Extraction of chitosan
Chitosan was extracted from 1 Kg waste residues of Parapaeneopsis stylifera (Fig. 3 & Fig. 4). Chitin obtained after the chemical process of demineralization and deproteinisation was further deacetylated to extract chitosan. In this process, 330 g of dried residues were used to extract about 9.0 g dry-1 weight chitosan. Quality test with acetic acid also indicated that chitosan was readily soluble in 1% acetic acid.
Determination of degree of deacetylation
Present study indicated that different concentrations of NaOH and demineralization with hydrochloric acid and acetic acid influenced the yield of the extraction process. It was also proved that the methods used also had an effect on the degree of deacetylation (Table 2) and to confirm that the biopolymer was chitosan, the product obtained was characterized and compared by infrared spectrometry. The degree of deacetylation of chitosan was calculated from its FT-IR spectra using the absorbance bands of 1320 and 1420. The degree of acetylation of the extracted sample chitosan is found to be 19.27, which is calculated from the Fourier Transform Infrared Spectrograph of the extracted sample. This degree of acetylation indicates that the purity of the extracted sample chitosan which is represented by its degree of deacetylation as 89.91%. The reference standard used for the whole process showed a degree of acetylation of about 11.91 and degree of deacetylation of 81.27%. The prepared chitosan nanoparticle exhibited degree of acetylation and decetylation as 9.57 and 90.42% respectively (Table 2). Hence the degree of deacetylation of extracted chitosan and chitosan nanoparticle were found to be 89.91% and 90.42% compared to 81.27% of commercial chitosan as determined by elemental analysis.
Table 2. Percentage DDA of chitosan samples
Samples
|
Absorbtion band
|
transmittance
|
absorbance
|
%DDA
|
Commercial chitosan
|
1320
|
98.1
|
0.0083
|
81.27
|
1420
|
99.5
|
0.0022
|
Sample chitosan
|
1320
|
98.2
|
0.0079
|
89.91
|
1420
|
99.7
|
0.0013
|
Chitosan nanoparticle
|
1320
|
98.2
|
0.0079
|
90.42
|
1420
|
99.4
|
0.0026
|
Chitosan nanoparticles
At the initial stage of the experiment, little amount of CNPs in the solution and the system showed the property as a clear solution. Gradually the amount of CNPs in the solution increased and the system changed from a clear solution to an opalescent emulsion, indicating the formation of CNPs (Fig. 5). Preparation of chitosan nanoparticles is based on an ionic gelation interaction between positively charged chitosan and negatively charged tripolyphosphate at room temperature. Interaction can be controlled by the charge density of TPP and chitosan, which is dependent on the pH of the solution and ultra-sonication time. There are many factors have effect on the size of chitosan nanoparticles such as chitosan concentration, sodium tri polyphosphate concentration, pH of solution and ultrasonication time. In the present study, kept the pH of solution and ultrasoincation time constant at pH 5.5 and 45 min respectively and changing the concentration of both chitosan and TPP. FESEM images of the biosynthesized nanoparticles are displayed in Fig.6. Spherical nanoparticles are seen that appear to be well separated and stable over the steps of the preparation process. FT-IR spectra of pure chitosan, reaction solution of chitosan with including CNPs are showed in Fig 5. Band at 3448.61 cm-1 corresponds to the combined peak of the NH2 and OH groups stretching vibration in chitosan. For reaction solution, the intensities of amide bands at 1560.77 cm-1, which also observed clearly in pure chitosan and decrease dramatically. One new absorption band at 1629.68 cm-1, which can be assigned to the absorption peak of the NH3+ absorption of chitosan is also observed.
Yield of chitosan and chitosan nanoparticle
The yield of chitosan was obtained by comparing the weight of the raw material to the weight of chitosan, which was obtained after the treatment, the weight of the dry shells taken for chitosan extraction was 780gm. After the process of demineralization, deproteinisation and deacetylation obtained 8.89 g of chitosan. The yield of chitosan nanoparticle was also calculated with the following equation.
Screening of antibacterial activity
The antibacterial screening of CNPs and extracted chitosan (1 mg/ml) were performed on pathogenic bacterial strains of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, Klebsiella pneumonia and Escherichia coli. The results indicated that CNPs inhibited most of the bacterial isolates to various degrees in agar disc diffusion method. According to the obtained results, Gram +ve strains exhibited most sensitivity compared to other Gram -ve isolates. However, most of the Gram -ve strains, did not exhibit a higher range of inhibition. The standard chitosan produced an inhibition zone of 12 mm towards Gram -ve Pseudomonas aeruginosa, 8 mm towards Klebsiella pneumonia and 9 mm towards Escherichia coli. The moderate level of antibacterial activity was produced by Gram -ve Klebsiella pneumonia against standard chitosan. It was also noted that among the Gram -ve bacteria, not all the target strains tested were equally susceptible to the antimicrobial metabolites against extracted chitosan and CNPs. Both Gram-positive and Gram-negative bacteria were susceptible to chitosan and chitosan nanoparticle greatly when compared with cefotaxime (30mg, USP). The most susceptible organisms was Gram +ve Bacillus subtilis against CNPs and the inhibition zone recorded was 23 mm (Table 3). It was also found that the inhibition zone size obtained by the disc containing chitosan powder solution around the bacteria is low compared to the disc containing biosynthesized CNPs. Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli was found to be inhibited by chitosan. It was also observed that extracted chitosan, which had a higher degree of deacetylation showed enhanced antimicrobial activity. The study highlights the need for standardized methods to be used in evaluating chitosan’s antimicrobial properties in future studies. Antibacterial screening of commercial antibiotics are depicted in Table 4.
Table 3 Results of Antibacterial screening
Samples
|
Bacterial pathogens (Inhibition zone in mm)
|
Staphylococcus aureus
|
Pseudomonas aeruginosa
|
Bacillus subtilis
|
Klebsiella pneumonia
|
Escherichia coli
|
Standard chitosan
|
10
|
12
|
11
|
8
|
9
|
Extracted chitosan
|
13
|
19
|
20
|
15
|
13
|
Chitosan nanoparticle
|
16
|
20
|
23
|
17
|
14
|
Table 4. Antibacterial screening of commercial antibiotics
Test microorganism
|
Cefotaxime (30 mg), usp
|
Ampicilin (10 mcg)
|
Staphylococcus Aureus
|
16
|
11
|
Pseudomonas aeruginosa
|
14
|
-
|
Bacillus Subtilis
|
18
|
-
|
Klebsiella pneumonia
|
14
|
-
|
Escherichia coli
|
15
|
12
|
Determination of micro algal flocculation efficiencies
Table 5 to Table 9 showing the flocculation efficiencies (calculated using OD 550) microalgae such as Pavlova lutheri, Nannochloropsis salina, Nannochloropsis oculata, Chlorella marina and Chlorella pyrenoidosa in different pH. The results indicated that the flocculation rate of chitosan depended both on the pH as well as the concentration of chitosan. Freshwater Chlorella, Chlorella marina and Pavlova lutheri showed maximum flocculation rate of 89%, 71% and 84% at pH 4.0 at a chitosan concentration level of 1% respectively. While Nannochloropsis salina showed maximum flocculation of 83% (pH 4) at a chitosan concentration of 4%. Nannochloropsis oculata exhibited maximum flocculation rate of 99% (pH 9.0) at the chitosan concentration of 1%. Among the microalgae Nannochloropsis oculata showed higher flocculation rate compared to other species.
The marine algae, Chlorella marina showed comparatively lower flocculation rate. Flocculation efficiency was not high for acidic pH values nevertheless; a great recovery was obtained at alkaline pH values. When pH value exceeds 9, turbidity of culture increases. In alkaline solutions, chitosan is able to produce large and dense flocks. However, in acidic solutions, it produces dispersed and small flocks. The results of flocculation tests at pH 4 in Nannochloropsis oculata (acidic solution) showed relatively low separation. The low separation efficiency is due to the changes in conformation of polymer chains. Relatively high results (99%) of flocculation at pH 9 also noted in the case of Nannochloropsis oculata. The results indicate that, given enough time, flocculation in alkaline solutions reaches the same figure regardless of the presence of flocculants. Therefore, pH 7 was the ideal solution for further analysis on the performance of chitosan. It was found that the flocculation rate of chitosan depended both on the pH as well as the concentration of chitosan. The results of conducted experiments also indicated the superior potential of magnetic chitosan in comparison to pure chitosan in separating microalgae cells from algal culture in a relatively shorter time. The flocculant dosage and solution pH proved to have significant effects on flocculation process. Thus, adjusting pH and dosage plays a major role in separation optimization. Practical application of chitosan as a water treatment coagulant is examined in the study presented here. From these results, it is suggested that chitosan aided flocculation could be used as an effective method with immense potential in water quality management.
Table 5. Rate of flocculation of freshwater Chlorella
Table 6. Rate of flocculation of Chlorella marina
Table 7. Rate of flocculation efficiency of Nanochloropsis salina
Table 8. Rate of flocculation efficiency of Nanochloropsis occulata
Table 9. Rate of flocculation efficiency of Pavalova lutheri
FT-IR spectroscopy
All the Infrared spectra were plotted on all specimen over the frequency range 4000-400cm-1 at resolution of 4cm-1. Degree of deacetylation (DDA) of chitosan was estimated with the produced spectra of sample. Properties of chitosan to a varying extent are strongly dependent on degree of N- deacetylation of chitin. The FTIR spectra of commercial chitosan are presented in Fig. 7. The graph represents wave number (cm-1) along the x-axis and percentage transmittance along y-axis and has several absoption bands. The graph shows the absoption band at 3450 cm-1 (–OH stretching) because OH has highly intense absorption band and at about 2870 cm-1 for the –C–H stretching because the intensity of peak is significant and the band does not involve in hydrogen bond. It also has CH2 bending at 1420cm-1 as well as the abortion band at about 1660cm-1 for the C=O in amide group (amide1 band). Rather than these bands, it shows absorption bands at 1030cm-1and 1070cm-1[C-O stretching]. The absorption bands also occur in 897cm-1 (glycoside linkage) and at 1660cm-1(asymetric C-O stretching). It also contains NH group- stretching vibration at about 3360cm-1and 1380 absorption band for the CH3 in amide group. The absorption band at about 1660cm-1 for C=O in amide group (amide 1 band) and the absorption band at about 1730cm-1 for carbonyl group vibration. The peaks of absorption band at about 1275cm-1for carbonyl group vibration. Another absorption band occurred at 1030cm-1 for C-O stretching.
Many peaks of chitosan were observed which shows a broad-OH stretching absorption band between 3450cm-1. Another major absorption band is between1220 and 1020 cm-1 which represents the free amino group (-NH2) at C2 position of glucosamine, a major peak present in chitosan. Peak at 1384 cm-1 represents the –C-O stretching of primary alcoholic group. Symmetric or asymmetric CH2 in both absorption bands at about 2920 and 2880cm-1. In the case of chitosan, TPP nanoparticles the tip of the peak of 3438 cm–1 has a shift to 3320 cm– 1 and becomes wider with increased relative intensity indicating an enhancement of hydrogen bonding. In nano particles, the peaks for N-H bending vibration of amine І at 1600 cm–1and amide ІІ carbonyl stretch at 1650cm–1 shifted to 1540 cm–1 and 1630 cm–1. The cross linked chitosan also showed a P=O peak at 1170 cm–1. These results also attributed to the linkage between phosphoric and ammonium ion. It is also noted that the tripolyphosphoric groups of TPP are linked with ammonium groups of chitosan. The terminal phosphate group of TPP binds with amine (NH2) group of chitosan by ionic bond. In chitosan nanoparticles the peak was shifted to 1564.03cm-1 due to the wagging of NH2 bond. The ionic interaction with the phosphate group of TPP indicated the conversion of chitosan polymer in the nano form, that forms a cross link with TPP. The strong and sharp peak of phosphate at 1092cm-1 in chitosan nanoparticles confirmed the involvement of TPP while making the nanoparticles.
In the FTIR spectra of chitosan nanoparticles, a strong wide peak in the 3500-3300 area shows hydrogen bonded (O-H) stretching vibrations. The peak for asymmetric stretching C-O-C was found at 1150cm-1. In the spectra, the tip of the peak of 3488 cm-1 has a shift to 3320 cm-1 and becomes wider with increased relative intensity showed enhancement of H-bonding. The cross linked chitosan also shows a P=O peak at 1170cm-1. It is because of the linkage between phosphoric and ammonium ions. So we can understand that the tripolyphosphoric groups of TPP are linked with ammonium groups of chitosan. In the FTIR spectra of chitosan the C-N stretching is shown between 1250-1375cm-1. But in C-N, the peak shifted to 1564 cm-1 due to wagging of NH2 bond. The strong and sharp peak of phosphate at 1092 cm-1 confirmed the involvement of TPP while making nanoparticle. The presence of P=O and P-O groups are at a frequency of 1180 cm-1. Results of preliminary investigations on the experimental conditions for the formation of chitosan nanoparticles showed that nanoparticles could be obtained by varying the concentrations of chitosan with concentration of TPP respectively (Fig. 5).
Field Emission Scanning electron microscopic (FE-SEM) studies of chitosan nanoparticles
The FE-SEM micrograph details of the pure chitosan nanoparticles was represented in Fig. 8. The pure chitosan texture is plain without pores having smooth, compact and homogeneous even surface structure with no gross effects, while the SEM micrograph of chitosan nanoparticles. Figure 6 revealed the rough surface morphology, with a solid dense cubical or rectangular structure and not aggregated. The spheres exhibited mean diameters around 500 nm. The nanoparticles dry powder consists of individual nanoparticles, which touched each other, but retained their original size and shape. The size variation also related to different conditions of sample preparation for SEM. The synthesized nanoparticles were found tobe nearly spherical shape with size in the range of 135-729 nm. The synthesized nanoparticles showed potent antibacterial activity against Gram positive and Gram negative bacteria and the results revealed that natural sources of materials such as shell wastes could be used for preparation of CNPs instead of use of chemical substances.
SEM micrograph of pure chitosan revealed that the texture is plain without pores having smooth, compact and homogeneous even surface structure with no gross effects. The SEM micrograph of chitosan nanoparticles (Fig. 6) revealed the rough surface morphology, has a solid dense cubical or rectangular structure and not aggregated. The spheres showed mean diameters around 500 nm. Nanoparticles dry powder consists of individual nanoparticles, which touched each other, but retained their original size and shape. The size variation also related to different conditions of sample preparation for SEM.