Bioconversion of chitin waste through Stenotrophomonas maltophilia for production of chitin derivatives as a Seabass enrichment diet

Preparation of colloidal chitin

A 5 g aliquot of chitin flakes was taken, mixed with 30 ml of 35.5% hydrochloric acid at 4 ºC, and incubated overnight. To this 250 ml of 50% chilled ethanol was added slowly with constant stirring at 4 °C. Colloids of chitin were observed in ethanol. This mixture was centrifuged at 10,000 rpm and the pellet was washed with distilled water till the pH reached 7.0¹⁰.

Sample collection

The soil samples were collected from shrimp and crab waste disposable sites in Parangipettai (Lat. 11°29′ N, Long. 79°46′ E), Cuddalore District, Tamil Nadu, India. Parangipettai coastal environment is a public area very close proximity to the institute Centre of Advanced Study in Marine Biology. Since it is a public domain no special permission is required to access it and also for research activities. All the experimental study was carried out under the approved guidelines.

Isolation and screening of chitinase producing bacteria

About 1 g of collected soil sample was diluted and plated in chitin minimal agar (colloidal chitin, 1%; KH₂PO4, 0.3 g; K₂HPO₄, 0.3 g; NaCl, 4 g; MgSO₄·7H₂O, 0.5 g; Agar, 1.5 g per liter). The inoculated plates were incubated at 28 °C for 72 h. After incubation, bacterial isolates which produced a zone of clearance around the colony were selected as the chitinase-producing bacteria for further studies^11,13.

Culture condition and production

A chitin minimal medium (CMM) containing 1% (w/v) colloidal chitin, 0.7% (w/v) K₂HPO₄; 4% (w/v) KH₂PO₄; 0.3% (w/v) NaCl; 0.5% (w/v) MgSO₄·7H₂O; and 0.5% (w/v) peptone, pH 7.2 was prepared for chitinase production and 50 ml of medium was dispensed in 250 ml of Erlenmeyer flasks and sterilized at 110 °C for 15 min. Each flask was inoculated with 1 ml aliquot of 18 h old seed culture and incubated at 37 °C at 100 rpm for 5 days. Culture broth has been centrifuged for 10 min at 8000 rpm (Sigma laboratory centrifuge 4K15), and the supernatant was used as a crude enzyme¹².

Chitinase assay

About 2.5 ml aliquot of phosphate buffer saline was added in 1% of colloidal chitin (substrate) followed by preparation of crude enzyme of 0.5 ml. The tubes were incubated at 45 °C for 1 h. After incubation, the reaction was stopped by adding 3 ml of 10% dinitro salicylic acid (DNSA) followed by heating in boiling bathwater. The colored solution was then centrifuged for 5 min at 8000g and the resulting supernatant was measured in a spectrophotometer at 540 nm wavelength. The reduction of sugar has been derived from a standard glucose curve. One unit of enzyme is known as the enzyme quantity that catalyzes the release of 1 μM of reduced sugar per minute under assay¹³.

Characterization of isolates

Chitin degrading isolates were characterized by studying their morphological and biochemical characteristics. The isolate results were compared with Bergey’s manual of determinative bacteriology. Further, a potential chitinase-producing bacterial strain was characterized by 16 S rRNA gene sequencing.

Molecular characterization

The phenol–chloroform technique was used to extract genomic DNA from the bacterial isolates. The agarose gel (0.8%) was used to analyze the isolated DNA and NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) was used to quantify it. PCR was used to amplify the bacterial strains 16S rRNA gene sequence using universal bacterial primers 27F and 1492R, as well as GeNeiTM PCR Master Mix (Genei, Bangalore, India). For the PCR protocol¹³, a thermal cycler (Genei, Bangalore, India) was used: 94 °C for 5 min (initial denaturation), 94 °C for 1 min (denaturation), 53 °C for 30 s (annealing), 72 °C for 90 s (elongation)—35 cycles and 72 °C for 7 min (final extension).

Phylogenetic analysis

The 16S rRNA gene sequences were amplified by polymerase chain reaction (PCR) using universal primers 27F and 1292R. The sequences of the amplified 16S rRNA genes CHI2 were obtained by an automated sequencer (Bioserve, Hyderabad) almost in full length. Clustal X mega program edited the sequences, and the National Center for Biotechnology Information (NCBI) database conducted a BLAST scan to find the nearest sequence. The sequencing findings were used to perform homology searches and the neighbor-joining method a phylogenetic tree was developed. The sequences of partially 16S rRNA gene sequences were deposited in the GenBank database, and it was given an accession number.

Extraction and purification of chitinase

At 4 °C, the crude enzyme was precipitated with varied doses of ammonium sulfate, ranging from 50 to 80% (w/v). The solution was slowly stirred and kept at 4 °C for 30 min to dissolve all concentrations of the ammonium sulfate. The precipitate was centrifuged at 10,000 rpm for 30 min at 4 °C; the precipitated protein was recovered and mixed with Tris–HCl buffer pH 7.5. The activity of chitinase and the amount of protein in the samples were examined. After precipitation, dialysis was employed to remove excess salt, organic solvent, and low molecular weight inhibitors. The protein sample was dialyzed overnight at 4 °C with 2–3 times buffer changes against 20 mM Tris HCl buffer at pH 7.5.

The dialyzed enzyme was put to a Sephadex 75 column (20 cm × 1.5 cm) and equilibrated with 20 mM Tris HCl buffer (pH 8.0). The enzyme was eluted with 20 mM Tris HCl and a flow rate of 1 ml/5 min was maintained. A 280 nm absorbance scale was used to determine the protein content. Peaks demonstrating enzyme activity were combined and identified as distilled enzymes. The effects of temperature and pH on the function and stability of the enzymes were investigated using this diluted enzyme solution.

Determination of optimal temperature and pH for enzyme activity

The reaction solution was incubated at different temperatures to determine the optimum temperature for enzyme activity. The thermostability of the enzyme was tested by incubating 0.2 ml of pure enzyme in 50 mM Tris–HCl (pH 9.0) for 1 h at various temperatures, including 30, 35, 40, 45, 50, 55, and 60 °C. To test the effect of pH on enzyme activity, researchers used five different buffer systems (all 50 mM): citric acid-Na₂HPO₄ (pH 5 to 6), phosphate buffer (pH 6 to 8), boric acid-NaOH buffer (pH 8 to 9.5), phosphate-NaOH buffer (pH 9.5 to 12), phosphate-NaOH buffer (pH 9.5 to 12), phosphate-NaOH buffer (pH 12–13 The stability of the enzyme was tested for 1 h by adding enzyme solution at various pH levels, and the activity was measured.

Experimental trial

Acclimatization of experimental fishes

Healthy Asian Seabass (Lates calcarifer) (20–30 g in weight) were procured from grow-out ponds of RGCA (Rajiv Gandhi Centre for Aquaculture), Karaikal, Tamil Nadu, India. The fishes (n = 300) were transferred to a 500-L fiber tank filled with UV treated seawater and continuous aeration was provided. Fishes were acclimated to these conditions for at least 7 days before initiating the experiment and fed with an artificial pellet two times a day. The experiment was carried out with the optimal water temperature of 27.2 °C ± 0.6, the salinity of 27 ± 2.2 ppt, the dissolved oxygen concentration of 5.6 ± 0.5 (mg/l), and pH 8.0 ± 0.3 respectively. The ammonia (0.01, 0.02, 0.03 and 0.04 mg/ml) and nitrite (0.01, 0.05, 0.05 and 0.06 mg/ml) contents in the water were maintained at low level. This study was carried out in compliance with the CPCSEA safety guidelines and following ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments. The study was approved by the Institutional Animal Ethics Committee (IAEC) 1793/PO/ReBi/S/2014/CPCSEA and by the Centre for Lab animal science, Sathybama Institute of Science and Technology.

Chitin degraded product enriched diet

Based on the chitinase optimization, the chitin degraded product was obtained. The degraded product was lyophilized and dispensed in a little volume of Milli-Q water. To prepare the experimental diet was mixed evenly to basal diet and air-dried aseptically. The control was sprayed with Milli-Q water. The prepared pellets were dried using an oven at 35 °C for 12 h and stored at − 20 °C for further use.

As indicated in Table 1, five dietary supplements including 0.0 (control), 0.5%, 1% , and 2% chitin degradation product feed were developed to give optimal 42% (w/w) crude protein. In addition, different quantities of the chitin derivatives feed supplement was mixed in 100 mL/kg feed and homogenized for 30 min with the other components to make fine consistency dough (Table 1). The dough was mixed in a mixer at the same time and pelleted using a paste extruder with a diameter of around 1 mm. The feed pellets were dried for 24 h at 55 °C and stored at − 20 °C for future use.

Experimental design

Four rectangular fiberglass tanks (100 L) were placed with continuous aeration. The fishes (20 ± 4 g) were randomly distributed in four experimental groups with triplicate. One tank was kept as control (T1), one with 0.5% chitin degraded product (CDP) (T2), 1% CDP (T3), and 2% CDP (T4), The fish were fed the basal feed during the acclimation period. The fish in the experiment were fed twice a day. After that, each tank was assigned to one of three duplicates of four different experimental meals at random. For 45 days, three groups of fish were fed one of the experimental diets twice daily (10:00 and 17:00 h) at about 4% of wet body weight per day at the start and 5% of wet body weight per day at the end of the feeding study. On the 14th day, fishes from the T3 and T4 group were inoculated intraperitoneally with 100 μl of Aeromonas hydrophilia (MTCC) suspension. Randomly 3 fishes were collected from each tank on the 1st, 2nd, and 3rd week of post-infection and anesthetized using MS-222.

Growth

Feed consumption measuring and growth rate

The fish fed with CDP/kg feed from each tank were taken, measured, and documented after the feeding trial analysis was completed on the 45th day. Enumerating the individuals in each tank was also used to calculate the fishes survival rate. Weight gain (WG), specific growth rate (SGR), feed intake (FI), feed conversion ratio (FCR), and protein efficiency ratio (PER) was used to calculate the efficiency of fish productivity and diet consumption using equations.

Non-specific immune response in Asian Seabass

RBC and WBC count

Haemocytometer was used to evaluate the number of RBC and WBC¹⁴. The number of cells per ml of the blood sample was determined using the formula.

Hematocrit p-value

The percentage of blood cells in the total volume of blood was determined as described by Goldenfarb et al.¹⁵.

Serum bactericidal activity

For evaluating serum anti-bactericidal activity, A. hydrophila was mixed with serum and incubated for 1 h at 37 °C. Then, 0.1 ml of the suspension was placed on nutrient agar incubated at 24 h at 37 °C. After incubation, the number of viable bacteria was calculated¹⁵.

Respiratory burst and lysozyme activity

The formation of oxidative radicals from neutrophils in blood during respiratory burst was evaluated by NBT assay, as described by Anderson and Siwick¹⁶. NBT assays were modified and used to quantify the formation of oxidative radicals by neutrophils in blood during the respiratory burst, In blood and 0.2% NBT were mixed in equal parts (1:1), incubated at room temperature for 30 min, and then 50 L were extracted and dispensed into Eppendorf tubes. 1 mL dimethylformamide (Sigma, USA) was used to solubilize the reduced formazan product, which was centrifuged for 5 min at 2000 rpm. Finally, using a micro reader and a supernatant, the extent of reduced NBT was evaluated at an optical density of 540 nm.

Lysozyme activity

Lysozyme activity was determined by using the turbidimetric method according to Abu-Elala et al.¹⁷. In a 96 well plate with 50 µl PBS, pH 5.8, 50 μl of serum were put in triplicate. The serum was serially diluted till the last well after mixing. Finally, in the last well, 50 µl of the sample was eliminated. Micrococcus lutius (75 mgml1in phosphate buffer) was introduced to each well in a volume of 125 µl. In an ELISA reader, the drop in absorbance at 450 nm was measured from 0 to 15 min at room temperature. Using hen egg-white lysozyme (Sigma, USA) as a standard, the lysozyme activity was converted to lysozyme concentration.

Disease resistance and survival experiment

The disease-resistant experiment was conducted following the methodology of Harikrishnan et al.¹⁸ with slight modifications. The fishes from the experimental and control group were inoculated with Aeromonas hydrophilia (1 × 10⁸ cells ml⁻¹). The fishes were observed for mortality and clinical symptoms.

Statistical analysis

All of the analyses were done in triplicate, and the results were expressed as mean standard error. Using SPSS software (version 17.0, SPSS Inc., USA), all of the collected data were subjected to one-way ANOVA analysis. ANOVA was performed to find differences between groups, and Duncan’s multiple range tests were employed to evaluate the difference in means.

Ethical approval

The present study follows institutional guidelines mandatory for human and animal treatment and complies with relevant legislation ethical approval from the institute for conducting the research. The study was approved by the Institutional Animal Ethics Committee (IAEC) 1793/PO/ReBi/S/2014/CPCSEA and by the Centre for Lab animal science, Sathybama Institute of Science and Technology.