Author’s Accepted Manuscript

Isolation and Characterisation of Urease-producing

Bacteria from Tropical Peat

Ignatius Ren Kai Phang, Yen San Chan, Kwong

Soon Wong, Sie Yon Lau

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PII: S1878-8181(17)30613-8
DOI: https://doi.org/10.1016/j.bcab.2017.12.006
Reference: BCAB672
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 27 November 2017
Revised date: 6 December 2017
Accepted date: 14 December 2017
Cite this article as: Ignatius Ren Kai Phang, Yen San Chan, Kwong Soon Wong
and Sie Yon Lau, Isolation and Characterisation of Urease-producing Bacteria
from Tropical Peat, Biocatalysis and Agricultural Biotechnology,
https://doi.org/10.1016/j.bcab.2017.12.006
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 Isolation and Characterisation of Urease-producing Bacteria from Tropical Peat
Ignatius Ren Kai Phang1, Yen San Chan2,*, Kwong Soon Wong1, Sie Yon Lau1
1
Department of Civil and Construction Engineering, Faculty of Engineering and Science, Curtin University
Malaysia, CDT 250, 98009 Miri, Sarawak, Malaysia.
2
Department of Chemical Engineering, Faculty of Engineering and Science, Curtin University Malaysia,
CDT 250, 98009 Miri, Sarawak, Malaysia
*Corresponding author: chanyensan@curtin.edu.my

Abstract
Urease were known to catalyze the conversion of urea to ammonia and carbon dioxide.
Microbial urease has demonstrated its benefits in wide biotechnological, agricultural, medicinal
and engineering application. There are number of diverse microbial species contribute to
urease activity in different natural habitats like soil, ocean and in various geological formation.
For this study, urease bacteria were screened and isolate from acidic peat in Sarawak, Malaysia.
Five distinct and diverse bacterial strains that were able to produce urease constitutively were
selected to be characterized with respect to morphology, biochemical test, growth conditions
and urease activity. The selected strain showed their capability to precipitate calcium carbonate
(CaCO3). Hence, the isolates could be potential source of acid ureases that can be use in various
industrial utilizations. 16S rRNA sequencing and phylogenetic analysis found that the selected
isolates belong to the genus of Bacillus.
Keywords: Urease; Bacteria; Peat; 16 rRNA sequencing
Highlights
 Urease producing bacteria were isolated from tropical peat in Sarawak, Malaysia
 Urease activity varies between isolated strain in acidic and alkaline pH
 Isolated strains capable of calcium carbonate precipitation
 16s RNA sequencing and phylogenetic analysis of selected isolates
1 Introduction
Urease or urea amidohydrolase (EC 3.5.1.5), a nickel metalloenzyme was known as the first enzyme to
be isolated as crystalline protein that catalyze the conversion of urea to ammonia and carbon dioxide
(Sumner, 1926; Dixon et al.,1980; Mobley et al. 1988). Microbial urease has been well studied from a
clinical perspective for its role in virulence factors in microorganisms contributing to urinary stones,
pyelonephritis and gastric ulceration (Collins and D'Orazio, 1993; Mobley et al., 1995; Lee and Calhoun,
1997; Rutherford, 2014). Regardless of its reputation as virulence mechanism, microbial urease has also
been shown extensive application in biotechnology and agriculture (Qin & Cabral, 2002). Urease has
demonstrated its benefits in medical uses including removal of urea for kidney failure and dental care
(Prakash and Chang, 1995; Clancy et al., 2000; Liu et al., 2012). Urease were immobilized and use as
biosensor for various target compound (Cullen et al., 1990; Sansubrino and Mascini, 1994; Senillou et al.,
1999; Soldatkin et al., 2000). Singh et al. (2017) has described the purification of urease enzyme from
Bacillus sphaericus for biosensor development. Urease were discussed to be use along with fertilizer due
to low cost production and handling which provide adequate nitrogen (Glibert et al., 2006). Traditionally
microbial urease has been used in fermented drinks and alcoholic beverage including sake, chinese
wine, red and white wine removing excess urea that leads to ethyl carbanate (EC) formation (Kobashi et
al.,1988; Ough & Trioli, 1988). For such purposes, acid urease was potentially studied from various
sources of bacteria strain including genus from Lactobacillus (including L. fermentum, L. reuteri, L.
animalis, L. salivarius, L. ruminis, L. delbrueckii), Streptococcus (including S. mitior S. salivarius, S.
thermophilus), Arthrobacter mobilis, Weissella (including W. cibaria, W. confusa, W. viridescens),
Enteroccocus (including E. faecalis, E. faecium, E. pseudoavium), Enterobacter sp., Providencia rettgeri
(Fujinawa et al., 1990; Yamazaki et al., 1990; Kaimoto et al., 1990; Miyagawa et al., 1999; Mora et al.,
2005; Fidaleo et al., 2006; Esti etl al., 2007; Zotta et al., 2008; Andrich et al., 2010; Liu et al., 2012; Zhang
et al., 2016; Arioli et al., 2017; Liu et al., 2017).

Microbial urease catalyzed biomineralization process also known as microbial induced calcite
precipitation (MICP) has been widely introduced in construction and materials, geotechnical engineering
and environmental applications (De Muynck et al., 2010; Phillips et al., 2013; Wang et al., 2017). For
example, Bacillus sphaericus and B. licheniformis were used for biosynthesis of calcium carbonate
(Seifan et al., 2016; Seifan at al., 2017). High urease production bacteria like Sporosarcina pasteurii
(formerly B. pasteurii) and B. megaterium were widely used for biomineralization sand and soil
(Hammad et al., 2013; Soon et al., 2013; Jiang et al., 2016). Apart from those above, engineering
application of other ureolytic microorganism including S. aquimarina, A. aerogenes, B. subtilis, B.
thuringiensis, D. halophila, H. eurihalina, H. pylori, K. flava, L. sphaericus, M. parvum, M. xanthus, P.
mirabilis, P. denitrificans, Spoloactobacillus sp., and S. ginsengisoli were discussed by Anbu et al. (2016).
Different bacteria strain has different capability of urease activity and its application (Achal et al.,
2009a).

Urease can be isolated in various bacteria, fungi, and higher plants where known ureases possessed
similar catalytic mechanism and structure (Karplus et al., 1997). Notably, urease is produced by diverse
soil microorganisms (Mobley & Hausinger 1989; Hammes et al. 2003). Ammonia, the product of urea
hydrolysis is the preferred nitrogen source which can be utilized by bacteria to assimilate variety of
nitrogenous compounds (Ferris et al. 2003; Burbank et al. 2012). In various studies, peat was observed
to possess urease activity due to the indigenous microbial community (Herlihy, 1971; Vetanovetz &
Peterson, 1987; Nannipieri et al., 2002) including peat from tropical regions (Siva et al., 1999). Limited
study was done on isolation of urease-producing bacteria from peat, characterization of urease activity
and calcium carbonate precipitation ability. The present work focuses on screening and isolation of
urease-producing bacteria from acidic tropical peat providing opportunity for acidic tolerance bacterial
strain isolation. Isolates were selected for characterization and preliminary test on their urease activity.
Selected strains were then used for 16S rRNA sequencing and phylogenetic study. Current study would
provide an insight on the potential alternative urease sources of bacteria from tropical peat. These
isolates capable to produce urease in acidic condition and precipitate carbonate which could be
employed in various application including biosensors, removal of urea, waste water removal of calcium
ions and in-situ soil stabilization resulting from MICP including in acidic soil environment.

2 Methodology
2.1 Collection, enrichment of sample and bacteria Isolation

Peat soil were collected aseptically in Curtin Malaysia (4°30'43.1"N 114°00'45.7"E) Miri, Sarawak,
Malaysia in radiation-sterilized polyethene zipper bag. The sample collected were from 1m depth of
peat where anoxic occurs and pH was recorded to be in the range of 3.8-4.9 indicating acidic
environment. Isolation of urease producing bacteria were done by enrichment method of peat which 1 g
peat was inoculated in 50 ml of nutrient broth (NB) (Sigma, USA) containing 2% sterile urea (Sigma, USA)
and incubated at incubation orbital shaker for 48 hours at 30oC with 140 rpm. Potential isolates were
obtained by the serial dilution method and by plating the samples on nutrient agar supplemented with
2% urea. The plates were incubated at 30oC and subsequent subculture was done until pure
distinguishable single bacterial colonies were obtained.

2.2 Selection of urease-producing bacteria

Potential urease producing isolates were screened using Christensen’s medium or urea agar (Himedia,
India). The agar medium contained the following; 1.0 g/l of Peptone, 1.0g/l of Dextrose, 5.0 g/l of
Sodium chloride, 2g/l of Monopotassium phosphate, 0.012 g/l of Phenol red and 15.0 g/l of agar. 2%
filter sterilized urea (Sigma, U5378) were supplemented. Single colony was selected and inoculated on
medium and then incubated at 30oC for 5 days. E. coli W (ATCC 9637) was used as a negative control.
The urease production was examined through visual observation every day throughout the incubation
period for color changes in medium from pale yellow to pink red. A change in color following incubation
period recorded as a urease-positive and their capability to rapidly produce urease qualitatively. Five
isolates designated as P1, P2, P3, P9, and P15, were selected for further studies based screening results
on urea agar.

2.3 Bacteria characterization

Characterization of colony and cell morphology was observed under light microscope (Nikon, JP) while
endospore staining, motility, catalase test and oxidase tests were performed by standard methods (Holt
et. al., 1994; Cowan & Steel, 2003; Mac Faddin, 1976). Growth tolerance of the five isolates were also
screened for parameters including pH (4-8), temperature (15oC - 50oC) and NaCl (0% - 12%) (Sigma, USA).

2.4 Cell culture and concentration measurement

Bacterial cells were pre-cultured in nutrient broth at 30oC and 140 rpm until log phase of the bacterial
growth. 1 mL of the preculture was inoculated into 100 mL of nutrient broth and incubated at
incubation orbital shaker at 30oC and 140 rpm. During the cultivation, cell concentration was
determined by measuring optical density (OD600) of bacterial suspension with a spectrophotometer
(Lambda 25 UV/Vis Double Beam, Perkin Elmer) at a wavelength of 600 nm (Harkes et al., 2010). Dilution
were performed if required to be within absorbance range of 0.2 to 1. Following the cell concentration
measurement, the urease activity was also measure as described below.

2.5 Urease activity measurement

One unit of urease activity is defined as the amount of enzyme that would hydrolyze 1 μmol urea per
minute (corresponding to 2 μmol of ammonia) per min under the assay conditions. Conversion of urea
to ammonia is stoichiometric, hence activity of urease was identified through ammonia formation by a
modified Nessler method (Nakano et al., 1984; Greenburg et al. 1992). The effect of cell biomass
concentration and urease activity was observed throughout incubation period. The bacteria pellet was
washed 3 times with buffer solution and re-suspended into 100 mM Tris/HCl buffer at pH 8 containing
300mM of urea and incubated at 30OC for 10 min. Then, the culture was centrifuged to obtain the
supernatant. 1 mL of supernatant was added with 100 μl of Nessler reagent (Fischer Scientific) in the
cuvette, and allowed to react for minute before taking the reading with spectrophotometer (Lambda 25
UV/Vis Double Beam, Perkin Elmer) at 425 nm. The absorbance readings were calibrated with using
different concentration of NH4Cl standards measured with same conditions as above. The sample was
diluted with deionized water in required range if necessary using a volumetric flask. As for the effect of
pH conditions on the urease activity, test was performed using 48 hours cultured cells at 30oC at
different pH values of 4, 7, 9 and 11.

2.6 Screening for calcium carbonate (CaCO3) precipitation

The ability of the isolated urease bacteria to precipitate carbonate were screened by culturing on the
calcium carbonate precipitation agar with calcium sources (Klement et al. 1990). The agar plates were
then incubated for 4 days at 28oC and the precipitation of crystals on agar were observed under light
microscopy. Calcite precipitation media which contained 3.0 g/l nutrient broth, 28.5 g/l CaCl 2, 2.12 g/l
NaHCO3 and 20 g/l urea (Ferris & Stehmeier 1992; Ferris et al. 1997) were prepared and sterile filtered
then pH was adjusted 6.5. The inoculated bacteria strains were incubated for 48 hours at 28oC. The test
were done in triplicates. CaCO3 that was precipitated were filtered with filter paper (Whatman filter
paper) followed by drying in 60°C oven for 48 hours before its weight was determine as estimation of
CaCO3 precipitation (Klement et al. 1990).

2.7 16s rRNA sequencing and analysis

For identification of the bacterial isolates, pure single colony was inoculated and grown overnight for
bacterial DNA extraction. The extraction was performed with MYgenTM Genomic DNA Prep Kit
(GeneXpress, MY) according to instruction provided. 16S rRNA gene was amplified by polymerase chain
reaction (PCR) using primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-
GGTTACCTTGTTACGACTT-3’) as the forward and reverse primers of 16S rDNA. The PCR program was
started with initial denaturation at 95 oC for 3 mins followed by 30 cycles of 20 s at 98 oC, 15 s at 64 oC,
50 s at 72 oC, and final extension at 72 oC for 50 s. The PCR products were then purified using MYgenTM
Gel & PCR Purification System (GeneXpress, MY) and sequencing were done at First BASE Laboratory
(Malaysia).
16S rRNA gene sequence was compared with database from GenBank using BLAST (Basic Local
Alignment Search Tool) available online at National Center for Biotechnology Information (NCBI)
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997). A phylogenetic tree was constructed by
the neighbor-joining method using MEGA 7.0 software with associated taxa clustered together in the
bootstrap test based on 500 replications for confidence values of the branches and evolutionary
distances computed using the Maximum Composite Likelihood method and are in the units of the
number of base substitutions per site (Felsenstein, 1985; Saitou & Nei, 1987; Tamura et al., 2004; Kumar
et al.,2016). The 16S rRNA gene sequences for P15 and P9 were then deposited in GenBank (NCBI) under
the accession numbers MG575433 and MG571570 respectively.

3 Results and discussion

3.1 Isolation and characterization of isolated bacteria

Among the isolated, only 5 bacterial strains were selected based on fast color formation of urease
activity within 24 hours from urease agar test (Figure. 1). Several studies have reported the use of urea
agar base as a quick differentiation screening of ureolytic bacteria can be utilized as a preliminary quick
screening method for isolation of urease producing bacteria (Achal et al., 2011; Burbank et al., 2011;
Hammad, et al., 2003). Urea agar base were supplemented with sterile urea and phenol red in the agar
as a pH indicator. Hence, when urea hydrolysis occurred, the process will increase the pH of the
environment render it alkaline thus altering the color of phenol red. However, false positive reactions
due to the utilization of peptones or other proteins may occur in which the pH were increased due to
protein hydrolysis and the release of excessive amino acid residues. Hence, the isolates were proceeded
for quantitative analysis.

Strain P1, P3, P9 and P15 was observed as rod shaped under light microscope and motile while P2 were
cocci and non-motile. No pigments were observed on any of these isolates when grown on nutrient
agar. Colony morphology differs among the isolates and were shown in Table 1. Strain P1, P2, P3, P9 and
P15 isolates showed growth between a pH range of 4-9. When subjected to salinity tests, there were
growth observed in P1, P2, and P9 in 0–12% NaCl concentration while for isolate P3 and P15, growth
was seen in 0-10% and 0-8% NaCl respectively. For temperature tolerance, five of the isolates seen to
grow in testing range of 15oC to 40oC respectively. Physiological features discussed above display
noticeable differences between the isolates as shown in Table. 1.

3.2 Urease activity of selected strain

Optical density (OD) or absorbance were known to be linearly proportional towards the cell
concentration in a solution based on Beer-Lambert Law (Parks 2009). The course of cell growth to
concentration by mean of measuring OD600 and urease activity of the selected isolates through a 24
hours incubation period in different time intervals at 0, 60, 180, 240, 360, 420, 480 and 540 min
respectively were shown in Figure. 2. The trend of each isolates was observed to be slightly different
between each other which distinguish each of them from each other. Urease activity of strain P1, P2, P9
and P15 peak at 240 min while following a decrease in activity and a lower second peak at 480 min. The
trend for P3 were different with peak urease activity at 360 min and a second lower peak activity at 540
min. The urease activity of the cell was seen to increase following the increasing cells growth in the
exponential phase until a point near the transitional phase of cell growth where it will drop following a
decrease in cell concentration. However, the trend does not suggest any obvious correlation between
cell biomass and urease activity. Different strain of bacteria will show different correlation of bacteria
biomass and its urease activity. Whiffin (2004) suggested that urease activity of P. vulgaris was produced
proportionally to biomass while higher growth for S. pasteurii does not directly induce increasing urease
production.

Urease activity of strain P1, P2, P3, P9 and P15 under 4 different pH conditions is shown in Figure. 3. The
graph displayed almost bell-shaped profile with its maximum enzyme activity around pH 9 except for
P15 which peak at pH 4. Minimum activity of urease was found for P1, P2, P3 and P9 under acidic (at pH
4) and alkaline (at pH 11) conditions as protein denaturation occurs under the harsh alkaline conditions.
However, it was interesting to note that urease activity for strain P2, P3, P9 and P15 were observed to
be higher in pH 4 compared to their activity in neutral condition (pH 7) which suggested acidic urease
compatibility. Generally, acidic environment will cause the denaturation of protein and its release of
nickel from the enzyme leading to irreversible loss of activity (Mobley & Hausinger, 1989). Acid urease
can be found in several genus including Arthrobacter, Bifidobacterium, Escherichia, Lactobacillus,
Staphylococcus, Streptococcus, Morganella, and Zoogloea species (Fidaleo et al., 2006). Certain bacteria
living in harsh environment like Helicobacter pylori will produce large amount of urease with exposure
to acidic condition (Bauerfeind et al., 1997) and certain chemolithotrophic ammonia-oxidizing bacteria
(AOB) will also contribute to higher urease activity at lower pH (Pommerening-Röser & Koops. 2009).
Generation of urease enzyme is an inducible event related to stress response of bacteria to survive at
acidic condition (Cotter and Hill, 2003). Hence, typical urea hydrolysis will neutralize an acidic
environment leading to the survival of the bacteria. Different strain of bacteria has different tolerance
towards pH changes. Strain P2 has a different trend compares to the others, urease activity was low in
pH 7 and pH 11 while it peaks at pH 9. Previous studies had shown that urease enzyme tend to be more
active in an alkaline environment (Stocks-Fischer et al.,1999; Anne et al., 2010). Such phenomena can be
seen on certain microbial urease like S. pasteurii and Klebsiella aerogenes that has a maximum activity
under weak alkaline conditions (Lauchnor et al., 2015; Park & Hausinger, 1993).

3.3 Calcium carbonate (CaCO3) precipitation

Calcite precipitation agar provided opportunity for the observation of Microbial-induced Calcite
Precipitation (MICP) by bacteria colony at a specific confined local micro-environment. Crystallization
were observed on all the selected urease strain on solid agar. Generally, the darkening observed on the
colony were known as initiation of precipitation which due to formation of amorphous CaCO3 between
24 hours and 3 days (varies by strain) and which then followed with the crystallization and the
maturation of the crystal which were also observed by Hammes et al. (2003). The crystal precipitation
was found to form within the bacteria colony on the agar surface as shown in Figure 4a. Such
phenomena of crystal generate within parent colony were also observed by Murai & Yoshida (2013). The
precipitation of crystal within the bacteria possibility leads to microbial incapacitation resulting from
encasement in calcite discussed by Cuthbert et al. (2012) and Tobler et al. (2012). However, for strain
P9, calcium carbonate crystals were found to form at surrounding of cell colony as shown in Figure. 4b.
The strains were then selected for precipitation study in broth medium. Figure 5 showed the amount of
CaCO3 precipitation was in such order: Strain P15 > P9 > P2 > P1 > P3. Microbial metabolism activity
leads to bio-cementation in generating intra-cellular or external insoluble compounds of inorganic and
organic. This can be seen such as those that produce organic protein like glycocalyx on their cell wall
which persist in the environment after cell death while others tend to cause the accumulation of
inorganic compounds which included carbonate, phosphate and silicate (MacLoad et al., 1988; Stocks-
Fischer et al.,1999). Certain bacteria have been known to their ability to increase environmental pH
through various physiological metabolism. For urease producing bacteria, urea was hydrolysed to
ammonia and carbonic acid which equilibrate in water to form bicarbonate, ammonium and hydroxide
ions. Hydroxide ions produced increases the surrounding pH and shifts the bicarbonate equilibrium
equation to the right resulting carbonate ions production (Fujita et al., 2008). Apart from environmental
pH alteration, bacterial surfaces with its were known to favors and contributes to carbonate
precipitation by attracting positively charged metal ions with its negatively charged surface acting
inducing heterogenous nucleation (Fortinetal.,1997; Bäuerlein,2003). Typical carbonates precipitation
occurred externally at surface of bacterial cells through successive stratification leading to the bacterial
cells trapped in the growing carbonate crystals (Rivadeneyra et al., 1998; Castanier et al., 1999).

3.4 Bacterial identification using 16S rRNA sequence analysis

Due to limitations of morphological identification as mentioned above, the ribosomal RNA (16S rRNA)
was also used for identification in this study. At present, the sequence analysis technique of 16S rRNA,
genes encoding small-subunit ribosomal RNA were commonly used for the phylogenetic classification
and reconstructing of prokaryotic phylogenies (Janda & Abott, 2007; Case et al., 2007). The comparative
analysis of 16S rRNA gene sequences also provides ways to investigate the dissimilarity between strains
collections and natural communities (Ferris et al., 1996; Weller et al., 1991). Isolates P9 and P15 were
subjected to 16s rRNA sequence method for bacteria strain identification. The 16s RNA nucleotide using
BLAST showed both isolates belong to family Bacillaceae and within the genus Bacillus. Phylogenetic
tree analysis was shown in Figure 6 and revealed that strain P9 and P15 falls into Bacillus cereus group
which are common spore-forming soil dwelling microorganism that are Gram-positive and has recorded
an independent branch within the Bacillus genus (Jiménez et al., 2013). Both P9 and P15 shown closely
related to the species B. cereus. B. cereus were well-known as ubiquitous soil bacterium and an
opportunistic pathogen which commonly associated with food poisoning (Helgason et al., 2013). Despite
its belonging to Risk group 1 (low individual and community risk), a variety of B. cereus were available
commercially for human probiotic (Duc et al., 2004). In nature, B. cereus benefits plants growth by
interacting with other pathogenic plants microbes in the rhizosphere and inhibiting plant disease caused
by protest pathogens (Jensen et al., 2003). Certain strain of B. cereus was found to produce antibiotics
zwittermicin A. and kanosamine which disrupt plant pathogen, oomycetes (fungus-like eukaryotic
microorganisms), variety of fungi and a certain bacterial species (Silo-Suh et al., 1994). B. cereus were
known to carry out ammonification and dissimilatory nitrate reduction which leads to bacterial
carbonatogenesis (Castanier et al. 2000). B. cereus isolated from surfaces of dolomitic (Karst
topographies) were found to precipitate calcium carbonate (Han et al., 2013). Urease activity of B.
cereus were found to increase the compressive strength of cement mortar by inducing biomineralization
(Maheswaran et al., 2014).

4 Conclusions

In conclusion, the findings indicate that there are various strains of bacteria in tropical peat capable to
produce urease. The urease activity of the isolates was inducible by environmental factor rather than
being govern by cell biomass. It was found that isolated strain from acidic peat environment were able
to produce urease activity in lower pH suggesting acidic urease compatibility. The isolates shown their
potential as a source of acid ureases in various industrial utilizations especially those that required
urease activity in acidic condition. The five isolated strains were also found capable to precipitate
calcium carbonate crystal in solid agar and with strain P15 producing highest precipitation compared to
the others in broth media. Although the test was not optimized, their ability to produce carbonate
crystal may provide an alternative sources of calcite precipitating bacteria for bio-cementation works
and soil stabilization resulting from MICP including in acidic soil. From 16s RNA bacteria identification,
Strain P9 and P15 showed that they belong to the genus Bacillus. The isolated strains have potential for
use in agriculture, health care and biotechnology applications. The isolates shown their potential as a
source of acid ureases in various industrial utilizations. Future recommended studies included
purification of ureases, optimum study for maximum enzyme activity and investigation of molecular
structure.

5 Acknowledgment

We acknowledge Curtin University Malaysia, Sarawak for providing the required facility for the research
work. The authors wish to thank the Ministry of Higher Education (Malaysia) for the financial support for
this project through the Fundamental Research Grant Scheme (FRGS)
(FRGS/1/2015/TK01/CURTIN/02/3). Authors wish to declare that there is no conflict of interest with
respect to this work.

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 a

Figure 1. Screening of urease positive bacteria from qualitative observation. Urease agar plate from 48 hours incubation. From
left to right: a. Urease negative (yellow formation); b. Blank (pale orange); c. urease positive (purple-pink).
Figure 2. Cell growth and urease activity of selected isolates plotted against time (incubation period). Result symbols indicates
average value with error bars representing standard deviations of triplicates.
Figure 3. Effect of pH towards urease activity of selected isolates. Error bars represent mean ± standard deviations of triplicates.

a
 Figure 4. Precipitation of CaCO3 crystal by strain P9 (a) and P15 (b) view under light microscope.

o
Figure 5. Carbonate precipitation in broth medium by isolated bacteria incubated for 48 hours at 28 C. The error bars indicate
standard deviations (n=3).
 Figure 6. Phylogenetic tree based on the nucleotide sequences of partial 16S rRNA. The isolate strain (bold) and their position based on the partial 16S rDNA sequence
comparison, according to the neighbor-joining method were shown. The number on the branches indicated bootstrap values and the accession numbers for the reference strains
are shown before the name of the strain.
Table 1. Comparison of some differential properties of the selected strain of urease positive isolated from peat.

Selected strains
Characteristic
P1 P2 P3 P9 P15

Cell shape Rod Cocci Rod Rod Rod

Colony Translucent Punctiform Smooth Creamy, Dull,

with flat clear opaque shiny opaque
elevation colony convex raised
colony colony

Motility + - + + +

Catalase + + - + -

Spore formation + - + - +

Growth tolerance

pH

4 + + + + +

7 + + + + +

9 + + + + +

Temperature (oC)

15 + + + + +

28 + + + + +

40 + + + + +

50 - - - - -

NaCl %

0 + + + + +

2 + + + + +

4 + + + + +

6 + + + + +

8 + + + + +

10 + + + + -

12 + + - + -
+ indicates positive and - indicates negative test