EXOPOLYSACCHARIDE PRODUCTION, EXTRACTION, AND CHARACTERIZATION FROM SOIL ISOLATE Bacillus spp.

1
EXOPOLYSACCHARIDE PRODUCTION,
EXTRACTION, AND CHARACTERIZATION FROM
SOIL ISOLATE Bacillus spp.
Ahmed Farag 1 ; Walaa Gamil 2 and Ehab Essawy 3
1Microbiology Department, Faculty of Science, Helwan University, 11795, Cairo,
Egypt.
2Biochemistry Master Program, Chemistry Department, Faculty of Science, Helwan
University, 11795, Cairo, Egypt.
3Chemistry Department, Faculty of Science, Helwan University, 11795, Cairo, Egypt.
Key Words: Exopolysaccharides, 16srRNA, HPLC, FTIR.
ABSTRACT
Exopolysaccharides (EPS) possess a reducing power that applied in
a wide range of biotechnological applications, like biosynthesis of
nanoparticles. In presented work, two samples from different plant soils
(clover and wheat) were collected for screening of EPS producing strain.
Our results showed that, only one colony of wheat soil formed a mucoid
surface on agar plates, this strain was identified morphologically and
confirmed by molecular characterization using 16srRNA as Bacillus pp.
The ethanolic extract of EPS had been characterized by High
performance liquid chromatography (HPLC) and Fourier transforminfrared
spectroscopy (FTIR). Our finding opens the door for utilizing
Bacillus Spp. as a promising natural source for EPS production.
INTRODUCTION
Microbial exopolysaccharides (EPS) are natural, non-toxic, and
biodegradable polymers produced and secreted by microbial cells to their
surrounding environment. Polysaccharides produced by microbes can be
generally classified by their biological functions into intracellular storage
polysaccharides (glycogen), capsular polysaccharides which are closely
linked to the cell surface (CPS) and extracellular bacterial
polysaccharides also termed exopolysaccharide (EPS) which form a
slime layer loosely attached to cell surface or secreted into environment
(for example, xanthan, sphingan, alginate, cellulose, etc.) .Various groups
of microorganisms are known to produce exopolysaccharides such as
bacteria (including extreme and marine bacteria) (Laurienzo, 2010;
Nicolaus et al., 2010) cyanobacteria (Laurienzo, 2010), fungi and yeasts
(Mahapatra & Banerjee, 2013). EPS synthesis play crucial biological
roles in maintaining viability of microbial cell. Among these functions
include adhesion to solid surfaces, cell-cell interactions , forming biofilm
on the surfaces to increase cell protection against environmental extremes
(Ates, 2015). The production of polysaccharides by microbes is achieved
within days and weeks as opposed to plants where production takes 3–6
months (Donot et al., 2012). Recently, the structure and properties of
microbial exopolysacchar`ide have attracted the scientific community,
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making them more suitable for bio nanotechnology and commercial
applications in different industrial sectors like food, petroleum, and
pharmaceuticals. The synthesis and stabilization of metal nanoparticles
using microbial exopolysaccharide was of interest because EPS contain
various functional groups that can act as reductive and stabilizing agents
via a chelating and capping process in the synthesis of metal
nanoparticles (Emam & Ahmed, 2016). The object of this study was to
screen and isolate efficient EPS producing strain and to characterize
extracted exopolysaccharide.
MATERIALS AND METHODS
1.Samples Collection
Two soil samples were collected from the village of Senourse,
Faiyum governorate, Egypt (Figure 1). The first clover soil sample and
the second wheat soil sample. All soil samples have been collected in
sterile plastic bags and stored at 4oC for further analysis.
Figure 1. Location of Senourse village, Fayoum.
29.3858558,30.8651253
2. Isolation, Screening and Identification of Exopolysaccharide
Producing Strain
The soil samples collected were used as the primary inoculum for
the isolation of bacterial strains. One gram of each soil was added to 9 ml of
sterile distilled water for soil suspension. Serial dilutions (10-1 to 10-7) were
performed and then inoculated on nutrient agar plates containing the
following components (g / l): peptone 5.0 g, beef extract 3.0 g, sodium
chloride 5.0 g and agar 15.0 g. For each soil sample, inoculated plates were
165 Egypt. J. of Appl. Sci., 35 (11) 2020
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incubated at 37 ° C for 24 h. Colonies with different morphological
characters have been collected and cultivated for further purification. The
pure exopolysaccharide (EPS) colonies producing bacteria have been spread
over modified nutrient agar media containing peptone 5.0 g, beef extract 3.0
g, agar 15.0 g, sodium chloride 5.0 g, sucrose 40 g and distilled 1000 ml of
water and incubated for 72 h at 30 ° C (Pawar et al., 2013).
Exopolysaccharide strain was selected by observation of mucoid colony
(Fusconi & Godinho, 2002) and was named SN-2.
3. Identification of Exopolysaccharide Producing strain
Gram staining was used to determine morphological properties such
as colony color, colony shape, gram stain and cell shape were observed
under light microscope. A strain was confirmed by molecular
characterization using 16s rRNA. Genomic DNA was extracted by the TE
boil extraction method for molecular characterization of the isolated strain
(Li et al., 2003). Two universal oligonucleotides bacterial primers (16S
rRNA forward primer: 5-GAG TAA TGT CTG GGA AAC TGC CT-3
and16S rRNA reverse primer: 5 CCA GTT TCG AAT GCA GTT CCC
AG-3) were used.
The PCR conditions used in the amplification of 16S rRNA genes
were the initial denaturation of 95 ° C for 5 min followed by 35 cycles of 95
° C for 1.5 min, 59 ° C for 1 min and 72 ° C for 1.5 min, with the final 10
min extension at 72 ° C. The 16S rRNA gene sequences were compared to
those of the GeneBank and EMBL databases by advanced BLAST
(Megablast) searches from the National Center for Biotechnology
Information (NCBI). The phylogenic relationship of the isolate was
determined by comparing the sequencing data with the related 16S rRNA
gene sequences in the GeneBank database of the National Center for
Biotechnology Information, via BLAST search. The phylogenetic tree was
constructed by the Geneious Pro 8.9 programs.
4. EPS Production and Extraction
Production of EPS was carried out by active growing inoculum of
isolated bacterial strains with inoculum size (100 μl) in a 250 ml bottle
containing 100 ml of 4.0 g sucrose NB, 0.5 g peptone, 0.3 g beef and 0.5 g
NaCl and pH adjusted to 7.5. Bacterial culture was incubated at Bulgara
Shaking incubator ® Model: VS-8480 at 35oC fo4.r 72 hours at 150 rpm.
EPS extraction was achieved by a (Pawar et al., 2013) minor modification
method, where the cells were removed by cooling centrifugation (Sigmas 3–
16 PK) at 10,000 rpm for 20 min at 4 ° C. The EPS was precipitated by
adding a double volume of refrigerated absolute ethanol to the cell-free
culture and kept at 4°C overnight. Ethanol extract was centrifuged at 10,000
rpm at 4°C for 20 min, crude EPS was collected and dried at 60oC.
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5. Characterization of EPS
Sugar monomers in EPS have been characterized by the use of highperformance
liquid chromatography (HPLC) Shimadzu Class-VPV 5.03
(Kyoto, Japan) equipped with a refractive index RID-10A Shimadzu
detector, L-C-16ADVP binary pump and PL Hi-Plex Pb column (Bebault
et al., 1973). EPS was subjected to hydrolysis by 2N sulfuric acid for 24 h.
The precipitate was filtered off at the end of the hydrolysis. Barium
carbonate was used for precipitation removal of sulfate from the filtrate. The
hydrolysate was evaporated under vacuum by means of a rotary evaporator
to produce residues that were dried and extracted with hot distilled pyridine.
The pyridine extract was evaporated to dryness and the residues were stored
at 4oC for HPLC analysis. Functional groups present in the purified EPS
were determined by Fourier transform infrared (FTIR) spectroscopy
(PerkinElmer ATR Sample Base Plate DIAMOND spectrometer) equipped
with KBr beam splitter with MIR TGS detector (4000-450 cm-1) at the
Central Lab, Faculty of Science, Helwan University, Egypt (Sterner et al.,
2008) .
RESULTS
1. Isolation and Screening of Exopolysaccharide producing bacteria
Two samples from different plant soils (clover and wheat) were used
to screen EPS strains, spread pure different colonies for each soil sample
over a modified nutrient agar medium containing 4 percent sucrose, and
incubated at 30oC for 72 hours. After incubation period the isolated five
colonies from clover soil didn’t show any mucoid surface on the nutrient
agar plates, however only one colony of wheat soil showed a mucoid surface
on the agar plates this an indication of EPS production as showed in Figure
2 this strain named as SN-2 was inoculated on nutrient slant and maintained
at 4oC further characterized by morphological and molecular
characterization.
Figure 2: Mucoid layer of isolated strain on nutrient plate.
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2. Morphological and Molecular Characterization of isolated strain
The strain-producing bacterial exopolysaccharide was identified on
the basis of morphological character as gram-positive rod-shaped
bacteria. Further confirmation was made using molecular
characterizations. The RNA of the strain was amplified with universal
primers. Purified PCR products have been sequenced in one direction
using forward universal primers. Based on the alignment of 16S rRNA
gene sequences from the GeneBank database and the phylogenetic tree,
the 16S rRNA gene sequence of the strain showed the highest similarity
to Bacillus spp with a 97.5 percent sequence identity.
3. Characterization of bacterial EPS
The functional groups of the EPS were characterized by Fourier
Transform Infrared (FTIR) spectroscopy. The spectrum (Figure
3) showed peaks at 3293, 2926, 2132, 1811, 1721, 1633, 885 and 724
cm−1. The strong band at 3289 cm−1 may correspond to broad stretching
O-H group; the peak at 2922 cm−1 is corresponding to C-H stretching
peak of methylene groups, and the band at 1727 cm−1 may due to C=O
vibrations. The monosaccharide units of the EPS extract were determined
by high performance liquid chromatography (HPLC). The extract
contains mainly galactose (80.3%) and glucose (19.7%). Standard peaks
of galactose and glucose correspond to peaks of tested EPS respectively
was found during the analysis (figure 4).
Figure 3: FTIR Chart of exopolysaccharide extracted from Bacillus spp .
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Figure 4: HPLC chromatogram of exopolysaccharide extracted from
Bacillus Spp.
DISCUSSION
Soil contains varieties of microorganism including bacteria that can be
established in any natural environment. A wide range of bacteria are
known to produce exopolysaccharides. Some studies approached to
produce exopolysaccharides (EPS) by bacterial species isolated from soil.
As appeared in study of (Sirajunnisa & Surendhiran, 2014) , this study
interested to use bacterial strain isolate (Pseudomonas fluorescens)
isolated from soil as source of exopolysaccharide and study of (Shukla
& Patel, 2015) which showed production of exopolysaccharide by
Bacillus coagulans isolated from soil (Benson, 2002) . During our
screening program for exopolysaccharide production from bacteria ,
only one bacterial strain showed a slimy surface , (an indication of EPS
production) (Fusconi & Godinho, 2002).The use of 16S rRNA gene
sequencing in the clinical laboratory play a very important role in
identifying biochemically unidentified bacteria or for providing reference
identifications for unusual strains (El-Batal et al., 2016) .The DNA of
the isolated strain was amplified with universal primers. showed that the
PCR product was 521 bp. Based on the alignment of 16S rRNA gene
sequences from the Gene Bank database and the phylogenetic tree, the
16S rRNA gene sequence of the strain showed the highest similarity to
Bacillus spp with a 97.5 percent sequence identity.
The functional groups of the EPS were characterized by FT-IR
spectroscopy. The spectrum showed the strong band at 3293 cm−1 may
correspond to broad stretching O-H group; the peak at 2926 cm−1 is
169 Egypt. J. of Appl. Sci., 35 (11) 2020
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corresponding to C-H stretching peak of methylene groups, and the band
at 1721 cm−1 may due to C=O vibrations. These bands are related to the
sugar ring and is accountable for the solubility of EPS in water (Galvez
et al., 2019). The detected C-H stretching peak is corresponding to the
hexoses such as glucose or galactose (Castellane et al., 2015). The set of
bands between 1047 and 616 cm-1 can be used to describe
polysaccharides (Wang et al., 2018). The monosaccharide units of the
EPS extract were determined by high performance liquid
chromatography (HPLC). The extract contains mainly galactose (80.3%)
and glucose (19.7%). It was reported that most of the exopolysaccharides
(EPS) mainly composed of galactose and glucose as well as some other
sugars like rhamnose, gluconic acids, and amino sugars (Roca et al.,
2015; Sathiyanarayanan et al., 2017).
CONCLUSION
The isolated strain from wheat plant soil showed a mucoid surface
on nutrient agar plate and used as source of EPS production. The
exopolysaccharide produced by bacteria in broth media was extracted by
ethanol and characterized by FTIR which shows the presence of O-H
group methylene groups, and C=O vibrations. The sugar monomers of
EPS were characterized by HPLC. The extract contains mainly galactose
(80.3%) and glucose (19.7%). Hence, the resultant product could be
utilized in biotechnological applications.
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استخلاص و توصيف السکريات المتعدده التي تم انتاجيا بواسطو السلالو
المعزولو من التربو الز ا رعيو Bacillus spp البکتريو
احمد حسن اب ا رىيم ف ا رج 1 ، ولاء جميل السيد محمد 2 ، ايياب عبد الرؤوف عيسوي 3
-1 مدرس الميکروبيولوجي- قسم النبات والميکروبيولوجي- کميو العموم – جامعو حموان
-2 طالبو ماجستير- قسم الکيمياء- کميو العموم- جامعو حموان
-3 مدرس الکيمياء الحيوي- قسم الکيمياء- کميو العموم – جامعو حموان
التي يتم انتاجيا بواسطو البکتريو احد التطبيقات التي يتم (EPS) تعتبر السکريات المتعدده
استخداميا في مجال التکنولوجيو الحيوي, وذلک يرجع الي الترکيب الکيميائي ليذه السکريات
التي يمکنيا ان تعمل کعامل مختزل في تخميق الجزيئات النانومتريو. في العمل المقدم تم
البحث عن عزل سلالو بکتيريو لدييا القدره عمي انتاج ىذه السکريات , حيث تم تجميع عينتين
من تربو نباتيو مختمفو ) القمح و البرسيم ( لفحص السلالو البکتريو المنتجو لمثل ىذه السکريات
Egypt. J. of Appl. Sci., 35 (11) 2020 172
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. اظيرت نتائجنا ان سلالو بکتيريو واحده فقط من تربو القمح لدييا القدره عمي انتاج السکريات
المتعدده عمي سطح الوسط الغذائي لمبکتريو , وقد تم التعرف عمي ىذه السلالو ظاىريا باستخدام
16 انيا srRNA الميکروسکوب الضوئي وتأکيدىا من خلال البيولوجيا الجزيئيو باستخدام
تم استخلاص السکريات المتعدده التي تم انتاجيا بواسطو ىذه ,Bacillus spp مطابقو ل
HPLC و FTIR spectroscopy السلالو باستخدام الکحول الايثيمي و توصيفيا باستخدام
لمعرفو الترکيب الکيميائي الخاص ليذه السکريات المستخمصو, ومن خلال chromatography
کمصدر طبيعيا واعد لانتاج Bacillus spp ىذه الد ا رسو يمکن فتح الباب الي استخدام
EPS
173 Egypt. J. of Appl. Sci., 35 (11) 2020