In this study, H3PO4 and KOH were used to remove minerals and proteins, instead of the traditionally used HCl and NaOH, respectively. The degree of demineralization (DDM) varied from 37.18% to 94.04% when the concentration of H3PO4 was increased from 10 to 30 g·L-1 (Table 1). Different solid-to-solvent ratios could influence the demineralization of shrimp shells. The DDM was over 90% when 30 g·L-1 H3PO4 with a solid-to-solvent ratio of 1:6 was used for 30 min. The degree of deproteinization (DDP) varied from 52.23% to 85.65% when increasing the concentration of KOH from 10 to 30 g·L-1 (Table 2), however, no significant increase was observed with a concentration above 30 g·L-1.
H3PO4 conc.(g·L-1) Solid/solvent ratio 10 30 50 1:4 1:6 1:8 DDM (%) 37.18 ± 1.36a 94.04 ± 0.52b 94.28 ± 0.36b 68.61 ± 0.19a 94.51 ± 0.53b 94.99 ± 1.31b Different letters indicate statistically significant results (P < 0.05) based on one-way ANOVA with Tukey's test. DDM is the degree of demineralization. Data are expressed as means ± SDs (n = 3)
Table 1. Effects of phosphoric acid concentration and the solid-to-solvent ratio on demineralization of shrimp shells
KOH conc.(g·L-1) Temperature (℃) 10 30 50 50 70 90 DDP (%) 52.23 ± 0.27a 85.65 ± 1.06b 84.10 ± 1.06b 75.61 ± 0.56a 84.58 ± 0.21b 83.87 ± 0.79b Different letters indicate statistically significant results (P < 0.05) based on one-way ANOVA with Tukey's test. DDP is the degree of deproteinization. Data are expressed as means ± SDs (n = 3)
Table 2. Effects of potassium hydroxide concentration and temperature on deproteinization of shrimp shells
It is necessary to remove the minerals (demineralization) and proteins (deproteinization) during the extraction of chitin from shrimp shells (Younes et al. 2014). Many industries extensively use chemicals for demineralization and deproteinization during the extraction process. In general, HCl and NaOH have been efficiently used for demineralization and deproteinization (Al Sagheer et al. 2009; Percot et al. 2003). The DDM was 93.8% with the use of 0.25 mol·L-1 HCl (John et al. 2006), and the DDP reached 80% with the use of 50 g·L-1 NaOH at 90 ℃ for 1 h (Holanda and Netto 2006). However, the liquid waste generated during the process is harmful to the growth of plants due to remaining chlorine in the chitin, thus limiting the further application of chitin and its derivatives as fertilizers.
Compared with earlier studies using HCl and NaOH, no discernable differences of DDM or DDP were observed for the treatment of H3PO4 and KOH in this study. Moreover, the liquid waste, generated during the demineralization and deproteinization using H3PO4 and KOH, was rich in phosphorus, potassium, and nitrogen, which are the main constituents of fertilizers. Process water in this study was totally recycled, which provided an eco-friendly method for the preparation of chitosan and its derivatives.
Microwave-assisted chemical reactions are rapid and efficient. In this study, the DDA of chitosans was enhanced by increased microwave heating time. Chitosan with four different DDAs (63.79%, 72.12%, 79.34%, and 88.15%) were prepared by microwave heating with 450 g·L-1 KOH for 6.0–8.5 min (Table 3). Compared with the traditional method using NaOH, the combination presented was more efficient (Yen et al. 2009; Viarsagh et al. 2010). Microwave heating is an important processing step for preparation of chitosan with specified molecular weight distribution. The molecular weight (17.4 × 105–5.7 × 105 Da) and viscosity of chitosans decreased with increased microwave heating time, indicating that the combination of microwave heating and alkali efficiently destroyed the carbohydrate chain (Bajaj et al. 2011).
Microwave heating time (min) 6 7 8 8.5 Degree of deacetylation (%) 63.79 72.12 79.34 88.15 [η] (mL·g-1) / 754 557 561 Mv (Da) 17.4 × 105 13.6 × 105 7.5 × 105 5.7 × 105
Table 3. The degree of deacetylation, viscosity [η], and molecular weight (Mv) of chitosans under different microwave heating times
According to the FTIR spectrum of chitin and chitosans (Fig. 1), the C=O bands were approximately at around 1626.61 cm-1 and 1660.29 cm-1 and N–H band was at 1557.99 cm-1. The characteristic absorption bands at 3445.62 cm-1 corresponded to OH stretching, while those at 3268.62 cm-1 and 3106.16 cm-1 corresponded to NH stretching. The absorption bands at 1417.07 cm-1 and 1378.51 cm-1 were attributed to CH2 and CH bending, respectively. Similar absorption bands were observed both with chitin (Fig. 1a) and chitosans with different DDA (Fig. 1b–e). Moreover, functional groups of chitosan, such as the C=O and N–H bands, were all presented (Fig. 1b–e).
Figure 1. FTIR spectra of chitin and chitosan with different degrees of deacetylation. a: Chitin; b–e: chitosans with different degrees of deacetylation (63.79%, 72.12%, 79.34%, and 88.15%)
Chitosans with different DDA (63.79%, 72.12%, 79.34%, and 88.15%) were oxidatively degraded with H2O2 to produce COS. HPLC analysis revealed that the molecular weights of the COS were 1559, 1553, 1599, and 1526 Da, respectively.
According to the component analysis results, whiteleg shrimp shell was rich in protein (49.61%), minerals (29.06%), and chitin (21.33%) (Table 4). In summary, 33.73 kg H3PO4, 12.77 kg, and 241.31 kg KOH were supplied during the processes of demineralization, deproteinization, and deacetylation for 100 kg shrimp shell waste, respectively. The whole process created a product with the fractions of N:P2O5:K2O:COS = 7.94:24.44:10.72:18.27. The supply of N, P, and K can also be adjusted according to plant requirements when used in an agricultural setting. Moreover, liquid waste and solid waste generated during the process was totally recycled, making COS valuable as fertilizer. The detailed process flow diagram of COS fertilizer from shrimp shells is presented in Fig. 2.
Components Mass (kg) Components of shrimp shell Shrimp shell 100 Protein 49.61 Mineral 29.06 Chitin 21.33 Input of raw materials H3PO4 (demineralization) 33.73 KOH (deproteinization) 12.77 KOH (deacetylation) 241.31 HAc (dissolved chitosan) 37.54 Water 2355.96 Components of products Chitooligosaccharide 18.27 N 7.94 P2O5 24.44 K2O 10.72 Waste 0
Table 4. The evaluation of input and output in process
Using chitosan oligomer (28 kDa, 78% DDA) to coat zucchini seeds has been shown to shorten germination time and increase germination percentage (Cristóbal et al. 2012). COS (70% DDA) has also been reported to be valuable as an elicitor enhancing the germination of barley seeds during seed priming (Lan et al. 2016). Additionally, Kananont and colleagues (2010) reported that chitosans with 70% and 80% DDA could significantly improve the germination of Dendrobium bigibbum var. compactum and that chitosan with 70% DDA could enhance the germination of Dendrobium formosum. This suggests that the DDA of chitosan may have a major impact on seed germination; however, limited data are available on the relationship between the DDA of COS and its effect on the germination of wheat seeds.
In this study, wheat seeds were soaked in COS with different DDA, and the germination percentage was recorded every day. Compared to the control group, a higher germination rate was observed with seeds soaked in COS (Fig. 3). During the first 24 h, no significant differences in germination percentage were observed among all groups. After 48 h, the highest germination percentage was observed with the treatment of 72.12% DDA, showing a significant difference compared to the control (P < 0.05).
Figure 3. Effects of COS with different degrees of deacetylation on the germination of wheat seeds. UN refers to the seeds treated with distilled water, used as the control. Data are expressed as means ± SDs (n = 3). Different letters indicate statistically significant results (P < 0.05) based on one-way ANOVA with Tukey's test
Germination involves complex physiological and biochemical processes (Bewley 1997). During germination, intrinsic enzymes, such as amylase and protease, are activated after the uptake of water by dry seeds. The low-molecular weight metabolites produced by the hydrolysis of starch and protein participate in respiratory events and provide energy for the seedlings (Müntz et al. 1998; Mohan et al. 2010; Zhu et al. 2017). Changes in enzyme activity and stored biochemical components significantly affect seed vigor (Nandi et al. 1995). In this study, changes in enzyme activity and contents of sugar, starch, and protein were assessed from 0 to 24 h during germination (Fig. 4).
Figure 4. Effect of COS with different degrees of deacetylation on the activity of enzymes and content of biochemical components during the early stages of germination. (a-c) The activities of β-amylase, α-amylase, and protease, respectively; (d-f) the contents of protein, starch, and sugar, respectively. UN refers to the seeds treated with distilled water, used as the control. Data are expressed as means ± SDs (n = 3). Different letters indicate statistically significant results (P < 0.05) based on one-way ANOVA with Tukey's test
During germination, the activity of β-amylase increased in the first 6 h, with its activity approximately 6–10 times higher than that of the α-amylase in the tested samples (Fig. 4a, b). COS with DDA of 63.79% and 72.12% significantly increased the β-amylase activity (P < 0.05), with the highest β-amylase activity (260.35 mg·min-1·g-1) observed in the 72.12% DDA-treated group. After 12 h, the β-amylase activity considerably decreased, which was analogous to an earlier report that the β-amylase activity significantly increased while the α-amylase activity had negligible change when nitric oxide was used as an inducer during the early stages of germination (Zhang et al. 2005). β-Amylase activity is a key indicator of germination potential, while α-amylase influences the speed of seedling growth in later stages of germination (Nandi et al. 1995). β-Amylase activity mainly depends on the release of free β-amylase, while bound β-amylase, which is linked to glutenin with a disulfide bond, has negligible activity (Sopanen and Laurièr 1989). Cysteine-endopeptidases have been reported to liberate the bound β-amylase from glutenin and increase its activity (Guerin et al. 1992). The increase in β-amylase activity in this study may be due to the release of β-amylase by endoprotease. Moreover, COS with different DDA were shown to enhance the protease activity compared to the control (P < 0.05), of which the COS with 72.12% DDA showed the best effect (Fig. 4c).
Soluble sugar, a substrate of the glycolytic pathway, participates in respiratory activity providing adequate energy to support metabolism during germination (Attucci et al. 1991). During the first 12 h, the soluble sugar content decreased. The seeds treated by COS with 72.12% DDA showed the lowest sugar content (Fig. 4e). Metabolism of protein and starch occur during the germination to support seedling growth (Kuraś 1986; Zhao et al. 2018). In this study, sugar consumption was more than the production of sugar by starch hydrolysis during germination. However, there was no regular change in the protein and starch contents during the first 24 h of germination when the seeds were treated with COS (Fig. 4d, f). This might be due to the dynamic balance between the consumption and synthesis of nutrients during early germination.