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 37C 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 37C for 2 days. The colonies on the plates

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were transferred on to another agar plate, which were again incubated at 37C 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 Bergeys 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: 96C for 10 sec,
50C for 5 sec, and 60C 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 37C 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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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 15-42C with optimum temperature 37C. 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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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 40C. 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.
REFERENCES
[1]
[2]
[3]
[4]
[5]

[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]

Beg QK, Kapoor M, Mahajan L, and Hoondal GS. 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol .Biotechnol. 56:
26-38.
Brock, T.D., Madigan, M.T., and Martinko, J.M. 1994. Biology of Microorganisme. 7th edition. Pretice-Hall International Inc.London.
Cano, A dan Palet, C. 2007. Xylooligosaccharide Recovery from Agricultural Biomass Waste Treatment with Enzymatic Polymeric Membranes and
Characterization of Products with MALDI-TOF-MS. Journal of Membrane Science 291: 96-105.
Dhanjal, S., dan Cameotra, S.S. 2010. Aerobic Biogenesis of Selenium Nanospheres by Bacillus cereus Isolated from Coalmine Soil. Licensee BioMed
Central Ltd.
Kim, J. J., Kwon, Y. K., Kim, J. H., Heo, S. J., Lee, Y., Lee, S. J., Shim, W. B., Jung, W. K., Hyun, J. H., Kwon, K. K., Kang, D. H., and Oh, C. 2014.
Effective Microwell Plate-Based Screening Method for Microbes Producing Cellulase and Xylanase and Its Application. J. Microbiol. Biotechnol. 24(11),
15591565.
Mashapso, N. 2005. The Microbial Composition of a Natural Methanogenic Consurtium. Thesis. Departement of Biotechnology, University of the Western
Cape.Western.
Meryandini, A., Widhyastuti, N., dan Lestari, Y. 2008. Purification and Characterization Xilanase Streptomyces sp. SKK1-8. Makara, Sains Vol.12: 55-60.
Muthezilan, R., Ashok, R., and Jayalakshmi, S. 2007. Production and Optimazation of Thermostable Alakine Xylanase by Penicilium oxalicum in Solid
State Fermentation. African Journal of Microbiology Research, 20-28.
Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R., and Hiroshi, K. 1993. Purification and Some Properties of an Alkaline Xylanase from Alkaliphilic
Bacillus sp. Strain 41M-1. Applied Enviromental Microbiology. 59(7): 2311-2316.
Ozcan, B.B., Coskun, A.,Ozcan, N., and Baylan,M.2011.Some Properties 0f a New Xylanase from Alkaliphilic and Thermophilic Bacillus sp. Isolate DM15.Journal of Animal and Veterinaryan Advances 10(2):138-143.
Panagan, A. T. 2011. Isolation Microbia Produced Antibiotica from Soil Unsri Indalaya with Soil Extract. Jurnal Penelitian Sains Volume 14.
Pelczar M. J. and Chan, E. C. S. 1988. Based of Microbiology. Jilid 2. Terjemahan Ratna Sri Hadioetomo, dkk., Penerbit Universitas Indonesia. Jakarta.
Viikari, L., Kantelinen, A., Sundqvist, J., and Linko, M. 2001. Xylanases in Bleaching from an Idea to the Industry. FEMS Microbiol. Rev. 13: 335-350.
Waluyo,L. 2007. Microbiology. Universitas Muhammadiyah Malang Press. Malang.
Zumrotiningrum, B. D., Susilowati, A., and Wiryanto. 2004. Lection and Identification of Fungi Selulotik and Lignoselulotik of Distillation Waste Leaves
White Wood (Melaleuca leucadendron L.) from KPH Gundih, Grobogan. Jurnal Biofarmasi 2 (1):24-28. ISSN: 1693-2242.
Sihwandini. 2007. Utilization of Rice Sraw as Media Xylanase Production of Aspergillus niger. Tesis. Departement of Biology, Sains Faculty. Jember
University. Indonesia.
Richana, N., Tun T. I., Anwar N., dan Khaswar S, 2008. Isolation Identification Xylanolitic Bacteria and Characterization Enzyme. Jurnal Agro Biogen.
Vol.4, No. 1, 24-34

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