Isolation, Identification and Characterization XYLANOLITIC Bacteria from Soil

IJSTE - International Journal of Science Technology & Engineering | Volume 3 | Issue 04 | October 2016 ISSN (online): 2349-784X

Isolation, Identification and Characterization Xylanolitic Bacteria from Soil Adista Cahyandika Student of Magister Program Department of Food Science & Technology Faculty of Agricultural Technology Brawijaya University, Malang. East Java

Yunianta Lecturer Department of Food Science & Technology Faculty of Agricultural Technology Brawijaya University, Malang. East Java

Agustin Krisna Wardani Lecturer Department of Food Science & Technology Faculty of Agricultural Technology Brawijaya University, Malang. East Java

Abstract The xylanase enzyme produced by microorganisms, can be used for food and non-food industries. Such as paper bleaching, waste processing, animal feed and area food industry such as for juice purification, bread making, producing xylitol sugar and ethanol. The type of xylanase enzymes produced by bacteria has more stability at high temperature, than xylanase enzymes produced by the mold class. This study was focused on enzymes produced by bacteria class. The purpose of this study was to determine the type of xylanolitic bacteria that can produce xylanase enzymes and characterization the bacteria. Types of xylanolitic bacteria were isolated based on molecular identification tests using 16S rRNA is bacterial type of Bacillus cereus strain CM 100B. It was a type of Gram-positive bacteria and the characterization test has a positive result on the test of indole, motility and had negative result on the methyl red test.

Keywords: Bacteria, Bacillus cereus, Isolation, Purification, Characterization, Xylanase ________________________________________________________________________________________________________ I.

INTRODUCTION

These day xylanase enzyme it is important to produce. Xylanase is a class of hemycellulose enzyme. That enzyme it has a function to degradating hemycellulose compound, especially xylan compound [9]. Xylanase can to produced by microorganism from class of bacteria and fungus. The xylanase enzyme producing by bacteria more thermostable than xylanase that producing by fungus, although the enzyme activity from the fungus higher than bacteria. The xylanase enzyme produced by microorganisms, can be used for food and non-food industries [8]. That enzyme in the food industrial can to used for baking process, juice purified, produced xilooligosaccharide that also prebiotic agent and to produced xylosa that is material of xylitol producing [1]. To applied nonfood industrial, xylanase enzyme can be used as paper bleaching [13], bioethanol [3], waste processing, animal feed and composting [7].

The type of bacteria that can to produce xylanase enzyme involved Pseudoalteromonas, Glaciecola, Staphylococcus, Xanthomonas, Rhodococcus, Bacillus [5] [14] [10]. The isolated of xylanolitic bacteria can be obtained from soil. Soil is a source of bacteria that are not limited. Soil has five main elements: water, mineral, gas, organic matter, and the place the bacteria of living [12]. Most microorganisms grow and multiply on the surface soil. Soil is a good medium for the growth and development of various kinds of microorganisms [11]. In this study the type of soil used to isolation xylanolitic bacteria from paddy field. Soil from paddy field has the potential presence of xylanolitic bacteria. Waste from paddy harvesting, contain a xylan compound. The xylan compound from rice husk 6,3% [17] and rice straw 20,4% [16]. In this study the type and characterization of bacteria in the soil that can produce xylanase enzymes need to know, to improve the producing of xylanase enzyme.

II. MATERIAL AND REAGENTS Soil samples and rice husk, collected from soil fields in Malang, Indonesian. The chemicals and reagents used in this study were beechwood xylan, yeast extract, NaCl, KH2PO4, NaH2PO4, NH4Cl and MgSO4. 7H2O, bacto agar, congo red.

Isolation and Screening of the Bacteria The soil sample give a enrichment treatment with rice husk to stimulant the xylanolitic bacteria for growing. Incubated treatment for the enrichment at 37â °C for 3 days. After the enrichment process, the sample was dilution system, and was isolation the bateria from the sample with spread plate method. These plates were incubated at 37â °C for 2 days. The colonies on the plates

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Isolation, Identification and Characterization Xylanolitic Bacteria from Soil (IJSTE/ Volume 3 / Issue 04 / 029)

were transferred on to another agar plate, which were again incubated at 37⁰C for 2 days. The colonies having clear zone in the plate were selected. Characterization, morphological properties and taxonomic characteristics of the bacteria was studied by the methods in Bergey’s manual of systematic bacteriology. The isolated bacteria were an aerobic, gram-positive, spore forming, rod-shaped organism. For knowing the strain of bacterium, used a molecular analysis system of 16S rRNA gene. Bacterial rRNA sequence was amplified using a pair of primers 8F and 1492R with 35 cycles of the following thermal program: 96⁰C for 10 sec, 50⁰C for 5 sec, and 60⁰C for 4 min. The nucleotide sequence was determined with a Big Dye terminator Cycle sequencing Kit (Applied Biosystems) using universal rRNA-specific primers.

III. RESULT AND DISCUSSION Isolation Bacteria The preliminary experiment of this study, bacteria samples were collected from soil field and giving enrichment treatment to stimulant xylanolitic bakteria for growth before isolation step. The sample was dilution, and isolation with spread plate method.

Collected and incubated at 37⁰C for 48 h. Observed and choose that colonies having a clear zone. From this step obtained three isolated having a clear zone, can be seen Fig 1.

Fig. 1: Clear zone from isolated bacterial (In a solid medium containing beech wood xylan substrate, the code (1) as x1, (2) as x2, and (3) as x3).

Identification Bacteria The selected isolates in this study is isolates x2, because it has a highest activity xylan hydrolisis (data not shown). Identified bacteria using 16S rRNA molecular to obtain specific types of bacteria. First step molecular identification test is extraction of DNA from bacteria followed by PCR stage, for the presence of DNA in PCR products which will continue to process sequencing. PCR products tested using DNA electrophoresis. The electrophoresis test results can be seen in Figure 2.

Fig. 2: Visualization of PCR products after electrophoresis of 16S rRNA (M = DNA Marker, 1 = other sample isolates, 2 = isolates x2, + = control positive, - = control negative)

The results by means of molecular identification using 16S rRNA sequencing get results with a length of about 600 sequences.

Using Primer 5'-AGAGTTTGATCCTGGCTCAG 8F-3 'and 5'-GGTTACCTTGTTACGACTT Primer 1492R-3'. The order of the sequences obtained table 1.

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Isolation, Identification and Characterization Xylanolitic Bacteria from Soil (IJSTE/ Volume 3 / Issue 04 / 029)

Table – 1 The Nucleotied sequences of the partial 16SrRNA gene of the isolate identified as B.cereus CM 100B 16S rRNA Gene Sequence GGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAG GTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCG ACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGAC GAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAAT AGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTC CGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAACTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAAC TGGGGAACTTGAGTG.

Based on sequence data obtained from the 16S gene sequencing of the samples x2, the order of the sequence be matched with gene sequences found in GenBank online to find the level of homology using the NCBI BLAST program www.ncbi.nlm.gov.

The sequence compared to the comparative sequences. Systematical sequence comparison performed by sequence alignment using ClustalW software contained on Bioedit. The alignment results are used to establish a tree phylogeny using software Mega 6. Results phylogeny tree can be seen in Figure 2 below.

Fig. 3: Tree of phylogenetic The phylogenetic tree shows that the rate of bacterial isolates kinship with other microorganisms based on primary sequence data from 8f and 1492r, x2 seen in isolates are closely related to the bacteria Bacillus cereus strain CM100B, which is based on the results of BLAST degree of similarity of 100%. According [6], when the degree of similarity below 95%, it can be said to be a new species. But the results of the bacterial isolates showed that the degree of similarity x2 reaches 100%, so it certainly is a type of bacteria Bacillus cereus strain CM100B. Characterization Bacteria Bacillus cereus strain CM100B is a type of bacteria that can survive temperatures 15-42ºC with optimum temperature 37ºC. In addition, this type of bacteria can survive at pH 5-11 with an optimum pH of 7.5 [4]. Therefore bacterial strains include species of bacteria mesophyll, which is optimum at neutral pH. According [4] type of bacteria Bacillus cereus strains isolated from soil CM100B around the coal mine environment can also hydrolyze starch, gelatin and casein. Further test phase of the bacteria Bacillus cereus strain CM100B is the characterization of the test bacteria with microscopic test and some kind of biochemical tests. Test characterization of isolates of Bacillus cereus strain CM100B can be seen in table 2.

Table – 2 The character of isolates x2 Characterization Results Indol (+) Motilitas (+) Metyl Red (-) Gram (+) Form Bacill

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Isolation, Identification and Characterization Xylanolitic Bacteria from Soil (IJSTE/ Volume 3 / Issue 04 / 029)

Bacillus cereus strain types CM100B have a characteristic shape or basil stems with stain results that include the type of Gram-positive bacteria. These types of Gram-positive bacteria have properties that hold or strong against physical treatments, one of which is the provision of a sufficiently high temperature treatment [14]. Therefore, even though the bacteria are classified as type mesophyll, but they can survive temperatures above 40ÂşC. The composition of the cell wall of gram-positive bacteria consists of peptidoglycan of 50% of the cell wall, but it also contains lipids in the cell walls are lower when compared to the type of Gram-negative bacteria [2].

IV. CONCLUSION The type of isolated xilanolitic bacteria based on molecular identification test using 16S rRNA is a type of Bacillus cereus strain CM 100B. Which is a Gram positive and have a positive result on the characterization indole, motility test? Whereas a negative result on the methyl red test.

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