Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 18, No. (1) : 2016 : 1-8© Global Science PublicationsISSN-0972-3005

*Corresponding author’s email: amrihprasetyo@yahoo.co.idAddress : Amrih Prasetyo, Faculty of Animal Science, Gadjah Mada University, Jl. Fauna 3, Kampus UGM, Bulaksumur,Yogyakarta 55281, Indonesia

Article-1

ISOLATION AND CHARACTERIZATION OF CHITINOLYTIC

PAENIBACILLUS SP. D10-2 FROM TROPICAL SHRIMP POND

WASTE WATER

1AMRIH PRASETYO*, 2LIES MIRA YUSIATI, 2RUSMAN, 2NANUNG AGUS FITRIYANTO,3TOMOYUKI NAKAGAWA, 3TAKASHI HAYAKAWA AND 2YUNY ERWANTO

1Indonesian Agency for Agricultural Research and Development Jl. Ragunan 29 Pasar Minggu JakartaSelatan 12540, Indonesia.

2Faculty of Animal Science, Gadjah Mada University, Bulaksumur, Yogyakarta 55281, Indonesia.3Faculty of Applied of Biological Sciences, Gifu University, Yanagido 1-1, Gifu 901-1135, Japan.

(Received 18 September, 2015; accepted 3 November, 2015)

Key words: Isolation, Chitinolytic Bacteria, Chitinase, Shrimp Waste

Abstract - The purpose of this study was to obtain superior chitinolytic bacteria capable of degrading chitinfrom the waste water of tropical shrimp pond. The bacterium characterization was conducted to yield high-activity chitnase isolates. While bacterium identification was carried out based on morphology, physiology,biochemistry and molecular. Screening was performed by colloidal chitin agar medium incubated at 30°C,five days, and flasks were incubated in an orbital incubator shaker at 30°C and 150 rpm. The enzyme activitywas measured at intervals for five days. The cultures were centrifuged at 10,000 g for 15 min at 4°C, and theenzyme solution (culture supernatant) was used for chitinase assay. The morphological, physiological andbiochemical characteristic of the VITEK system. The 16SrRNA method was used to identify strain. Resultsof morphological identification, biochemical and molecular identified as Paenibacillus sp. D10-2 includingthe genus Bacillus. Showed chitinase activity in medium containing chitin as a carbon source. The enzymechitinase produced by Paenibacillus sp. D10-2 chitinase contained 29.46 (U / mg protein). Chitinolytic bacteriaPaenibacillus sp. are able to degrade chitin specifically into the monomer in the form of glucosamine. Theyare widely used in in the fields of agriculture, food and health industries.

INTRODUCTION

The United Nation’s Food and AgricultureOrganization (FAO) in 2010 ranked Indonesia at 4th

around the world in terms of the shrimp production(FAO, 2010). The shrimp industry in Indonesiaprocessed about 352,000 tons in 2010 andsignificantly increased to 381,288 tons in 2011.Ninety percent of shrimp production is the exportedto other countries in the form of frozen shrimps.During processing of shrimps into food, 40–50% ofthe total weight is by-products, consisting of shrimphead and the skin of the shrimp. About 40% of theby-product consists of chitin, encrusted withcalcium carbonate and astaxanthin (Gimeno et al.,2007).

Chitin, the homopolymer of β-1,4 linked N-

acetylglucosamine (NAG), is the most widespreadstructural bio-polymers in nature. Among thenatural resources, crustacean bio-wastes especiallyshrimp and crab shells have the highest chitincontent (Dahiya et al., 2006). In the global scenario ofescalating demand for shrimp products, Asiancountries contribute about 80% of world’s shrimpmarket (Kandra et al., 2012). During the shrimpprocessing, the shells are considered as wastes anddiscarded through ocean dumping, incineration andlandfilling. The aquatic environment is a vastaccumulation of chitin in marine sediments aschitinolytic bacteria in marine ecosystems can todegrade chitin (Halder et al., 2013).

Shrimp by-product in Indonesia reaches 240,000tons/year (Data from The Indonesian Central Bureauof Statistics, 2004). The by-product is abundantly

2 PRASETYO ET AL.

dumped into the sea, whereas a small part is driedand used as poultry feed without any technologyapproaches. Chitin is a homopolymer of N-acetylglucosamine. Nitrogen content in this compoundreaches 52.70%, it discards the protein of about 0.527X 152,515 tons = 80,375.405 tons (Mahata et al., 2008).This by-product always pollutes the environment.

Chitinase (EC 3.2.11.14) enzyme can hydrolyzeinsoluble chitin from oligomers and monomer.Chitin can be found in a variety of organismsincluding bacteria, fungi, insects, higher plants, andanimals. They play important physiological rolesdepending on their origin (Gooday, 1990). Chitinasehas a wide-range of applications such as preparationof pharmaceutically relevant chitooligosaccharides.Chitin waste can be altered into N-acetyl-D-glucosamine, treatment of chitinase waste, andfunctional food (Dahiya et al., 2006). Chito-oligomers produced by enzymatic hydrolysis ofchitin are used in various function for agriculturaland industrial applications, such as antibacterial,antifungal, and as a food quality enhancer(Bhattacharya et al., 2007).

Some researcher have described the isolation,characterization and genetic mapping of potentialbacteria for chitinase production such as Aeromonassp. from the soil in the various area in Lucknow,India (Kuddus and Ahmad, 2013). Waldeck)conducted a similar study in Taiwan where Bacilluslicheniformis. was isolated to degrade shrimp shells.Anuradha and Revathi (2013) found similarfindings. Although some research in isolation ofchitinolytic bacteria has been conducted, there wasa limited study in the isolation and characterizationof chitinase from the marine area especially in thetropical shrimp pond.

The purpose of this study was to obtain superiorisolates chitinolytic bacteria capable of degradingchitin from the waste water of tropical shrimp pond.Characterizing based on morphology, biochemistryand physiology were intended to yield bacteria withthe highest chitinase activity. While identificationbased on molecular was intended to yield partialgenome of isolates

MATERIALS AND METHODS

Sample Collection and Isolation of ChitinolyticBacteria

Forty-five isolates from the waste water of tropicalshrimp pond in Bantul, Yogyakarta, Indonesia were

used. Samples were taken from the tank wasteshrimp ponds, samples collected at a depth of 0.5 mfrom the surface of the water, the water pH 8, witha water temperature of 27oC. Geographical locationlatitude -07.98306, longitude 110.22958. In the wastewater tank of shrimp ponds, many shrimps died,thus, it is possible that chitinolytic bacteria grow.Samples were isolated by serial dilutions of watersamples and plated on colloidal chitin agar medium.The agar medium amended with colloidal chitinwas used to screen the chitinolytic bacteria. Bacterialisolates were detected by the appearance of clearzones around colonies grown on colloidal chitinagar plates after five days. The medium consists of(g/L): KH

2PO

4, 3; Na

2HPO

4, 6; NH

4Cl, 1; NaCl, 0.5;

yeast extract, 0.05; agar, 18 and colloidal chitin 1%(w/v) incubation at 30ºC, 5 days. (Monreal andReese, 1969). The colonies showing clearance zoneson a squeamish background were considered aschitinase-producing bacteria.

Preparation of Colloidal Chitin

Colloidal chitin was prepared from the chitin(Sigma, USA) by the modified method used byGomaa (2012). In brief, chitin powder (40 g) wasslowly added with 600 ml of 10 N HCl and keptovernight at 4°C with vigorous stirring. Thesuspension was added to cold 50% ethanol withrapid stirring and kept overnight at 25°C. Theprecipitate was collected by centrifugation at 10,000rpm for 20 min and washed with sterile distilledwater until the colloidal chitin became neutral (pH7.0). It was freeze dried to powder and stored at 4°Cuntil further use.

Chitinase Produced by Bacteria

Those strains forming bigger zones were selectedand inoculated into Erlenmeyer flasks containing 20mL of medium culture. The medium supplemented1% colloidal chitin as a carbon source (g/L):Na

2HPO

4, 0.65; KH

2PO

4, 1.5; NaCl, 0.25; NH

4CL,

and 0.5; MgSO4, 0.12. The final pH was adjusted to

6.5. The medium culture was incubated in anincubator shaker at 30°C and 150 rpm for three days.The inoculum culture (1 mL) was added to culturemedium (500 mL) containing 1% colloidal chitin, 1%tryptone and 1% NaCl (pH 7). The enzyme activitywas measured for five days at time intervals. Thecultures were centrifuged at 10,000 X g for 15 min at4°C. The enzyme solution (culture supernatant) wasused for chitinase assay, aliquots of the reactionmixture were taken every 24 h (Chang et al., 2004).

Isolation and Characterization of Chitinolytic Paenibacillus sp. D10-2 from Tropical Shrimp Pond 3

Identification of Selected Bacterial Isolates

Identification of the bacterial isolates was performedbased on their morphological, physiological, andbiochemical characteristics, as described in Bergey’sManual of Systematic Bacteriology (Claus andBerkeley, 1986). The morphological characteristics ofthe isolates were observed with a Olympusbiological microscope, high-class system BX51Olympus, Tokyo) using cells grown on LB media.The VITEK 2 Compact 60 system, bioMerieux Inc.Hazelwood, MO, USA was used to characterize theBacillus isolates further via enzymatic, acidification,alkalinization, assimilation test. The experimentswere performed in duplicate with an inoculum sub-cultured at least once under the same testconditions.

Enzyme Assay

Chitinase enzyme assay was carried out withcolloidal chitin as a substrate. The assay mixturecontained 0.3 mL of 1% colloidal chitin, 200 μLsodium phosphate buffer (100 mM, pH 7.0) and 0.3mL of supernatant as an enzyme source, which wasincubated for 2 h at 45°C. The sugar was tested bymeans of the DNSA method at 540 nm (Waghmareand Ghosh, 2010). The released sugar during thereaction was quantified by using N-acetyl-â-D-glucosamine standards with concentrations rangingfrom 0.1 to 1.3 mg/mL by the dinitro salicylic acid(DNSA) method (Miller, 1959). β-N-acetylglucosamidase was determined by using thereaction mixture contained 100 μL N-acetyl-β-D-glucosamine (5 mM), 100 μL enzyme solution(culture supernatant) appropriately diluted in bufferand 200 μL sodium phosphate buffer 100 mM, (pH7.0). After incubation at 2 h, 45°C, 1 mL glycine-NaOH (0.2 mM pH 10.5) was added to stop thereaction. The absorbance of the N-acetylglucosamine released was measured at 540 nm. Oneunit of enzyme activity was defined as the amountof enzyme that required releasing 1 μmol of N-acetyl glucosamine per hour (U/mg protein). Proteinconcentration of the crude enzyme solution wasdetermined by Lowry’s method (Lowry et al., 1951),using Bovine Serum Albumin as a standard.

Strain Identification

16S rRNA gene sequencing and analysis: Theisolates were analyzed for species identity using the16S rRNA gene sequencing method according toRochelle et al., (1995). The gene sequencing wasperformed at Genomics Research (Gifu Univ). DNA

sequences were aligned using DNA star & DataCollection v3.1 Communication Patch1. Thegenomic DNA to extract, bacterial colonies werepicked with a sterilized and suspended in 0.5 μL ofsterilized saline, then centrifuged at 10,000 X g for 10min. After the removal of supernatant, the pellet wassuspended in 0.5 μL of Insta Gene Matrix (Bio-Rad,USA), incubated at 56°C for 30 min and then boiledto 100°C for 10 min. After heating, the supernatantcan be used for the PCR reaction. Bacterial 16SrRNAs were amplified by using the followinguniversal bacterial 16S rRNA primers. Forwardprimer 27 F (5’-AGAGTTTGATCMTGGCTCAG-3’)and reverse primer 1792 R (5’-TACGGYTACCTTGTTACGACTT-3’) (Gomaa,2012). Polymerase chain reaction was performedusing kits with Ampli Taq DNA polymerase(FSenzyme) (Applied Biosystems, USA). From asingle colony, agar plate was taken and then addedto 50 μl of PCR reaction solution. Amplification wasperformed using temperature program at 55°C, 30cycles at 94°C for 30 min, 55°C for 30 min, and 72°Cfor 1 h (TAKARA, Japan). Sequencing wasperformed using a Big Dye terminator cyclesequencing kit (FastGene Gel/PCR Extraction Kit,Japan). Sequencing products were sequenced on anApplied Biosystems model ABI PRISM 3100Hitachi, automated DNA sequencing system(Japan). All the sequence of strain were analyzed byusing Basic Local Alignment Search Tool forNucleotides (BLASTN, NCBI, USA) .

RESULTS AND DISCUSSION

Screening of Chitinolytic bacteria

In the selection step, chitinolytic bacteria strainswere identified by clear zones forming abilityaround their colonies on minimal medium agarplates supplemented with 1% colloidal chitin as asole carbon source. In this step, we obtained varioustypes of microbial colonies from more than 45isolates. Twenty-five isolates showed activitychitinolytic on colloidal chitin agar. Five isolateswere selected, based on the largest chitinolyticindex. Screening is done by growing in mediacolloidal chitin that were incubated at 30 ° C, for 5days, up to 4 times. The clearing zones weremeasured after the five days incubation. Amongthose strains, five strains, which gave the biggestchitinolytic ratio of clearing zones, were selected(Fig. 1). These strains have enough ability toproduce chitinase activities. The sample collected

4 PRASETYO ET AL.

from the local area: Srandakan, Bantul, ProvinceYogyakarta, Indonesia, showed the highest chitinaseactivity among five chitinolytic strains (Table 1).

Identification of the Isolates

Identification of the isolates based on biochemical,morphological and moleculer. Morphological testthe isolates D10-2 are rod-shaped with a length ofup to 0.7 ìm and a width of up to 2 ìm, gram-positive, spore and motile. Catalase positivebiochemical test results, using the substrate glucose

and β-N-Acetyl-Glucosamine and oxygenrequirements are aerobic and facultative anaerobic.Taxonomic was carried out according to themethods describes in Bergey’s Manual of SystematicBacteriology (Claus and Berkeley, 1986). Thephysiological and biochemical characteristics ofstrain D10-2 are shown in Table 2. The phylogenetictree showed that D10-2 is closely related to membersof the species Paenibacillus (Fig. 3). Based on of thenucleotide sequence of the partial 16S rRNA gene,D10-2 was classified as the genus Bacillus. Apairwise alignment (BLASTn) analysis of 16S rRNAsequence of isolate D10-2 indicated that the DNAsequence had the highest similarity withPaenibacillus (more than 99% similarity at the level ofDNA).

Growth of Cell and chitinase production

The production of extracellular chitinase wasmonitored by observing the growth of D10-2 (Fig.3). The bacteria enzymes production at 24 h ofincubation and produce maximum enzyme at 48 hand the bacteria attained its stationary phase ofgrowth after 48 h. The results indicated that thechitinase production started from the beginning of

Fig. 2. a). Paenibacillus D10-2 grown on chitin agar plates 1% and showed clear zones. The culture was incubated at 30°Cfor 120 h b). Gram staining of bacteria showed bacillus and gram-positive

Table 1. The strains selected based on chitinoltic ration, mineral medium added 1% chitin coloidal.

Strains Outer diameters (mm) Iner diameters (mm) Chitinolytic ratio

D10-2 9,92 ±0,35 6,01 ±0,45 1,66 ±0,06A10-1 15,03 ±0,12 8,50 ±0,25 1,77 ±0,07B10-51 12,00 ±0,00 7,25 ±0,50 1,67 ±0,12B10-13 12,56 ±0,19 7,87 ±0,39 1,60 ±0,05A-2 16,32 ±0,69 7,88 ±0,38 2,07 ±0,01

Fig. 1. The strain selected based on clear zones mineralmedium added 1% colloidal chitin.

Isolation and Characterization of Chitinolytic Paenibacillus sp. D10-2 from Tropical Shrimp Pond 5

Table 2. Morphological, biochemical, and physiological characteristics of strain D10-2.

Shape

Gram stainSpore formationMotilityColony morphology

CatalaseOxidaseEllmanEsculin hydrolaseIndol productionD-RiboseLeucin arylamydase

VITEX system was used; +, positive; , negative,

Rod(0.50.7×1.21.7μm) + + +CircularSmoothLow convexYellowSemitranslucent +

+ + – + +

Urease –Eslrinehydrolysis –Gelatine hydrolysis –Galactosidase –

Substrate utilizationD-Glucose +D-Ribose +D-Mannitol –N-Acetyl-D-glucosamine +Maltotriose +D-Gluconate –D-Trehalose +β-N-Acetyl-glucosamine +

Phenyllalanine Arylamydase +

Fig. 3. Phylogenetic tree based on partial 16S rRNA sequences analysis of strain D10-2 showed 99% identityto the bacteria most closely related. The accession number are in parentheses. The Scale barcorresponds to 0.02 subscriptions per nucleotide position.

the logarithmic phase and increased linearly withthe increase up to 48 h.

Chitinolytic of bacteria strain D10-2 grown onchitin agar plates and showed clear zones due tochitinase activity (Fig. 4). The chitinase production

could be detected by reducing sugar estimation after24 h of incubation and progressively increase to thehighest value of 120 h (29.463 U/mg). Similarobservations (Jami Al Ahmadi et al., 2008) in case ofPaenibacillus chitinolyticus JK2 showed that the

6 PRASETYO ET AL.

highest production of chitinase happened at 72 h.Many chitinase were produced by Bacillus sp. (Wanget al. 2001; Thamthiankul et al. 2001).

Temperature and Effect of pH

The effect of temperature and pH on enzymeactivity is shown in Fig. 5a. The crude enzymeshowed an optimum activity at pH 5. Chitinaseactivity showed 93.6 and 100% at pH 4 and 5,indicating that enzyme tends to be active in acidiccondition respectively. This is similar to the pH 5reported for Bacillus circulans WL-12 (Watanabe etal., 1992). So similar to pH 6 for Vibrio sp. strain98CJ11027 (Park et al., 2000), pH 5 and 7 for Bacillussp. Strain JK2 (Jami Al Ahmadi et al., 2008) and pH5-8 for Aeromonas hydrophila H-2330 (Hiraga et al.,1997). The temperature effect of the chitinaseactivity was also performed which indicated that theactivity was increased to the temperature below theoptimum temperature at 30ºC and decrease at ahigher temperature. The enzyme showed 100% ofthe maximum activity at 30ºC gave the highest yieldof chitinase Fig. 5b.

DISCUSSION

The novelty of the research was the findingsPaenibacillus D10-2, producing chitinase in tropicalarea. The strain was isolated from wastewater

Fig. 4. The cells growth and chitinase production ofbacteria strain D10-2, the cells growth microbialstrains were cultivated in medium composed of1% NaCl, 1% tryptone, and 1% colloidal chitin(30°C, 150 rpm, 5 days). The culture supernatantwas obtained by centrifugation (10,000 g, 15 min).The culture supernatant was used for chitinaseassay after overnight dialysis against 10 mMsodium acetate buffer (pH 5.0). Cell growth wasmeasured by absorbance at 600 nm (Kuk et al.2005).

Fig. 5. Enzymatic activities of strain D10-2 measured atoptimum temperature 30ºC and pH 5. Datashown are mean values ± SD from threeexperiments. (a) Optimum pH of the enzyme wasdetermined in various pH buffers (pH 3.5 – 5.0),sodium phosphate buffer (pH 5.0 – 8.0). (b)Optimum temperature for enzyme activity wasdetermined by incubating the reaction mixtures atdifferent temperature range of 30-50ºC.

tropical shrimp pond sample collected from thelocal area: Srandakan, Bantul, Province Yogyakarta,Indonesia. Taxonomic studies showed that strainD10-2 belonged to the genus Bacillus sp. Bacillus sp.is one of the most efficient bacteria for thedegradation of chitin (Laribi-Habchi et al., 2015)

The use of optimal culture competition, the effectof initial pH, temperature and cultivation time onthe production of chitinase were also investigated.Maximum chitinase production was achieved at pH5 and 30ºC. Bacillus licheniformis LHH 100 wasreported for having chitinases activity optimal at pH4. By adding calcium at 75ºC the activity of theenzyme was optimum (Laribi-Habchi et al., 2015).The effect of cultivation time on the production ofchitinase was investigated by showing the enzymeactivity every 24 h for 5 days. The incubation time toachieve maximum chitinase product was stationeryfrom 48 h to 120 h. The same observation was

Isolation and Characterization of Chitinolytic Paenibacillus sp. D10-2 from Tropical Shrimp Pond 7

reported for Paenibacillus chitininolyticus JK2 (JamiAl Ahmadi et al., 2008), Bacillus sp., (Joo et al., 2007),Paenibacillus ehimensis KWN38 (Naing et al., 2014),Bacillus circulans No.4.1, (Wiwat et al., 1999), Bacilluscereus TKU030, (Liang et al., 2014).

The present results and comparison with our bestknowledge about other chitinase producers, thisstrain has the capability for the production ofchitinase on an industrial scale in the future. Thismicroorganism may be useful for the treatment ofchitinous waste and also for production manyproducts of hydrolyzed chitin for variousapplications.

CONCLUSION

The strain D10-2 was selected among those givingmaximum enzyme production in the shortest time.It was further identified as Paenibacillus D10-2.Detected chitinase activities (29.463 U/mg).werefound in chitin as a carbon source. The chitinaseenzyme that was produced by this strain haschitinase activities. Further studies on the propertiesof D10-2 isolate and its chitinase could determine itspotential for industrial application especially forwaste treatment programs.

ACKNOWLEDGMENTS

This work was supported by Indonesian Agency forAgricultural Research and Development Ministry ofAgriculture, Faculty of Animal Science UniversitasGadjah Mada and Faculty Applied Biology ofScience, Gifu University Japan.

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