Biotransformation of Chitin to Chitosan using Bacillus cereus JCM44 isolated from Kanyakumari, India and its Characterization

Bacillus cereus JCM44 was isolated from the soil samples collected from the Kanyakumari district of Tamil Nadu, using a minimal agar medium. It was characterized by its ability to biotransform chitin into chitosan at 30°C for 48 h. The Chitosan (CS) was recovered by using 0.1N Sodium hydroxide and 2% acetic acid. It was confirmed by iodine solution with dark purple colouration and qualitatively identified by acetone and ethanol. CS formation has been identified from X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR). Scanning Electron Microscopy (SEM) analysis elucidated the formation of an average size of 349 nm to 1µm with the morphology of a non-porous, smooth membrane with irregular shape and crystallite. The Zeta potential study of the bacterially synthesized chitosan showed their excellent stability with a surface charge of -2.28 mV. Bacterially Synthesized Chitosan (BSC), with a polydispersity index (PDI) value of 0.626 indicates the aggregation of particles. The peaks of higher wavenumber confirm the formation of blend in Bacterially Synthesized Chitosan (BCS) at OH and NH was denoted. The XRD analysis of the BSC detected the crystallinity of the samples when compared to the commercial chitosan.


INTRODUCTION
Chitosan (CS) is a degradable biopolymer and it's a derivative of chitin. 1 Though, the supply of marine resources is variable, seasonal and very expensive. 2] However, CS is a polysaccharide generated by the deacetylation of chitin, using harsh chemicals may decrease the quality of CS and high in its processing rate.][9] Fermentation technologies have been used, recently for culturing organisms that contain CS.This approach could be an attractive method for the production of biopolymer CS.In most fungi, CS is found in the outer surface (cell wall), where it likely shows an essential part as a glycan.Chitin deacetylase (CDA) has a role in biotransformation of chitin to chitosan by the hydrolysis of acetamido group. 10Some of the investigators studied the production and the isolation of CS from microbes broadly as an alternative to marine shell wastederived product, as in Aspergillus sp. 11Most of the fungal strains have low CDA-producing capacity and their fermentation requirements are much more difficult.Therefore, new appropriate potential strains of bacteria which produce CDA should be required to replace the fungal strains.Numerous bacterial species which produce CS were reported early, including Bacillus thermoleovora. 12he problems of chemical processing s h o u l d b e t ra n sfo r m e d u s i n g va r i o u s microorganisms, [13][14][15] and proteolytic enzymes [16][17] used to remove the proteins and mineral content.The production of microbial enzymes either in pure or partially purified form is an important aspect of bio-based industries.The applications of microbial enzymes in the industry are very diverse. 18Therefore, the use of specific microbial CDAs to catalyze the conversion of chitin to CS was considered interesting due to certain advantages over chemical reactions.In microbial fermentation, deproteinization follows through the activity of microbial proteases and demineralization by the acid produced by microorganisms during fermentation. 19In this present study, CS was produced by fermentation using Bacillus cereus JCM44 and the characteristics of synthesized CS were analyzed.

Materials
Chitosan (Shrimp shell) with the deacetylation degree of ≥ 75%, chitin (CAS 1398-61-4, degree of deacetylation: 65-70%) and all the chemicals with analytical grade were purchased and used from Himedia Laboratories Pvt. Ltd.The purchased chemicals were used as it is without any further purification.

Isolation of CDA Producing Bacteria
Coastal soil samples of 5 to 10 cm depth were collected from different locations in Kanyakumari district, Tamil Nadu, India.One gram of soil sample was taken and dissolved in 9mL of sterile distilled water and serially diluted up to 10 -9 .The Minimal medium (MM) containing (w/v) 1% chitin was added as a sole source of carbon, 0.2% Sodium Nitrate (NaNO 3 ), 0.1% Dipotassium hydrogen Phosphate (K 2 HPO 4 ), 0.1% Potassium Dihydrogen Phosphate (KH 2 PO 4 ), 0.05% Magnesium Sulphate (MgSO 4 ) and 2% Agar were used for the isolation of bacterial isolates and incubated at 35°C for 24 to 48 h. 20,21sing a diagnostic strip test, the bacterial isolates producing CDA were screened for CDA activity.A potent bacterial isolate (JCM44) was selected based on quantitative screening.Physiological and biochemical tests of JCM44 like catalase, oxidase, indole, Methyl Red-Voges Proskauer (MR-VP) and citrate utilization tests were performed.16S rDNA sequencing was done by 27F (5'-AGAGTTTGATCMTGGCTCAG -3') and 1492R (5'-TACGGYTACCTTGTTACGACTT -3'). 22The PCR product was purified, sequenced and submitted to National center for Biotechnology Information (NCBI).An accession number was obtained.

Chitin to CS-Biotransformation (JCM44)
For the biotransformation of Chitin to CS the medium containing (1g of yeast extract, 0.4g of Ammonium Sulfate (NH 4 ) 2 SO 4 , 0.15g of KH 2 PO 4 with a pH of 8.0 and chitin (50 mg) was added along with it.The medium was taken in a conical flask (250 mL) and (1%) inoculum of the positive isolate JCM44 was added along with it and one of the flasks without inoculum was taken as control.The flasks were incubated for 2 days.Flasks were taken after incubation for CS recovery. 23

CS Recovery
The incubated flasks were taken for CS recovery.The broth (fermented) was taken and centrifuged (12000 rpm) for 15 min.The pellet attain was taken and 10mL of (0.1N) sodium hydroxide was added along with it.After the content was mixed, it was autoclaved for 15 min.The tubes were taken and allowed for cooling.In the alkaline action, all the cell debris got solubilized.Again the tubes were centrifuged (12000 rpm) for 15 min and the pellet was collected.Acetic acid (2% 10 mL) was added to the pellet and the pellet was left overnight at room temperature for the solubilization of CS.Again the tubes were centrifuged (12000 rpm) for 15 min the very next day.The pellet was collected and (1N) sodium hydroxide was added along with it.A white precipitate was formed which shows the presence of CS. 21

Qualitative Estimation of CS
Centrifuged (5000 rpm) the precipitate again for 15 min.and washed using distilled water twice to attain pH 7. Again the precipitate was resuspended in 0.5mL dis.H 2 O and it was kept for drying at 55°C for 2 to 4 h.The confirmatory test was done to the attained precipitate.To confirm the precipitate as CS, iodine solution (a few drops) was added to the precipitate, it turns the precipitate dark brown and to acidify, 1% Sulphuric Acid (a few drops) was added along with it.It changes its colour from dark brown to purple, which demonstrates the presence of CS. 21

Quantitative Estimation of CS
The clean Petri plates were taken and weighed.The precipitated CS was washed thoroughly with distilled water (pH 7) and resuspended in 1 mL of dis.H 2 O (pH 7).1mL of chitosan suspension was poured on the clean Petri plates and kept for drying at 55°C for (2-6) h.The precipitate CS was weighted after drying. 21

Characterization of Bacterially Synthesized Chitosan (BSC) Scanning Electron Microscope (SEM)
The extracted BSC was analyzed with Scanning Electron Microscope (SEM) (Jeol JSM-  Model SEM 6390, Japan).The dried samples of BSC images were observed and compared with the commercial chitosan at 10,000x magnification at an accelerated voltage of 20 kV.

Dynamic Light Scattering (DLS)
Zeta potential and particle size were measured for BSC, (Zetasizer, Nano ZS, Malvern Instruments Ltd.,) based on a DLS technique.The stability of the BSC was measured at a stable measurement temperature of 25°C, and a scattering angle of 90°.Photon Correlation Spectroscopy (PCS) was used for the size evaluation of BSC.

The Polydispersity Index (PDI)
The homogeneity dispersion of BSC is a measure of PDI (0 to 1).The homogeneity is shown by values closer to 0 (zero) and non-uniformity values (> 0.5).

X-ray Diffraction Method (XRD)
XRD data of the diffraction pattern of BSC was obtained with Shimadzu-model XRD 6000, operated at a voltage of 40 kV and a current of 30 mA with Cu, Kα radiation in a 2θ configuration from 10-60°.

Fourier-transform Infrared Spectroscopy (FT-IR) Analysis
The IR spectra of BSC were analyzed with FT-IR Spectrophotometer, Perkin Elmer Make-Model spectrum, RX over the frequency series of 4000 -400 cm −1 .The results were compared to commercial (Shrimp shell) chitosan.

Isolation and Identification
In this study, the highest CDA-producing bacterial isolate JCM44 Figure 1a.The morphology of strain JCM44 was milky-white with a smooth surface, rod and Gram-positive.CDA-producing bacterial isolate JCM44 was exposed to a strip test where p-nitroacetanilide was used as an indicator.The bacterial isolate JCM44 showed a positive result by producing yellow colour in chitin agar broth supplied with indicator p-nitroacetanilide Figure 1b.Also, the isolate was positive for catalase, oxidase, MR-VP, citrate utilization test and negative for indole.16S rDNA sequencing was done to isolate JCM44 and deposited in the NCBI with accession number MG917734.1.The sequence has high homology to Bacillus cereus and with greater than 99% of sequence identity and a phylogenetic tree was constructed.Various researchers have found that marine bacteria were reported that major producers of CDA including Bacillus sp. 21,24,25

Production of CS
The selected bacterial isolate Bacillus cereus JCM44 has shown the highest chitin degradation was cultivated in a production  .Once the isolate was proven to release CS from the chitin, its yield was also determined.The yield of chitosan by Bacillus cereus JCM44 was 60mg/L as maximum Figure 2(b).Early reports of CS production from certain fungi shows, 0.12 g/L of CS in liquid medium, 26 78.3 mg/L of CS by A. niger, 1.8g/L of CS by Absidia coerulea. 27,28In this study, the production of CS by the bacterial isolate Bacillus cereus JCM44 is not much more when compared to fungal isolates but by enhancing the medium conditions of CDA production will be increased for the production of CS.Hence, this bacterial isolate can be used on an industrial scale for the biotransformation of chitin to CS evidencing it to be a hopeful isolate for a biologically approachable process.

SEM
The SEM analysis of BSC showed that it has a non-porous, smooth membrane with irregular shape and crystallite in nature; this structural modification may be due to aggregation of the particles, this was due to their high surface charge Figure 3 (b&d).This was in agreement with the previous data reported in CS from B. portentous. 29The morphology of the BSC was different from that of commercial CS, Figure(3a) which has only the fibrous exterior could be owing to the extraordinary molecular lining.Rough layers of flakes and pores were seen in lower magnification (×2,500) Figure 3b.At ×10,000 magnification, BSC has pores with smooth and round surfaces; crumbling flasks have seen in Figure 3d.1][32][33][34] SEM images showed larger pores in insects and commercial chitin due to the separation of chitin and CS synthesis from various parts of the insects.

DLS
DLS is used to determine the stability and particle size in the suspension based on surface charge.The stability of the BSC particles synthesized was related to the commercial CS in the present study and was measured by using Zetasizer and is shown in Figure 4 (a & b).The BSC particles and commercial CS were diluted The Zeta potential of -2.28 mV of BSC was perfect for the physical constancy of any suspension when compared to the commercial CS which has a zeta potential of +58.9 mV.The results conclude that the BSC is more stable than the commercial chitosan.A similar trend was observed by 35 works, which presented the zeta potential value (4mV) of EA@CSNP, which shows the nanoparticle's optimistic surface charge with less stability.Due to the presence of drugs on the polymer surface, the Zeta potential values should be decreased.For the physical stability, the particle charge shows > + 30mV and < -30mV is ideal of any suspension.Similar trends were found for chitosan nanoparticles (CSNPs) synthesis by 36 shown + 30mV to -5mV which might be the occurrence of alkali on the particles.

Polydispersity Index (PDI)
The PDI describes the degree of "nonuniformity", and simplifies thoughtful of scattering and accumulation of the particles in the medium.
The dimension scattering of BSC and commercial CS is shown in Figure 5 (a & b).PDI values reflect the dimension scattering.PDI values will be shown based on the higher and lower range of particles.The PDI value of BSC is shown as 0.0626 and the PDI value for commercial CS was 0.534; this shows the heterogeneous nature of the particles.It was reported that PDI more than 0.5 is indicative of aggregation of CS particles. 37This reveals the aggregation of the particles also size distribution of BSC is smaller than the commercial CS.Similar particle size distribution for CSNPs with heterogeneous dispersion was reported. 38

FT-IR Analysis
FT-IR spectra were done to detect the structural groups of BSC.The CS from Bacillus cereus JCM44, as related to the physical property with the commercial CS used as a control Figure 6   (aromatic amine and alkane groups).Also, the IR spectral peaks of commercial CS of 3364, 2884, 2356, 2151, and 1653 cm -1 were comparable to BSC.This demonstrates the confirmation of CS.Similar reports have been reported in 39 and also similar results of IR stretching peaks observed in polysaccharide (Chitosan) prepared by processing prawn waste (shell) were revealed by. 40

XRD
The crystalline nature and phase purity of the prepared and commercially obtained sample were analyzed using powder XRD diffraction.The high intense peak was observed at 19.3°and 19.1° of 110 planes for the CS and BSC, respectively, which correspond to the characteristic peak of chitosan.Comparatively, a slight decrement in the intensity of the peak for BSC sample was observed which might be due to the low crystalline nature of the synthesized chitosan.Thus, this confirms the formation of CS Figure 7 (a & b).In addition, a few peaks other than the characteristic peak of the chitosan were also observed.The presence of such peaks reveals the trace amount of materials in the sample which were used to prepare BSC.[43][44]

CONCLUSION
It can be inferred from the present study that CDA secreted by Bacillus cereus JCM44 appears to be a prospective strain for the biotechnological application for the production of CS.The BSC was characterized by FT-IR, XRD, SEM, DLS (Zeta potential) and polydispersity index (PDI) analysis and compared with commercial CS.The BSC was irregular in shape with a smooth surface.FTIR reveals the presence of OH, NH groups, alkenes and amine groups of chitosan.The crystalline nature of synthesized chitosan was confirmed with XRD analysis.The Zeta potential and the Polydispersity index showed the stability of the synthesized bacterial chitosan and it revealed that the particle was heterogeneous which indicates the aggregation of the particles.Significant production of CS by enhancing bacterial isolates helps the financial, feasible and ecologically approachable process.Such synthesized CS is biocompatible, having the assets to be recycled for bio-based requirements.The vital data attained contributes to our knowledge of the capabilities and possibilities of Bacillus cereus JCM, 44 and successfully reduced the expenses of fabrication of CS.

Figure 5 (
Figure 5 (a & b).Dynamic Light Scattering of commercial CS and BSC

Figure 6 (
Figure 6 (a & b).FTIR spectrum of the prepared samplesCommercial CS and BSC

Table .
Qualitative Estimation of BCS Chitosan recovered in Neutralization 10 mL of 2% Acetic acid 10 mL of 2% Acetic acid (2% acetic acid) with (1N) NaOH White precipitate observed No precipitate observed Iodine reaction Dark purple coloration NIL