9, 21.1, 13.6 lm and 160.2, 114.2, 185.3 lm, respectively, revealing that these adsorbents should possess poor surface area properties toward the probe molecule (i.e., nitrogen). 3.2. Physical characterization of the eggshell samples The data in Table 1 indicate the BET surface area, total pore volume, densities and porosity of eggshell and eggshell membrane particles. It can be found that pore properties between eggshell and eggshell membrane are very similar according to the data of BET surface area and total pore volume. From the N2 isotherm in Fig. 1, these samples possess poor pore properties

6
eggshell:

Volume adsorbed (cm3/g STP)

5 4 3 2 1 0 0

eggshell membrane:

Adsorption Desorption Adsorption Desorption

0.2

0.4 0.6 0.8 Relative pressure (P/P0)

1.2

Fig. 1. Nitrogen adsorption/desorption isotherms of eggshell and eggshell membrane particles.

Table 1 Main pore properties of eggshell and eggshell membrane particles Sample Eggshell Eggshell membrane
a b c d e f

SBETa (m2 g1) 1.023 0.339f 1.294 0.424

Vtb (cm3 g1) 0.0065 0.0025 0.0063 0.0016

qsc (g cm3) 2.532 0.021 1.358 0.001

qpd (g cm3) 2.491 1.346

epe () 0.0162 0.0088

BET surface area. Total pore volume. True density. Particle density, calculated by: qp = 1/[Vt + (1/qs)]. Particle porosity, computed by: ep = 1 (qp/qs). Denote the mean standard deviation for two measurements.

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towards probe molecule (i.e., nitrogen), showing that the desorption hysteresis loop does not occur practically on the N2 isotherm. According to the classication of Brunauer, Deming, Deming and Teller (BDDT) (Gregg and Sing, 1982), the adsorption isotherm should belong to a typical Type II, which is frequently encountered when adsorption occurs on nonporous materials or on materials with macropores or open voids. However, it is noted that the true density of eggshell is signicantly larger than that of eggshell membrane (2.532 g cm3 vs. 1.358 g cm3, listed in Table 1), implying that the crystalline shell is mainly composed of calcium carbonate in the form of calcite because true density of calcite is 2.710 g cm3 (Dean, 1987). On the other hand, eggshell membrane is mainly consisted of brous proteins or collagen-like proteins (Tullett, 1987), indicating that the organic matrix derived from the eggshell membrane is not rigid like inorganic minerals. It would be expected to result in the more macropore structure in eggshell particles because of the intrinsic existence of pore canal (Poland and Sheldon, 2001). Therefore, the porosity of eggshell was estimated to be larger than that of eggshell membrane as seen in Table 1. The textural structure examination of eggshell and eggshell membrane particles can be observed from the SEM photographs. From Fig. 2, it can be clearly seen that these two samples do not indicate well-dened pore structures. It should be noted that the observation was consistent with the results from the nitrogen adsorptiondesorption measurements in Table 1. The crystalline structure of eggshell particle exhibited an angular pattern of fracture as seen in Fig. 2(a). In contrast with

Fig. 2(b), eggshell membrane particle appeared as rodlike brous fractures (Yang et al., 2003). 3.3. Chemical characterization of the eggshell samples The results of ultimate analysis of eggshell and eggshell membrane particles in this study are given in Table 2. The elemental contents (including C, H, N and S) of eggshell membrane sample are obviously larger than those of eggshell sample. It is reasonable that the composition of bers in eggshell membrane has been determined to be approximately 95% protein with a small amount of polysaccharide (Parsons, 1982). On the other hand, the residual contents (i.e., H, N and S) of eggshell sample are relatively low, showing that the eggshell particle should be almost composed of carbonate minerals (e.g., calcite). The observation described above was also demonstrated by the FTIR spectra, shown in Fig. 3. Fig. 3 shows the most signicant peak of intensity of eggshell particle at 1417 cm1, strongly associated with the presence of carbonate minerals within the eggshell matrix (Dean, 1987). There are also two observable peaks at about 712 cm1 and 875 cm1, respectively, which should be associated with the in-plane deformation and out-plane deformation modes, respectively, in the presence of calcium carbonate (Busca and Resini, 2000). On the other hand, the presences of amines and amides in the eggshell membrane particle were found in Fig. 3 (Yang et al., 2003), exhibiting signicant peaks at intensity of 32003500, 1651, 1538 and 1384 cm1, respectively (Busca and Resini, 2000). Therefore, the

Fig. 2. SEM photographs (1000) of (a) eggshell, and (b) eggshell membrane particles.

Table 2 Elemental analyses of eggshell and eggshell membrane particles Sample Eggshell Eggshell membrane C (wt.%) 13.09 0.00 47.50 0.07 H (wt.%) 0.35 0.01 6.78 0.08 N (wt.%) 0.54 0.01 15.34 0.06 O (wt.%) 29.46 0.08 12.03 0.04 S (wt.%) 0.03 0.00 3.00 0.04

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Fig. 3. FTIR spectra of eggshell and eggshell membrane particles.

eggshell membrane particle would be expected to contain positively charged functional groups such as NH and CON+H2, which are dependent on the 3 pH of the aqueous solution (Koumanova et al., 2002). 3.4. Adsorption of methylene blue Methylene blue (i.e., basic blue 9) was chosen as adsorbate to evaluate the adsorpability and eectiveness of the resulting eggshell particles. To facilitate the estimation of adsorption capacities at various liquid-phase concentrations of methylene blue, the two well-known equilibrium adsorption isotherm models (Al Duri, 1996), Langmuir and Freundlich, were employed as follows: Langmuir : qe qm K L C e =1 K L C e Freundlich : qe
1=n K FCe

can be determined by its slope and intercept. In Eq. (2), KF is a constant for the system, related to the bonding energy. KF can be dened as adsorption or distribution coecient and represents the quantity of dye adsorbed onto adsorbents for a unit equilibrium concentration (i.e., Ce = 1 mg dm3). The slope 1/n, ranging between 0 and 1, is a measure for the adsorption intensity or surface heterogeneity. A plot of ln qe vs. ln Ce enables the empirical constants KF and 1/n to be determined by the intercept and slope of the linear regression. Table 3 presents the results of Langmuir and Freundlich isotherm ts by using the adsorption capacity data at 25 C. Obviously, it can be seen that the Freundlich model yields a somewhat better t than the Langmuir model, as reected with correlation coecients (R2) of 0.939 vs. 0.794 and 0898 vs. 0.715. The adsorption isotherms revealed the eggshell and eggshell membrane particles can only uptake 0.80 and 0.24 mg g1, respectively, in relatively low concentration of the basic dye in aqueous medium, which is indicative of their poor pore properties in comparison to adsorbents for methylene blue (Banat et al., 2003). As also illustrated in Table 3, the value of 1/n is less than 1.0 that indicates a slight favorable adsorption isotherm.

4. Conclusions The hen eggshell and eggshell membrane particles were prepared and analyzed to characterize their physical and chemical properties. The resulting solids were further examined in the adsorption of basic dye (i.e., methylene blue) from aqueous solution. The following conclusions can be drawn: Based on the BET surface area and total pore volume, the pore properties of the two biomaterials are similar each other. However, the true density of eggshell is signicantly larger than that of eggshell membrane, implying their signicant dierences in the textural composition. From the nitrogen adsorptiondesorption isotherm, the pore structures of the two biomaterials should belong to a typical Type II, which is clearly indicative of the nonporous and/or macropores nature.

1 2

In Eq. (1), Ce and qe are the concentration (mg dm3) and amount (mg g1) of methylene blue adsorbed at equilibrium, respectively, KL is a direct measure for the intensity of the adsorption process (dm3 mg1), and qm is a constant related to the area occupied by a monolayer of absorbate, reecting the adsorption capacity (mg g1). From a plot of 1/qe vs. 1/Ce, qm and KL

Table 3 Parameters in Langmuir and Freundlich adsorption isotherm models of methylene blue onto eggshell and eggshell membrane particles at 25 Ca Sample Langmuir qm (mg g1) Eggshell Eggshell membrane 0.80 0.24 KL (dm3 mg1) 0.35 0.97 R2 0.898 0.745 Freundlich KF (mg g1 (dm3 mg1)1/n) 0.18 0.10 1/n () 0.80 0.47 R2 0.939 0.794

a Batch adsorption conditions: initial concentration = 110 mg dm3, adsorbent dosage = 0.1 g; 0.05 dm3, agitation speed = 110 rpm, initial pH = 7.0, and temperature = 25 C.

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By the observations in the FTIR spectra, the chemical composition of resulting eggshell particle is strongly associated with the presence of carbonate minerals within the eggshell matrix. In contrast to the resulting eggshell membrane particle, the presences of amines and amides would be expected to contain positively charged functional groups such as NH and CO 3 N+H2, depending on the pH of the aqueous solution. From the isotherm data of methylene blue at 25 C, the Freundlich model yielded a somewhat better t than the Langmuir model. The adsorption isotherms revealed the two eggshell biosorbents can only uptake the basic dye of less than 1.0 mg/g in aqueous medium, as reected with their poor pore properties.

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