Ultrasound-assisted treatment of chitin: evaluation of physicochemical characteristics and dye removal potential

2.1 Chitin obtainment

The shrimp (Penaeus brasiliensis) wastes were obtained from a fishery industry located in southern Brazil and stored in freezer (-18°C) until its use. Chitin was obtained from shrimp wastes by the following steps (15): demineralization (7 kg of shrimp wastes and 14 l of HCl solution 2.5 g l^-1 in stirred tank during 2 h at room temperature), deproteinization (21 l of NaOH solution 5.0 g l^-1 in stirred tank during 2 h at room temperature) and deodorization (35 l of NaClO solution 0.4 g l^-1 under agitation during 3 h). After each above step, washing was done with water to remove the remaining solutions. Finally, chitin was dried (16), ground (Wiley Mill Standard Model 03, Thomas Scientific, Swedesboro, USA) and sieved until the discrete particle size ranging from 425 to 850 μm.

2.2 Ultrasound-assisted treatment of chitin

Chitin samples (5.00 g) were mixed with 300 ml of deionized water. The mixture (water/chitin) was treated in an ultrasonic processor (UP400S, Hielscher, Teltow, Germany) of 400 W equiped with a titanium sonotrode during 1 h at 24 kHz. The ultrasound-assisted treatment of chitin was performed under different ultrasound cycles (0.50, 0.75 and 1.00) (for example, considering 1 min of treatment, when the cycle is 0.50, the suspension remained under ultrasound during 0.50 min) and amplitudes (20, 60 and 100%) without temperature control. After the ultrasonic treatment, the chitin slurry was separated by filtration, oven dried (40°C, 24 h) and stored for further characterization and use. These conditions were determined by preliminary tests. All experiments were performed in replicate (n=3)

2.3 Determination of chitin characteristics

Chitin, before and after the ultrasonic treatments, was characterized according to the moisture content, ashes content (17), Sauter diameter (D_Sauter), deacetylation degree (DD), crystallinity index (CI), specific surface area (S_BET) and scanning electron microscopy (SEM).

The chitin grain-size analysis was carried out in a standardized mesh screen. The average diameter was calculated by (18):

where, D_Sauter is the average diameter of Sauter (μm), D_mi is the arithmetic average diameter between two screens (μm) and X_i is weight fraction of particle size D_mi (%).

The identification of the chitin characteristic functional groups was performed by Fourier transform infrared spectroscopy (FTIR) (Shimadzu, Prestige 21, Kyoto, Japan), using the technique of diffuse reflectance in potassium bromide (19). From the absorbance of amide I band (A_C=O) at 1655 cm^-1 and hydroxyl groups (A_–OH) at 3450 cm^-1, the deacetylation degree (DD, %) was determined by (20):

The amorphous/crystalline structure of chitin was assessed by X-ray powder diffractometry (XRD). Chitin samples were analyzed in a diffractometer (Rigaku, Miniflex 300, Tokyo, Japan) equipped with Ni–filtered Cu K<alpha> radiation (λ=1.54051 Å), with 2θ=5–60°. The diffractometer was operated with 1° diverging and receiving slits at 30 kV and 10 mA. The crystallinity index (CI) was calculated from the normalized diffractograms after mathematical treatment of the peaks, corresponding to its deconvolution and application of the Lorentzian function. The intensities of the peaks at 110 lattices (I₁₁₀, at 2θ≈20° corresponding to maximum intensity) and at 2θ≈16° (amorphous diffraction, I_am) were used to calculate CI, as follows (21):

The specific surface area (S_BET) was determined in a adsorption analyzer (Micromeritcs, ASAP 2020, Norcross, USA) at 77 K. Samples were pre-treated at 473 K for 24 h under a nitrogen atmosphere in order to eliminate the moisture adsorbed on the solid sample surface. The samples were then submitted to 298 K in vacuum, reaching the residual pressure of 10^-4 Pa. The multi-point BET method was used to estimate the specific surface area (S_BET) (22).

Scanning electron microscopy (SEM) was carried out to verify the possible structural, morphological and textural changes, which were caused by ultrasound-assisted treatment. The images were obtained at 15 kV with magnification of 300× (Jeol, JSM-6060, Tokyo, Japan) (23).

2.4 Evaluation of dye removal potential

The dye removal potential of chitin (untreated and treated in all ultrasonic conditions) was evaluated using batch adsorption experiments with MB and P4R dyes. The cationic dye, MB (color index 52015, molar weight of 319.8 g mol^-1, λ_max=664 nm) and the anionic dye P4R (color index 16255, molar weight of 604.5 g mol^-1, λ_max=505 nm) were purchased from Plury Chemical Ltd. (Diadema, Brazil). All other reagents utilized were of analytical grade (Vetec, Duque de Caxias, Brazil). Deionized water was used to prepare all solutions.

The batch adsorption experiments were performed in a thermostated agitator (Marconi, MA 093, Piracicaba, Brazil) as follows: 250 mg of chitin (untreated and treated in all ultrasonic conditions) were added in Erlenmeyer flasks containing 50 ml of solution with initial dye concentration of 50 mg l^-1. The flasks were stirred at 200 rpm for 120 min at 303 K [these experimental conditions were determined by preliminary tests and the literature (3–7)]. The initial pH of dye solutions was not adjusted, being 7.00 for MB and 6.00 for P4R. After the experiments, the solid phase was separated by filtration (Whatmann no 40) (the amount of dye retained in the filter was considered for the calculations) and the remaining dye concentration in the liquid phase was measured by a spectrophotometer (Shimadzu, UV1800, Kyoto, Japan) at 664 nm for MB and 505 nm for P4R. The experiments were performed in replicate (n=3) and blanks were realized. The dye removal percentage (R, %) and adsorption capacity (q, mg g^-1) were determined as follows:

where, C₀ is the initial dye concentration in liquid phase (mg l^-1), C_f is the final dye concentration in liquid phase (mg l^-1), m is the chitin amount (g) and V is the volume of solution (L).

3.1 Physicochemical characteristics of chitin

In this study, chitin was ultrasonically treated to improve its dye adsorption characteristics. It is know that the dye adsorption potential of chitin/chitosan is dependent of its physicochemical characteristics, such as, moisture content, ashes content (24), Sauter diameter (25) deacetylation degree (26), crystallinity index (27) and specific surface area (24). In this way, the effects of ultrasound cycle (0.50, 0.75 and 1.00) and amplitude (20, 60 and 100%) on the above mentioned characteristics were evaluated.

The chitin moisture content before and after all ultrasonic treatments was 6.0±1.2% (wet basis). The ashes content decreased from 4.8±1.0% (untreated chitin) to 0.5±0.1% (ultrasonic treated chitin). This can be occurred because chitin was exposed to high frequency mechanical waves during the ultrasound-assisted treatment. These waves can have caused the leaching of inorganic salts and minerals, decreasing the chitin ashes content. For comparison, the commercial α-chitin has moisture content of 5.8% (wet basis) and ashes content of 2.4% (28). These results revealed that the ultrasonic treatment provides a highly pure chitin.

Figure 1 shows the influence of ultrasonic treatments on the chitin Sauter diameter. It can be seen in Figure 1 that chitin D_Sauter decreased from 625 to 250 μm after the ultrasonic treatment and this decrease was independent of the ultrasonic condition (cycle or amplitude). This result is important from the dyes adsorption viewpoint, since, lower D_Sauter values provide higher free surface area and make the adsorption sites more accessible. This phenomenon can be explained according to the “hot spot” theory, i.e. cavities are formed on the liquid, and the implosion of these cavities releases liquid jets at high velocities, fragmenting the chitin particles (29, 30). Jambrak et al. (31) verified that a significant particle size decrease occurred during the ultrasound treatment of whey proteins.

Figure 1:

Influence of ultrasonic treatment on the chitin Sauter diameter (D_Sauter).

FTIR was used to identify the chitin characteristic functional groups and to estimate the deacetylation degree (DD), before and after the ultrasound-assisted treatment. Figure 2 shows the FTIR vibrational spectrums of (A) untreated chitin and (B) ultrasonic treated chitin (cycle 1.0 and amplitude 60%) (the spectrums after ultrasonic treatment were similar for all conditions of cycle and amplitude). For untreated chitin (Figure 2A), the OH stretching vibration can be observed in a broad band centered at 3450 cm^-1. The NH stretching vibrations can be verified at 3250 and 3100 cm^-1. The C-H stretching band around 2850 cm^-1 was observed. The bands of 1650 and 1550 cm^-1 can be assigned to the amide I (vibration of carbonyl C=O stretching of amide) and amide II (N-H deformation of amide II), respectively. The CH_X deformation can be noted at 1450 cm^-1. The bands in the region 1250–950 cm^-1 can be attributed to the C-O and C-O-C bonds (20). After the ultrasonic treatment (Figure 2B), some bands were slightly modified. This indicated that modifications and rearrangements on the chitin polymeric chains can be occurred during the ultrasound assisted treatment. Chitin DD was not influenced by the ultrasound treatment, remaining in the range of 42.3–45.8%.

Figure 2:

FTIR vibrational spectrums of (A) untreated chitin and (B) ultrasonic treated chitin.

The CI reduction can be visualized in Figure 3A and B. In Figure 3A (untreated chitin), the diffraction planes at (020) (2θ≈9–10°) and (110) (2θ≈20°) of the shrimp type of α-form orthorhombic crystal structure can be visualized. However, for ultrasonic treated chitin (cycle 1.0 and amplitude 60%) (Figure 3B), the diffraction planes were less intense, confirming that chitin was decrystallized. Figure 3C shows the influence of ultrasonic treatment on the chitin crystallinity index (CI). It was found from Figure 3C that, for all ultrasonic conditions, the chitin CI values decreased from 86.3% (untreated chitin) to 70.0%. This CI reduction is probably related to a rearrangement of chitin chains during the ultrasonic treatment. Also, the CI reduction can be attributed to the leaching out of chitin inorganic compounds. Similar trend was verified by Villa-Lerma et al. (32) when used ultrasonication as pretreatment for chitin oligosaccharide production by chitinases. They found, for native chitin, CI of 88.13%, and after the ultrasound treatment, CI decreased to 85.50%. Regarding to the dyes adsorption, low crystalline chitin is preferred, since the accessibility to the internal sites is facilitated (24).

Figure 3:

XRD diffractograms of untreated chitin (A) and ultrasonic treated chitin (B) and (C), influence of ultrasonic treatment on the chitin crystallinity index (CI).

The influence of ultrasonic treatment on the chitin specific surface area (S_BET) is presented in Figure 4. It was found that, at lower amplitudes (20%), the specific surface area was not influenced by the ultrasonic treatment. However, at 60% of amplitude, the S_BET values, increased until 38.5 m² g^-1. A further increase to 100% of amplitude presented no influence in the S_BET values. Thus, the use of ultrasound cycle of 1.0 and amplitude of 60% is sufficient to increase 17.5 times the chitin specific surface area. This behavior can be also attributed to the “hot spot” theory (above mentioned) (29, 30). The increase in S values is essential for dyes adsorption purposes, since facilitates the access of the large dye molecules to the adsorption sites (4, 22 , 24).

Figure 4:

Influence of ultrasonic treatment on the chitin specific surface area (S_BET).

The possible structural, morphological and textural changes caused by the ultrasound-assisted treatment on the chitin surface were assessed by scanning electron microscopy (SEM). SEM images of untreated chitin and ultrasonic treated chitin (cycle 1.0 and amplitude 60%) are show in Figure 5A and B, respectively. As can be seen in Figure 5A, chitin has a smooth, rigid and non porous surface. However, after the ultrasound-assisted treatment (Figure 5B), a rough surface with cavities and protuberances was observed. These cavities and protuberances are desirable for adsorption purposes, since improves the transfer of the dye solution to the inner and lower pores of the adsorbent.

Figure 5:

SEM images of (A) untreated chitin and (B) ultrasonic treated chitin.

In summary, it was verified that the ultrasound assisted treatment provides a highly pure and fine chitin, without changes in deacetylation degree, with lower crystallinity and higher specific surface area. Some important modifications on chitin surface were also observed after the ultrasound assisted treatment, such as, formation of cavities and protuberances. These structural modifications can be favorable for the adsorption processes.

3.2 Dye removal potential of chitin

The dye removal potential of chitin (untreated and treated in all ultrasonic conditions) was evaluated for MB (cationic dye) and P4R (anionic dye) dyes. The results, in terms of dye removal percentage (R, %) and adsorption capacity (q, mg g^-1) are shown in Figure 6.

Figure 6:

Potential of untreated and ultrasonic treated chitin for the adsorption of MB [(A) Dye removal percentage, (B) adsorption capacity] and P4R [(C) Dye removal percentage and (D) adsorption capacity].

In general lines, it can be seen in Figure 6 that the ultrasonic treated chitins were more efficient to remove MB and P4R than untreated chitin. For MB (Figure 6A), the R values increased from 53.2 (untreated chitin) to 74.3% (ultrasonic treated chitin at cycle of 1.0 and amplitude of 60%). In the same way, the q values increased from 5.3 to 7.4 mg g^-1 (Figure 6B). For P4R (Figure 6C), the R values increased from 3.3 (untreated chitin) to 20.2% (ultrasonic treated chitin at cycle of 1.0 and amplitude of 60%). In the same way, the q values increased from 0.3 to 2.0 mg g^-1 (Figure 6D). This behavior can be attributed to the better adsorbent characteristics of ultrasonic treated chitin, such as, higher purity and specific surface area; and lower crystallinity index and Sauter diameter (20–23). Similar trend was found by Küncek and Şener (30) in the adsorption of Methylene Blue (MB) onto sonicated sepiolite. They verified that the adsorption capacity of sepiolite for MB increased from 79.37 to 128.21 mg g^-1 after the sonication.

Comparing Figure 6A and C, it can be seen that chitin is more efficient for the removal MB than P4R. This occurred because the point of zero charge of chitin is 5.4 (experimentally measured). So, chitin is positively charged at pH values lower than 5.4 and negatively charged at pH values higher than 5.4. Since the experiments were made at pH>6.00, electrostatic attraction occurred between MB (cationic dye) and chitin, increasing the R and q values. On the contrary, electrostatic repulsion occurred between P4R (anionic dye) and chitin, decreasing the R and q values. In this work, the maximum values for the chitin adsorption capacity (q) were 7.4 mg g^-1 and 2.0 mg g^-1 for MB and P4R, respectively. These values can be compared with the following systems in the literature: chitin-Methylene blue (2.60 mg g^-1) and chitin-yellow croconate (7.69 mg g^-1) (33); chitin-indigo carmine (5.78 mg g^-1) (34) and chitin-Ponceau S (3.3 mg g^-1) (5). Furthermore, a detailed comparison between the (q) values is presented in Table 1.

3.3 Desorption and reuse

According to the literature (24), desorption and reuse studies are fundamental to verify the possible industrial application of the adsorbents. Desorption experiments were performed for MB and P4R dyes. For MB dye (cationic), the testes were performed using HCl at different concentrations (from 0.1 to 1.0 mol l^-1) and, for P4R dye (anionic), the testes were performed using NaOH at different concentrations (from 0.1 to 1.0 mol l^-1). The fixed conditions were 250 mg of dye loaded chitin (raw and ultrasonic treated in the more adequate condition), 50 ml of solution, 200 rpm, 120 min and 303 K. The results revealed that for raw and modified chitins, the MB desorption was complete using 0.3 mol l^-1 HCl. For P4R, desorption was complete using 0.5 mol l^-1 NaOH. For both dyes, using raw and treated chitins, ten adsorption-desorption cycles were performed. The most promising results were found for treated chitin with MB, where, the adsorbent could be used seven times maintaining the same performance.