See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/323932705

Isolation, identification and characterization of Bacillus subtilis from tap water

Article  in  Asian Journal of Microbiology, Biotechnology and Environmental Sciences · January 2017

CITATION READS
1 607

1 author:

Khaled E El-Gayar
Jazan University
22 PUBLICATIONS   29 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

domestic wastewater treatment View project

Dairy industry waste treatment View project

All content following this page was uploaded by Khaled E El-Gayar on 17 September 2018.

The user has requested enhancement of the downloaded file.

Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 19, No. (4) : 2017 : 817-830
© Global Science Publications
ISSN-0972-3005

ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF

BACILLUS SUBTILIS FROM TAP WATER
KHALED E. EL-GAY AR 1, 2
1
Department of Biology, Faculty of Science, Jazan University, KSA
2
The Holding Company for Biological Products & Vaccines (VACSERA),Cairo, Egypt

(Received 4 March, 2017; accepted 5 April, 2017)

Key words : Tap water, Bacillus subtilis, Antibiotics, Heavy metals, Protease, Amylase, Fungicide.

Abstract–A total of three tap water and four commercial packaged water samples were randomly collected
from Jazan district, KSA. The bacterial total count of all samples was calculated. Using biochemical tests and
16S rRNA gene sequencing, one bacterial isolate was identified as Bacillus subtilis. Antibiotic susceptibility
test was performed using disc diffusion technique. Bacillus subtilis showed high resistance to Rifampicin
only. Heavy metal sensitivity test of Bacillus subtilis was determined. Bacillus subtilis was resistant to
Chromium and sensitive to Mercury, Cadmium, and Silver at minimum concentration (25mg/L). It was
tolerant to Zinc, Cupper and Lead up to 100mg/L. The biomass maximum yield from bacterial culture was
the highest in the cultures containing 1% yeast extract. The optimum salinity for bacterial growth was at
0.5% NaCl. Also, Bacillus subtilis showed maximum growth at 37oC and pH 7and it was tolerant up to 48oC.
Bacillus subtilis posed amylolytic and proteolytic with gelatinolytic activities. Protease activity was gradually
increased with the increasing of incubation temperature of Bacillus subtilis growth up to 50oC but the
optimum temperature for amylase activity was at 37%. Optimal protease activity for Bacillus subtilis strain
was increased at 20% skim milk. The maximum amylase activity was at 0.2% starch. The protease activity
was increased with increasing of Calcium salt concentration up to 20 mg/L. However it was increased with
increasing of Magnesium salt concentration up to 40 mg/L. Protease activity was slightly increased with
addition of 80 mg/L from Manganese salt concentration. Amylase activity was increased with increasing
of Calcium and Manganese salts concentrations up to 20 mg/L. However the optimum enzyme activity was
increased with increasing of magnesium salt concentration up to 40 mg/L. Clear antifungal potentiality was
reported by Bacillus subtilis strain against Curvularia lunata. The obtained result revealed that Bacillus subtilis
has a noticeable antagonistic action in opposition to the tested pathogen.

INTRODUCTION
Water is considered the most important elements for
all forms of life and essential for the composition
and cells renewal. Water represents 70% of the body,
participates in the composition of our tissues, and
transports the most diverse substances throughout
the organism (Penna et al., 2002). Drinking water
should be safe enough to be used by human where;
the biological contamination is a major source in the
public health in the world (Suthar et al., 2009;
Igwemmar et al., 2013). It was estimated that
nearly1.5 billion people lack safe drinking water and
about 5 million deaths every year can be attributed
to water -borne disease (Sharmin et al., 2013). So
reduction of microbial diseases is the most public
health aim in all countries (Mulamattathil et al.,
2014).
The pathogenic effect of G-ve bacteria associates
with the lipopolysaccharide layer of the bacterial
cell envelope. So G –ve bacteria are more resistant
against antibiotics. This resistance can cause serious
diseases to human and public health. It was listed
some genera and species of Gram –ve bacteria that
contain the most important pathogenic bacteria to
human. Some of these genera are cocci as Bordetella
pertussis, Brucella melitensis, and Brucella canis and
some are bacilli as Helicobacter pylori, Neisseria
meningitidis, Rickettsia rickettsii, Salmonella typhi and
Shigella sonnei (Baron et al., 1996; Convert et al., 2008).
On the other hand, the anaerobic Gram-positive
cocci and anaerobic Gram-positive non-
sporeforming rods are considered to be of relatively
low virulence (Brook and Frazier, 1993). An

*Corresponding author’s email: k_elgayar@yahoo.com

818 KHALED E. EL-GAYAR
important analysis of the clinical microbiology
laboratory is the performance of antimicrobial
susceptibility testing of bacterial isolates. The goal of
testing is to assure susceptibility to drugs of choice
for particular infections and for proper therapy
(Barth et al., 2009; Poonia et al., 2014).
Heavy metals are a group of metals with density
greater than 5 g/cm3. They are non-degradable and
persist in nature and consequently tend to
accumulate in food chains (Lima et al., 2012; Syed
and Chinthala, 2015). So Heavy metals are toxic
because they interfere with the normal biochemical
reactions of the human body (Hussein and Joo,
2013). Different microbes have varied capacity of
metal uptake in differing concentrations based on
their relative tolerance levels (Vijayadeep and Sastry,
2014). Microorganisms are considered to be the best
indicators of changes in environmental conditions.
They are very sensitive to low concentration of
heavy metals but rapidly adapt to the specific
habitat conditions (Kamala-Kannan and Lee, 2008).
Proteases are present in all living organisms, but
microbial proteases are most exploited group of
enzymes (Singh et al., 2010). Proteases are among the
most important class of industrial enzymes, which
constitute more than 65% of the total industrial
applications, as laundry detergent, leather
preparation, meat tenderization, peptide synthesis,
food industry, dehairing process, pharmaceutical
industry and in bioremediation process. Proteases
are also used for removing the stiff and dull gum
layer of sericine from the fiber of raw silk (El-Shafei
et al., 2010; El-Gayar et al., 2012).
Amylases have been reported to occur in
microorganisms, animals and plants. α-amylase and
glucoamylase have been identified in
microorganisms (Pandey et al., 2000). Both enzymes
account for about 30 % of the world’s enzyme
production. Amylases are among the most
important enzymes represent a great significance for
biotechnology. Amylases have potential application
in many industrial processes as food, textile,
detergent, paper, and pharmaceutical industries.
However, with the advances in biotechnology, the
amylase application has expanded in many fields
such as clinical, medicinal and analytical chemistry,
as well as their widespread application in starch
saccharification and distilling industries
(Ramachandran et al., 2004; Akcando, 2011).
Bacillus subtilis cells are rod-shaped, Gram-
positive bacteria that are naturally found in soil and
vegetation. Bacillus subtilis grow in the mesophilic
temperature range. Starvation and stress are
common in this environment; therefore, Bacillus
subtilis has evolved a set of strategies that allow
survival under the harsh conditions as formation of
stress-resistant endospores. Another strategy is the
uptake of external DNA, to adapt bacteria by
recombination. However, these strategies are time-
consuming. Bacillus subtilis can also gain protection
more quickly against many stress situations as
acidic, alkaline, osmotic, or oxidative conditions and
heat (Bandow et al., 2002). B. subtilis has no known
pathogenic interaction with human or animals;
unlike products of Gram negative bacteria,
recombinant products produced by B. subtilis cells
would not be contaminated with lipopolysaccharide
endotoxins (Zaghloul et al., 1994).
Also B.subtilis can be used as biological agents
against wide range of phytopathogens (Selim et al.,
2017). Studies on antifungal metabolites secreted by
the bacteria confirmed the presence of siderophore
and several hydrolytic enzymes like protease,
chitinase, amylase and lipase. B. subtilis strains
exhibited several traits beneficial to the host and
showed promising results when applied as
bioinoculants, they may be used to develop safer
and effective formulations as an alternative to
chemical fungicides (Saha et al., 2012).
The aim of this study is: 1) Isolation and
identification of a bacterial strain from tap water.
2)Determinationthe antibiotic and heavy metals
resistance profiles for the isolate. 3) Enzymes
production from Bacillus subtilis. 4) Effect of Bacillus
subtilison phytopathogens.
MATERIALS AND METHODS
Samples and isolation of bacterial strain
Three tap water samples and four commercial water
samples were collected in sterilized glass bottles
aseptically. They were collected from different areas
in Jazan district; KSA. The total plate count was
conducted by plating technique on nutrient agar
plate and MacConkey agar plate. Counting the
colonies was done after incubation at 37°C for 24
hours (Pelczar and Chan, 1977). The viable bacterial
count (colony forming units, CFU/mL) was carried
out as described earlier (Aftab et al., 2006). Selected
colonies were purified by sub-culturing using the
streaking method.
Identification of the bacterial isolate
Using the high power magnifying lens of light
microscope, the bacterial isolate was observed after
 Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water
Gram staining. The standard microbiological
methods were done as described in Bergy’s Manual
of Systematic Microbiology (Hensyl et al., 1994). The
physiological and biochemical characters were used
for identification of bacterial isolate using GEN III
Micro Plate kit according to its manufacturer
instructions.
The bacterial isolate was identified using 16S
rRNA gene sequencing technique. The genomic
DNA was extracted using TIANamp genomic DNA
kit (Tiagen, Korea) according to its manufacturer
instructions. The primers used in amplification of
the 16S rRNA gene were forward primer (F; AGA
GTT TGA TCC TGG CTC AG) and reverse primer
(R; GGT TAC CTT GTT ACG ACT T). PCR was
performed according to (Sambrook and Russel,
2001) with some modifications for 35 cycles under
the following conditions: denaturation step at 94°C
for 40 sec, annealing step at 55°C for 1 min,
extension step at 72°C for 2 min and final extension
at 72°C for 10 min. Sequencing of the amplified
fragments was done at Vacsera, Egypt. Sequence
analysis was performed using the sequence
alignment software BLASTn with the NCBI
database (National Center for Biotechnology
Information), http://www.ncbi.nlm.nih.gov/.
Antimicrobial susceptibility testing
An antibiotic susceptibility test was performed
using disc diffusion method (Mulamattathil et al.,
2014). A single bacterial colony was streaked on the
nutrient agar plate using a sterile loop. The bacterial
isolate were investigated using antibiotics disc
containing Amikacin (AK,30µg), Gentamicin
(GM,10µg), Cefepime (CPM,30µg), Ticarcillin
(TC,75µg), Piperacillin (PRL,100µg), Imipenem
(IMI,10µg), Colistin (CL,10µg), Rifampicin (RIF,5µg),
PenicillinG(PG,10µg), Erythromycin (E,15µg),
Cephalothin (KF,30µg), Clindamycin (CD,2µg),
Cotrimoxazole (TS,25µg). Using a sterilized forceps,
the discs were placed and fixed on the nutrient agar
medium then incubated O.N. at 35°C (Cheesbrough,
2000). The different zones of inhibition were
measured to the nearest millimetre and interpreted
as sensitive, moderate sensitive and resistant based
on the interpretation table recommended by the disc
manufacturer (Agwa et al., 2012).
Tolerance testing against heavy metals
The following seven tested heavy metals CuSO4,
AgNO3, CdCl 2, HgCl 2, Pb(CH 3COO) 2, K2Cr 2O 7 ,
ZnSO4 stock solutions were made in distilled water,
819
sterilized by filtration through membrane filters and
stored in sterile flasks in the dark at 4°C. Tolerance
test were conducted in a nutrient agar medium. The
metal concentrations tested were 25, 50 and 100mg/
l for Cr, Ag, Cd and Hg metals while they were 50,
100 and 150 mg/L for Pb, Cu and Zn metals. For all
media, the pHs were adjusted to 7 but the copper-
containing medium pH was adjusted to 5.5 to avoid
precipitation. Heavy metal sensitivity test was
determined by the appearance of halo zone in the
bacterial growth after incubating the plates at 37°C
temperature for 24-48h. The distance of the halo
zone around the colonies were measured (Jeanthon
and Prieur, 1990).
Factors affecting on bacterial growth
For optimum growth of Bacillus subtilis, four
parameters were considered. Bacterial cells were
activated by growing them overnight on soyabean
casein broth. Fresh soyabean casein broth was
inoculated with activated cells then incubated at
room temp., 30, 37 and 50°C. To study the effect of
different concentrations of salinity on bacterial
growth; soyabean casein broth was supplemented
with 0.5% (control), 2% and 5% NaCl. Also the effect
of yeast extract as carbon source for bacterial growth
with different concentrations (0.1, 0.5 and 1%) was
studied. In another experiment, three fresh soyabean
casein broth flasks were prepared at different pH
(pH4, pH7 and pH10). A hundred µL of each
dilution of last cultures was plated on nutrient agar
plates separately, incubated overnight at 37°C and
the number of developed colonies was processed to
obtain the colony forming units per ml culture
(CFU/mL).
Screening of Bacillus subtilis for enzymes
production
Skim milk nutrient agar medium was used for
protease screening for several colonies by streaking
method using sterile tooth pick and incubated at
37°C (Zaghloul et al., 2004; El-Gayar et al., 2012).
Colonies forming transparent zones, because of
partial hydrolysis of milk casein, were selected. The
clear zone surrounding the colony was measured in
mm from the edge of the colony to the limit of
clearing and also the diameter of colony was
recorded.
The gelatin hydrolysis test was used to detect the
ability of isolated microorganism to produce the
enzyme gelatinase. The procedures were carried out
by inoculation a heavy inoculum of Bacillus subtilis
820 KHALED E. EL-GAYAR

by stabbing on the tube containing nutrient gelatin millimeter (Selim et al., 2017).
medium. After that, incubation the inoculated tube
along with an uninoculated medium at 37°C for 14 RESULTS
days. Removing the tubes daily from the incubator
and placing in ice bath or refrigerator (4°C) for 15- In the current study, the bacterial total count of all
30 minutes (until control is gelled) every day to samples of the commercial water was zero CFU/ml.
check for gelatin liquefaction (Acharya, 2014). The bacterial count of tap water samples was
Screening of Bacillus subtilis for amylase averaged; 1700, 5600 and 410, CFU/ml. MacConkey
production was done. Starch agar media plates were agar was used to isolate Gram – negative bacteria in
streaked with Bacillus subtilis colony using sterile parallel with isolation on nutrient agar. There wasn’t
tooth pick. After incubation at 37oC for 48 hours, the any growth on MacConkey agar, indicated that; the
Petri dishes were flooded with 5.0 mL of iodine samples were free from Gram – ve bacteria or
solution. The clear zone surrounding the colony was sewage wastes.
measured in mm from the edge of the colony to the After purification of selected several colonies
limit of clearing and also diameter of colony was using streaking techniques, the morphology graph
recorded (Qadeer et al, 1989). Semi-quantitative of isolated bacteria showed that; The isolates were
estimation was possible using the following short chains forming, Gram-positive and rod shaped
formula: EA = D-d; were D is the diameter of the bacteria. According to the biochemical identification
clearing zone; and d is the microbial colony diameter tests (Table 1), they were determined as Bacillus sp.
(Sicuia et al., 2015). To confirm the biochemical identification result, the
sequencing of the gene for 16S rRNA was performed
Factors affecting on protease and amylase activity
(Song et al., 2005; Tasic et al., 2014). The sequence of
Effect of temperature on both of protease and 16S rRNA of isolate from tap water was compared
amylase enzymes activity was studied at different with the sequences in the NCBI gene bank data for
temperatures (30, 37 and 50oC). Also, the effect of 16S rRNA, with maximum homology (99% identity)
substrate concentration on enzymes activity was of Bacillus subtilis BS-1 strain (accession number
studied. For protease activity; the skim milk AY172514.1; Figures 1 and 2). From the results of
substrate concentrations were 5%, 10% and 20%. For biochemical identification tests and DNA
amylase activity; the starch substrate concentrations alignments of the 16S rRNA sequence of the isolate
were 0.2%, 0.4% and 0.6%. Effect of trace elements
on both enzyme activities was studied. Calcium,
Magnesium and Manganese trace elements were
used at concentrations 20, 40 and 80 mg/l. The
enzymes activity was determined as described
above.
Antimicrobial activity of Bacillus subtilis
The antimicrobial activity of Bacillus subtilis bacteria
was carried out against Curvularia lunata. The fungal
strain was afforded by Prof. Dr. Tarek M.
Abdelghany, Department of Biology, Faculty of
science, Jazan University, KSA. The fungal strain
was cultured on potato dextrose agar (PDA)
meanwhile bacterial strain was cultured on nutrient
agar (N.A.). For antifungal assay, Bacillus subtilis was
grown on nutrient agar plates at 37oC for 24 hrs. 100
μL of spore suspension from fungus was spread on
PDA plates. At equal places of PDA plates, nutrient
agar discs of Bacillus subtilis were placed. Duplicate
Fig. 1. Partial DNA sequences of the 16S rRNA gene of
dual-inoculated plates, with the fungus alone as a the bacterial strain isolated from Tap water, Jazan,
control were incubated at 28oC for 7 days. Then the KSA and the corresponding gene of Bacillus subtilis
diameters of inhibition zones were measured in BS-1 (accession number AY172514).
Table 1. Biochemical analysis of bacterial strain isolated from tap water.
Character Results Character Results Character Results Character Results
Dextrin +ve D-Fructose +ve L-Arginine +ve L-Lactic Acid +ve
D-Maltose +ve D-Galactose -ve L-Aspartic Acid +/- Citric Acid +ve
D-Cellobiose +ve 3-Methyl Glucose -ve L-Glutamic Acid +/- α-Keto-Glutaric Acid -ve
D-Trehalose +ve D-Fucose -ve L-Histidine +ve D-Malic Acid -ve
Sucrose +ve L-Fucose -ve L-Pyroglutamic -ve L-Malic Acid +ve
Gentiobiose D- +ve L-Rhamnose -ve Acid L-Serine -ve Bromo-Succinic Acid -ve
Turanose +ve Inosine -ve lincomycin -ve Nalidixic acid -ve
Stachyose -ve 1% sodium lactate +ve Niaproof 4 -ve Lithium chloride +ve
pH5 +ve Fusidic acid -ve Guanidine HCl -ve Potassium tellurite +ve
pH6 -ve D-serine +/- Pectin +ve Tween 40 -ve
D-Raffinose +ve D-Sorbitol +ve D-Galacturonic Acid +ve γ-Amino-Butryric Acid -ve
α-D-Lactose -ve D-Mannitol +ve L-Galactonic Acid Lactone +/- α-Hydroxy-Butyric Acid -ve
D-Melibiose +ve D-Arabitol -ve D-Gluconic Acid +ve β-Hydroxy-D,L-Butyric Acid -ve
β-Methyl-D-Glucoside +ve Myo-Inositol +ve D-Glucuronic Acid -ve α-Keto-Butyric Acid -ve
D-Salicin +ve Glycerol +ve Glucuronamide -ve Acetoacetic Acid -ve
N-Acetyl-D-Glucosamine +/- D-Glucose- 6-PO4 -ve Mucic Acid +ve Propionic Acid -ve
N-Acetyl-β-D-Mannosamine -ve D-Fructose- 6-PO4 -ve Quinic Acid -ve Acetic Acid -ve
N-Acetyl-D-Galactosamine -ve D-Aspartic Acid -ve D-Saccharic Acid +ve Formic Acid -ve
N-Acetyl Neuraminic Acid -ve Troleandomycine -ve Vancomycine -ve aztreonam +ve
1% Nacl +ve Rifamycine SV -ve Tetrazolium violet -ve Sodium butyrate +ve
4% Nacl +ve Minocycline -ve Tetrazolium blue -ve Sodium bromate +ve
8% Nacl +ve Gelatin +/- p-Hydroxy- Phenylacetic Acid +/- Positive control +ve
α-D-Glucose +ve Glycyl-L-Proline -ve Methyl Pyruvate +/- Negative Control -ve
Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water
821
822 KHALED E. EL-GAYAR

Fig. 3. The effect of some antibiotics on Bacillus subtilis

Fig. 2. Electropherogram data of B. subtilis isolated from
tap water
and the genes for 16S rRNA deposited in all DNA
databases, it was possible to conclude that the
isolate from tap water was Bacillus subtilis.
The current results proved that, Bacillus subtilis
isolated from tap water is sensitive to most
antibiotics. The results obtained are showed in Table
2 and Fig. 3. The results revealed that the isolate
was sensitive to Gentamicin, Amikacin, Colistin,
Piperacillin, Ticarcillin, Imipenem, Cefepime,
Clindamycin, Cephalothin, Cotrimoxazole,
Erythromycin, Penicillin G with different sensitivity
degrees represented about 92% of the antibiotics
tested. It was resistant to Rifampicin only.
To assess the heavy metals susceptibility profile
of Bacillus subtilis isolated from tap water. Bacillus
subtilis was screened against seven heavy metals as
shown in Table (3). The results indicated that the
isolate; Gram +ve Bacillus subtilis was tolerant to
Chromium even at high concentration. Lead,
Copper and Zinc affected on the growth with
increasing the concentration up to 150 mg/L. It was
isolated from tap water. Where, Figure (A)
contains 1: Gentamicin (GM), 2:Amikacin (AK), 3:
Colistin(CL), 4: Rifampicin (RIF); Figure (B)
contains 5: Piperacillin (PRL), 6: Ticarcillin (TC), 7:
Imipenem (IMI), 8:Cefepime (CPM) and Figure (C)
contains 9:Clindamycin (CD), 10:Cephalothin
(KF), 11: Cotrimoxazole (TS), 12: Erythromycin (E),
13: Penicillin G(PG).
sensitive to Silver, Cadmium and Mercury at
minimum inhibitory concentration (25 mg/L)
tested.
As shown in Figure (4), the biomass maximum
yield from bacterial culture was the highest in the
cultures containing 1% yeast extract. The optimum
salinity (NaCl%) for bacterial growth was at 0.5%
NaCl. Also, Bacillus subtilis showed maximum
growth at pH 7 and sustained up to pH10 and very
moderate growth was observed at pH 4. Bacillus
subtilis was affected by the temperature at which
cultures were grown and it was tolerant up to 48oC.
Generally, the growth efficiency was in the following
order: 37oC>30oC >48oC > RToC.
In preliminary experiments, Bacillus subtilis
colonies were streaked for protease producing
ability on skim milk nutrient agar plate and nutrient
gelatin tube. As shown in Figure 5, after being
incubated for 24 hrs, a plate containing skim milk

Table 2. The antibiotic sensitivity test for B. subtilis isolated from water.
No. Antibiotic conc. (μg/ disc) Status of halo zone
susceptibility diameter/mm
1 Gentamicin (GM) 10µg S+++ 21
2 Amikacin (AK) 30µg S++++ 25
3 Colistin (CL) 10µg S+ 12
4 Rifampicin (RIF) 5µg R 0
5 Piperacillin (PRL) 100µg S++++ 25
6 Ticarcillin (TC) 75µg S++ 20
7 Imipenem (IMI) 10µg S+++ 22
8 Cefepime (CPM) 30µg S++++ 25
9 Clindamycin (CD) 2µg S++ 20
10 Cephalothin (KF) 30µg S++++ 30
11 Cotrimoxazole (TS) 25µg S++++ 30
12 Erythromycin (E) 15µg S++++ 30
13 Penicillin G (PG) 10 µg S++++ 30
R = Resistance, S= sensitive
 Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water 823

Log CFU/ml

Fig. 4A. Monitoring the effect of addition of Log CFU/ml

yeast extract(with concentration 0.1%,
0.5% and 1%) on the growth of Bacillus
subtilis, O/N at 37oC.
Log CFU/ml
Fig. 4B. Monitoring the effect of Salinity
(NaCl%) on the growth of Bacillus
subtilis at 2%, 5% and 10% , O/N at
37oC.

Log CFU/ml

Fig. 4C. Monitoring the effect of pH on the Fig. 4D. Monitoring the effect of temperature on
growth of Bacillus subtilis at pH4, pH7 the growth of Bacillus subtilis O/N at room
and pH10 O/N at 37oC. temperature, 30oC, 37oC and at 48oC.

Table 3. The heavy metals sensitivity test for Bacillus subtilis isolated from tap water.
Heavy metals Mercury Cadmium Silver Chromium Zinc Cupper Lead
concentration Hg Cd Ag Cr Zn Cu Pb
(Halozone–Control)

25 mg/L 9 19 14 0 ND ND ND
diameter/mm

50 mg/L 17 25 15 0 2 0 0
100 mg/L 20 27 20 0 2 0 2
150 mg/L ND ND ND ND 4 2 10
824 KHALED E. EL-GAYAR
Fig. 5. Represents Qualitative determination of protease
by streaking of different Bacillus subtilis colonies
on skim milk nutrient agar and incubated O/N at
37 °C.
nutrient agar showed zone formation around the
bacterial colony indicated the protease positive
strain which may be due to hydrolysis of casein. In
the experiment, after being incubated for 14 days,
the tube containing gelatin was liquefied (Fig. 6). So
Bacillus subtilis may possess collagenolytic or
gelatinolytic activities. Hence the strain was
identified as a protease producer and it was taken
for further experimental studies.
Fig. 6. Represents Qualitative determination of protease
by inoculating of Bacillus subtilis colony on
nutrient gelatin agar tube. A: Represents nutrient
gelatin agar only as control incubated at 37°C for
14 days. B: Represents nutrient gelatin agar
liquefied after inoculation with Bacillus subtilis and
incubated at 37°C for 14 days.
Screening for amylolytic properties of Bacillus
subtilis was done on starch agar plates. After
flooding the petri dishes with 5.0 mL of iodine
solution. The clear zone surrounding the growth
was noticed and more active colony was kept for
further work (Fig. 7). This amylase enzyme can be
used as the novel amylase to hydrolyze starch.
As shown in Table 4, protease activity was
gradually increased with the increasing of
incubation temperature of Bacillus subtilis growth on
skim milk up to 50 oC with 22 mm halozone
diameter. But the maximum temperature for
amylase activity was at 37oC with 20mm halozone
diameter then decreased at 50 oC to 10mm halozone
diameter.
Fig. 7. Represents Qualitative determination of amylase
by streaking of Bacillus subtilis on starch agar and
incubated O/N at 37°C then flooded with iodine
solution to appear the hydrolysis of starch.
In cultures supplemented with different
concentrations from substrates, the optimal protease
activity for Bacillus subtilis strain was increased at
20% skim milk to 24mm halozone diameter. The
maximum amylase activity reached to 20mm
halozone at 0.2% starch as substrate then decreased
to 10mm halozone diameter at 0.4 and 0.6% starch.
The protease activity was increased with
increasing of calcium salt concentration up to 20mg/
l with 24mm halozone diameter then stabled up to
80 mg/L. However the optimum enzyme activity
was increased with increasing of magnesium salt
concentration up to 40 mg/L with 27mm halozone
diameter then decreased to 24mm at 80 mg/L.
Protease activity was slightly increased to 27mm
halozone diameter with addition of 80 mg/L from
manganese salt concentration. The amylase activity
was increased with increasing of calcium salt
concentration up to 20 mg/L with 25mm halozone
diameter then stabled up to 80 mg/L. However the
optimum enzyme activity was increased with
increasing of magnesium salt concentration up to 40
mg/L with 25mm halozone diameter then stabled
up to 80 mg/L. Amylase activity was increased at 20
mg/L manganese salt concentration registered
29mm halozone diameter then stabled up to 80 mg/
L. The halozone was 16mm for protease activity of
control culture for all factors while the halozone of
amylase activity for control culture formed 20mm
for all factors.
In preliminary study to apply Bacillus subtilis as
fungicide, Clear antifungal potentiality was
reported by Bacillus subtilis strain against Curvularia
lunata (Fig. 8). The obtained result revealed that
Bacillus subtilis has a noticeable antagonistic action in
 Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water 825

Agency (EPA) has determined that the presence of

Fig. 8. Shows clear antifungal potentiality with the use of
B. subtilis as fungicide against Curvularia lunata.
opposition to the tested pathogen.
DISCUSSION
Drinking water is a major source of microbial
pathogens. Poor water quality, sanitation and
hygiene account for 1.7 million deaths a year world-
wide, mainly through infectious diarrhea (Ashbolt,
2004). The United States Environmental Protection
fecal coliforms or E. coli is generally not harmful
themselves, but their presence in drinking water is
very serious because they are associated with
sewage wastes. The presence of these bacteria in
drinking water is a result of a problem with water
treatment and indicates that the water may be
contaminated (Doyle and Erickson, 2006).
MacConkey agar was used to isolate Gram-negative
and enteric bacilli. Enteric bacteria that have the
ability to ferment lactose can be detected using the
carbohydrate lactose (Anderson and Cindy, 2013).
Reduction in the number of bacteria in the treated
water could be due to the treatment process
(Mulamattathil et al., 2014).
Antibiotics resistance in bacteria is a major health
problem in many countries (Samra et al., 2009).With
agreement of previous study, an evaluation of the
bacteriological quality of drinking water confirmed
the presence of Bacillus subtilis bacterial species
sensitive to several classes of antibiotics (Jaysankar
et al., 2008). Also in another study Bacillus sp. isolates

Table 4. Effect of temperature, substrate concentration in addition to calcium, magnesium and manganese
concentrations on the production of protease and amylase enzymes from Bacillus subtilis isolated from tap
water by measuring the halozones (millimeter). The results at 37oC was control for all factors for both enzymes.
Effect of temperature
Temperature Halozone diameter for Halozone diameter for
protease production amylase production
30oC 14 12
37oC 16 20
50oC 22 10
Effect of substrate
Substrate % Halozone diameter for Substrate % Halozone diameter for
protease production amylase production
5%skim milk 16 0.2%starch 20
10%skim milk 20 0.4%starch 10
20%skim milk 24 0.6%starch 10
Effect of trace elements concentration
Trace elements Halozone diameter for Halozone diameter for
concentration protease production amylase production
20 mg/l CaCl2 24 25
40 mg/l CaCl2 24 25
80 mg/l CaCl2 24 25
20 mg/l MgSO4 26 20
40 mg/l MgSO4 27 25
80 mg/l MgSO4 24 25
20 mg/l MnCl2 23 29
40 mg/l MnCl2 25 29
80 mg/l MnCl2 27 29
826 KHALED E. EL-GAYAR
tested were found to be susceptible to Rifampicin,
Chloramphenicol , Erythromycin, Ciprofloxacin,
Streptomycin, Gentamycin and Lincocin and 100%
resistance against Norfloxapin, Floxapen and
Ampiclox (Agwa et al., 2012). On the contrary the
current results, E.coli isolates showed higher
multiple antibiotic resistance (MAR) to Cephalothin,
Cephoxithin, Clindamycin, Metronidazole,
Penicillin and Vancomycin indicated its human
origin in drinking water. Resistance to antibiotics is
acquired by a change in the gene makeup of
bacterium, which can occur by either a gene
mutation or by transfer of antibiotic resistance genes
between bacteria in the environment (Kawane,
2012).
The threat of environmental pollution due to
enhanced availability of both essential and toxic
metals is a matter of great concern (Srivastava et al.,
2005). Environmental pollution with heavy metals
presents a real threat to wildlife because the metals
cannot be naturally decomposed as in the case with
organic pollutants (Stavreva-Veslinovska, 2011).
Heavy metal resistant microorganisms play an
important role in the bioremediation of heavy metal
contaminated environment (Singhet al., 2013).
Responses to varying concentrations of Hg, Cd, and
Zn were studied on two strains of bacterial isolates
(Flavobacterium sp. and Bacillus sp.) from Indian
coastal waters. Growth responses showed inhibition
and the order of inhibition was Hg>Zn>Cd in the
case of Bacillus sp. and Hg> Cd > Zn in the case of
Flavobacterium sp., indicated that the Gram positive
isolate was less adaptable to metals than the Gram
negative (Nair et al., 1993). Also it was proved that
Gram –ve organisms like E. coli exhibited more
resistance to metals like Zn, Cu and Hg in relative
comparison with Gram +ve organisms like Bacillus
(Vijayadeep and Sastry, 2014). It was suggested that,
increasing industrialization has resulted in an
alarming increase in the discharge of heavy metals
and other pollutants into the environment including
water resources (Syed and Chinthala, 2015).
Industrial sectors frequently use Bacillus subtilis
for the production of various enzymes. It is a rod-
shaped organism, which can form a tough,
protective endospore and can withstand extreme
environmental conditions. Bacillus species are
obligate aerobes or facultative anaerobe and include
both free-living and pathogenic species (Pant et al.,
2015).The optimization of fermentation conditions,
particularly physical and chemical parameters are
important in the development of fermentation
processes due to their impact on the economy and
practicability of the process. The growth and
enzyme production of the organism are strongly
influenced by medium composition thus
optimization of media components and cultural
parameters is the primary task in a biological
process (Akcan, 2011). Optimum growth conditions
are done to large scale biomass production for
further applications. The carbon/energy source and
nitrogen sources as yeast extract were necessary for
the growth and product formation in microbial
cultivation. The nature and characteristics of these
substrates has a predominant role to play in the
metabolism of microorganism (Anderson and
Jayaraman, 2003). Under all conditions, increasing
NaCl concentrations caused increasing, albeit
reversible, inhibition of germination. High salinity
delayed and increased the heterogeneity of
germination initiation, slowed the germination
kinetics of individual spores and the whole spore
population, and decreased the overall germination
efficiency (Nagler et al., 2014). Most natural
environments have pH values between 5 – 9 and
most organisms have pH optima in this range.
Temperature is probably the most important
environmental factor affecting growth. The
minimum and maximum temperatures for microbial
growth vary widely among microorganisms and are
usually a reflection of the temperature range and
average temperature of their habitat (El-Gayaret al.,
2012). In previous study, The bacterial isolates
including Bacillus spp. were screened for enzyme
production. Yeast extract was found to be the
optimum nitrogen source with temperature of 50°C
was found to be optimum for enzyme production
(Boominadhan and Rajakumar, 2009).In another
study; they have got similar results to the current
study. Bacillus subtilis could group up to 40ºC and
pH range 6-9 with optimal growth temperature and
pH at 37ºC and 8.0 respectively. It was also
optimized for carbon test and nitrogen test with
optimal growth in dextrose and peptone
respectively (Krishnaveniet al., 2012).
In similar results to the current study, Bacillus sp
isolated from local marine samples collected from
Saudi Arabia to produce protease enzyme. Six
bacterial isolates were screened for protease
production on the basis of gelatin hydrolyses
(Alnahdi, 2012). It was suggested that; those
gelatinolytic enzymes can be used as the novel
protease capable of hydrolyzing gelatin (Sai-Ut et al.,
2013). Previous studies on the protease
 Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water
characterization revealed that the optimum
temperature of this enzyme was 60ºC (Nascimento
and Martins, 2004). Also, it was found that capable
of producing an extracellular protease extracted
from Bacillus sp showed optimum activity at 50 °C
and pH 11.0 (Tekinet al.,2012). In previous
investigation it was concluded that the thermostable
protease has potential applications in various
industrial processes (Lakshmi et al., 2014). A-
amylase production from Bacillus cereus using solid-
state fermentation has been investigated and the
enzyme is reported to show activity at high
temperature (75%C) (Anto et al., 2006). In another
study, the optimum temperature value of a purified
amylase was found to be 45°C (Demirkan, 2011).
In the current study, the substrate concentration
affected on the enzymes activity.It has been shown
experimentally that if the amount of the enzyme is
kept constant and the substrate concentration is then
gradually increased, the reaction velocity will
increase until it reaches a maximum. After this
point, increases in substrate concentration will not
increase the velocity (Worthington, 2017).
The metal ions in media are an important factor
that affects enzyme production due to act as
inducers (Sevic and Demirkan, 2011). In agreement
with the current study it was proved that, Calcium
and Magnesium as antagonistic agents that may
regulate the proteolytic activity of some species
(Robinson, 2000). Also it was found the optimum
proteolytic activity was accelerated by the addition
of Mg2+ , Ca2+ and Mn2+ , where as it was inhibited
by Hg2+ (Abu Sayem, 2006). In previous study the
Cobalt, Magnesium, Cadmium, and Manganese
increased amylase activity. On the other hand, Iron
and Sodium decreased residual activity to different
extents, while Calcium, Zinc and Copper inhibited
amylase activity (Al-Quadanet al., 2011). Also they
revealed that amylase activity was affected by
Calcium and Magnesium concentration. In presence
of Calcium and Magnesium ion in a specific range
the enzyme activity increased (Khanra, 2016).
Curvularia infections in humans are relatively
uncommon despite the ubiquitous presence of this
soil-dwelling dematiaceous fungus in the
environment. Originally thought to be solely a
pathogen of plants, Curvularia has been described as
a pathogen of humans and animals in the last half-
century, causing respiratory tract, cutaneous, and
corneal infections (Carter and Boudreaux, 2004). The
suppressive impact of Bacillus subtilis against the
fungal pathogens was attributed to the capability of
827
this strain to produce bioactive molecules that may
act as antimicrobial compounds. The biocontrol
activity of bacterial strain was ascribed to the impact
of alkaline serine proteolytic enzyme in addition to
the induction of host systemic acquired resistance
(Selim et al., 2017).
CONCLUSION
In conclusion, the current results demonstrate that
(i) Some tap water from Jazan are free from G-ve
bacteria but contains G +ve bacteria (Bacillus
subtilis). (ii) Bacillus subtilis was sensitive to various
classes of antibiotics and some heavy metals. (iii)
The metals tolerant Bacillus subtilis can be used for
heavy metals bioremediation. (iv) B. subtilis can be
used for large-scale production of many enzymes as
protease, amylase and gelatinase to meet the needs
in the industrial sector. (v) Some factors affected on
production of protease and amylase by B. subtilis.
(vi) B. subtilis has a biological control against some
phytopathogen. The future work will be: a) Scale up
fermentation to produce enzymes in mass
production. b) Enzymes purification using
chromatography techniques. d) Application of B.
subtilis against many phytopathogens as biology
control.
ACKNOWLEDGMENT
Author wishes to extend thanks to all members of
Biology Department, Faculty of Science, Jazan
University, KSA, especially prof. Dr. Tarek M.
Abdelghany and Dr. Ashraf M. Essa, and my
colleagues in Holding Company for Biological
Products & Vaccines (VACSERA), Egypt. I extend
my thanks from deep of my heart to my family
members and friends for their moral support.
REFERENCES
Abu Sayem, S. M. Alam, M. J. and Hoq, M. 2006. Effect of
temperature, pH and metal ions on the activity and
stability of alkaline protease from novel Bacillus
lichiniformis MZK03. Proc. Pakistan Acad. Sci. 43(4) :
257-262.
Acharya, T. 2014. Gelatin Hydrolysis Test: Principle,
procedure and expected results. Microbe on line.
https://microbeonline.com/gelatin-hydrolysis-test-
principle-procedure-expected-results/
Aftab, S. Ahmed, S. Saeed, S. and Razoo, S. A. 2006.
Screening, isolation and characterization of alkaline
protease producing bacteria. Pak. J. Biol. Sci. 9 : 2122-
2126.
828 KHALED E. EL-GAYAR
Agwa, O. K. Uzoigwe, C. I. and Wokoma, E. C. 2012.
Incidence and antibiotic sensitivity of Bacillus cereus
isolated from ready to eat foods sold in some markets
in Portharcourt, Rivers state, Nigeria. Asian Jr. of
Microbiol. Biotech. Env. Sc. 14 (1) : 13-18.
Akcan, N.2011.High Level production of extracellular á-
Amylase from Bacillus licheniformis ATCC 12759 in
submerged fermentation. Romanian Biotechnological
Letters. 16(6).
Alkando, A. A. Elamin, H.B. and Ibrahim, H. 2011. A
Thermostable extracellular alpha-amylase from
Bacillus Licheniformis Isolated from soil in Sudan .
Sudan Academy of Science Journal (in Press).
Alnahdi, H. S. 2012. Isolation and screening of
extracellular proteases produced by new isolated
Bacillus sp. Journal of Applied Pharmaceutical Science. 2
(9) : 071-074.
Al-Quadan, F. Akel, H. and Natshi, R. 2011. Characteristics
of a novel, highly acid- and thermo-stable amylase
from thermophilic Bacillus strain HUTBS62 under
different environmental conditions. Ann Microbiol .
Anderson and Cindy. 2013. Great Adventures in the
Microbiology Laboratory (7th ed.). Pearson. 175–176.
ISBN 978-1-269-39068-2.
Anderson, R. K. I. and Jayaraman, K. 2003. Influence of
carbon and nitrogen sources on the growth and
sporulation of Bacillus thuringiensis var Galleriae for
Biopesticide production. Chem. Biochem. 17 (3): 225–
231.
Anto, H. Trivedi, U. and Patel, K. 2006. Alpha amylase
production by Bacillus cereus MTCC 1305 Using solid-
state fermentation. Food Technol. Biotechnol. 44(2) :
241–245.
Ashbolt, N. J. 2004. Microbial contamination of drinking
water and disease outcomes in developing regions.
Toxicology. 198(1–3): 229–238.
Baron, S. Salton, M.R.J. and Kim, K.S. 1996. Structure. In
Baron’s Medical Microbiology (4th ed.). Univ of Texas
Medical Branch. ISBN 0-9631172-1-1. PMID 21413343.
Barth, R. Weinstein, M. Jorgensen, J. and Ferraro, M. J.
2009. Antimicrobial Susceptibility Testing: A Review
of General Principles and Contemporary Practices.
Clin Infect Dis. 49 (11) : 1749-1755.
Bandow, J. E. Britz, H. and Hecker, M. 2002. Bacillus subtilis
tolerance of moderate concentrations of rifampin
involves the B-dependent general and multiple stress
response. Journal of Bacteriology. 184(2): 459-467.
Boominadhan, U. and Rajakumar, R.2009. Optimization
of protease enzyme production using Bacillus Sp.
isolated from different wastes. Botany Research
International. 2 (2): 83-87.
Brook, I. and Frazier E.H.1993. Significant recovery of
nonsporulating anaerobic rods from clinical
specimens. Clin Infect Dis. 16 : 476-480.
Carter, E. and Boudreaux, C. 2004. Fatal Cerebral
Phaeohyphomycosis Due to Curvularia lunata in an
immunocompetent patient. J. Clin. Microbiol. 42 (11):
5419-5423.
Cheesbrough, M. 2000. District Laboratory Practice in
Tropical Countries, part 2.Cambridge,University press.
Convert, N. F. M. Piffaretti, J. C. Gurny, R. and Lange, N.
2008. Effects on Gram-Negative and Gram-positive
bacteria mediated by 5-aminolevulinic acid and 5-
aminolevulinic acid derivatives. Antimicrobial Agents
and Chemotherapy. 52(4) : 1366-1373.
Demirkan, E. 2011. Production, purification, and
characterization of α-amylase by Bacillus subtilis and
its mutant derivatives. Turk J Biol. 35 : 705-712.
Doyle, M.P. and Erickson, M. C. 2006. Closing the door
on the fecal coliform assay. Microbe. 1 : 162-163.
El-Gayar, K. E. Zaghloul, T. I. Haroun, M. A. and Saeed,
H.M. 2012. Purification of alkaline protease from
hydrolyzed chicken feather waste using recombinant
B. subtilis strain. Scientific Journal of King Faisal
University, KSA. 13 (1): 1433.
El-Shafei, H. A. Abdel-Aziz, M. S. Ghaly, M. F. and Abdalla,
A.H. 2010. Optimizing some factors affecting alkaline
protease production by a marine bacterium
Streptomyces albidoflavus. Proceeding of fifth scientific
environmental conference, Zagazig Uni. 125-142.
Hensyl, W.R. 1994. Bergey’s Manual of Systematic
Bacteriology 9 th edition. John. G. Holt and Stanley, T.
Williams (Eds.) Williams and Wilkins, Baltimore,
Philadeiphia, Hong kong, London, Munich, Sydney,
Tokyo.
Hussein, K. A. and Joo, J. 2013. Heavy metal resistance of
bacteria and its impact on the production of
antioxidant enzymes. African Journal of Microbiology
Research. 7(20): 2288-2296.
Igwemmar, N. C. Kolawole, S. A. and Okunoye, L. K.
2013. Physical and chemical assessment of some
selected borehole water in Gwagwalada, Abuja.
International Journal of Scientific & Technology Research.
2 (11).
Jaysankar, D. Ramaiah, N. and Vardanyan, L.2008.
Detoxification of toxic heavy metals by marine
bacteria highly resistant to mercury. Marine
Biotechnology. 10(4): 471-477.
Jeanthon, C. and Prieur, D.1990. Susceptibility to heavy
metals and characterization of heterotrophic bacteria
isolated from two hydrothermal vent polychaete
annelids, Alvinellapompejana and Alvinella caudate.
Applied and Environmental Microbiology. 3308-3314.
Kamala-Kannan, S. and Lee, K. J. 2008. Research article:
Metal tolerance and antibiotic resistance of Bacillus
species isolated from Sunchon Bay sediments, South
Korea. Biotechnology. 7: 149-152.
Kawane, R. S. 2012. Studies on antibiotics and heavy metal
resistance profiling of Escherichia coliform drinking
water and clinical specimens. Bioscience Discovery.
3(3) : 292-295.
Khanra, K. 2016. Partial purification and biochemical
characterization of amylase from Aeromonascaviae
NK1 isolated from industrial waste of India. Journal
of Biology and Life Science. 7(185): 2157-6076.
Krishnaveni, K. Mukesh, K.D.J. Balakumaran, M. D.
Ramesh, S. and Kalaichelvan, P. T. 2012. Production
and optimization of extracellular Alkaline Protease
 Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water
from Bacillus subtilis isolated from dairy effluent. Der
Pharmacia Lettre.4 (1) : 98-109.
Lakshmi, B. K. M. Ratna, S.P.V. Ambika, D.K. and
Hemalatha, K. P. J. 2014. Media optimization of
protease production by Bacillus licheniformis and
partial characterization of Alkaline protease. Int. J.
Curr. Microbiol. App. Sci . 3(5): 650-659
Lima, D.A, Ribeiro, D. Carvalho, M. A. De Souza, S. L.Dias,
P. M. Da Silva, R. G. Saramago, C. S. De Melo,
B.Hofer, E.2012. Heavy metal tolerance(Cr, Ag and
Hg) in Bacteria isolated from sewage. Brazilian Journal
of Microbiology. 1620-1631.
Mulamattathil, S. G. Bezuidenhout, C. Mbewe, M. and
Ateba, C. N. 2014. Isolation of environmental bacteria
from surface and drinking water in mafikeng, South
Africa, and characterization using their antibiotic
resistance profiles. Journal of Pathogens. 2014:11.
Nagler, K. Setlow, P. Li, Y. and Moellera, R. 2014. High
Salinity Alters the Germination Behavior of Bacillus
subtilis spores with nutrient and non-nutrient
germinant. Applied and Environmental Microbiology.
80 (4): 1314 –1321.
Nair, S. Bharathi, P. A. L. and Chandramohan, D.1993.
Effect of heavy metals on marine Bacillus sp. and
Flavobacterium sp. Ecotoxicology. 2: 220-229.
Nascimento, W. C. and Martins, M. L. L.2004. Production
and properties of an extracellular protease from
thermophilic Bacillus SP. Brazilian Journal of
Microbiology. 35 : 91-96.
Pandey, A. Nigam, P. Soccol, C. R. Soccol, V. T. Singh, D.
and Mohan, R. 2000. Advances in microbial
amylases. Biotechnol. Appl. Biochem. 31 : 135–152
Pant, G. Prakasha, A. Pavania, J. V. P. Beraa, G. V. N.
Devirama, S. Kumara, A. Panchpurib, M. and
Prasuna, R. G.2015. Production, optimization and
partial purification of protease from Bacillus subtilis.
Journal of Taibah University for Science. 9 (1):50–55.
Pelczar, M. J. and Chan, E.C. 1977. Laboratory Exercises in
Microbiology, 4th edition, McGraw Hill, Inc.
Penna, V.T.C. Martins, S.A.M. and Mazzola, P.G.2002.
Identification of bacteria in drinking and purified
water during the monitoring of a typical water
purification system. BMC Public Health. 2:13.
Poonia, S. Singh, T. S. Tsering, D. C.2014.Antibiotic
susceptibility profile of bacteria isolated from natural
sources of water from rural areas of east sikkim.
Indian J Community Med. 39(3): 156–160.
Qadeer, M. A. Aurangzeb, M. and Igbal, J.1989.
Production of raw starch hydrolyzing enzymes by
mould cultures. Proceeding of the international
symposium on biotechnology for energy. Dec.16-
21.Malik, K. A.; Nagvi, S.H. M. and Aleem, M.I. H.
(eds.) Faisalabad, Pakistan.119-128
Ramachandran, S. Patel, A. K. Nampoothiri, K. M.
Chandran, S. Szakacs, G. Soccol, C. R. and Pandey,
A. 2004. Alpha amylase from a fungal culture grown
on oil cakes and its properties. Braz. Arch. Biol.
Technol. 47 :309–317.
Robinson, J.J.2000. Effects of calcium and magnesium on
829
a 41-kDa serine-dependent protease possessing
collagen-cleavage activity. J Cell Biochem. 18; 80(1):
139-145.
Saha, D. Purkayastha, G. D. Ghosh, A. Isha, M. and Saha,
A. 2012. Isolation and characterization of two new
Bacillus subtilis strains from the rhizosphere of
Eggplant potential biocontrol agents. Journal of Plant
Pathology. 94 (1): 109-118.
Sai-Ut, S. Benjakul, S. and Sumpavapon, P. 2013. Screening
of gelatinolytic enzyme producing bacteria for
production of hydrolysate with antioxidative
activity. 2nd International Conference on Nutrition and
Food Sciences, IPCBEEl.53:11.IACSIT Press,
Singapore.
Sambrook, J. and Russel, D.2001.Molecular cloning: A
Laboratory Manual.3rd ed. Cold Spring Harbor, NY:
Cold Spring Harbor laboratory.
Samra, Z. Q. Naseem, M. Khan, S. J. Dar, N. and Athar, M.
A. 2009. PCR targeting of antibiotic resistant bacteria
in public drinking water of Lahore metropolitan,
Pakistan. Biomedical and Environmental Sciences. 22:
458463.
Selim, H. M. M. ,Gomaa, N. M. and Essa A. M.2017.
Application of endophytic bacteria for biocontrol of
Rhizoctoniasolani (Cantharellales:
Ceratobasidiaceae) damping-off disease in cotton
seedlings. Biocontrol Sci Technol. 27(1): 81-95.
Sevinc, N. and Demirkan, E.2011. Production of protease
by Bacillus sp. N-40 isolated from soil and its
enzymatic properties. J. Biol. Environ. Sci. 5(14): 95-
103.
Sharmin, S. Lutful Kabir, S. M. Enamul Hoque Kayesh,
M. Ziaul Haque, A. K. Mufizur,
R.M.2013.Bacteriological quality of tap water
samples obtained from different sources in and
around Mymensingh city of Bangladesh with
particular focus on antimicrobial resistance of
Escherichia coli. Advanced Research Journal of
Microbiology.1(2):10-17.
Sicuia, O. Grosu, I. Constantecscu, F. Voaides, C. and
Cornea, C. P. 2015. Enzymatic and genetic variability
in Bacillus spp. Strains with plant beneficial qualties.
AgroLife Scientific Journal.4(2).
Singh, S. K. Tripathi, V. R. Jain, R. K. Vikram, S. and Garg,
S. K.2010. An antibiotic, heavy metal resistant and
halotolerant Bacillus cereus SIU1 and its
thermoalkaline protease. Microbial Cell Factories. 9:59.
Singh, Y. Ramteke, P. W.Tripathy, A. and Shukla,P. K.2013.
Isolation and characterization of Bacillus resistant to
multiple heavy metals. Int. J. Curr. Microbiol. App.
Sci. 2(11): 525-530.
Song, B. and Leff, L. G.2005.Identification and
characterization of bacterial isolates from the Mir
space station. Microbiological Research. 160:111—117.
Srivastava, S. Singh, P. Bhagat, R. and Tripathi, V. N. 2005.
Application of bacterial biomass as a potential metal
indicator. Current science.89(7).
Stavreva –Veselinovska, S. 2011. Microorganisms
indicators of the level of soil pollution with lead. 1st
 830 KHALED E. EL-GAYAR
National Agriculture Congress and Exposition on behalf
of Ali Numan Kýraç with International Participation.
April 27-30, 2011.
Suthar, S. Chhimpa, V. and Singh. S. 2009.Bacterial
contamination in drinking water:a case study in rural
areas of northern Rajasthan, India. Environ Monit
Assess.159(1-4):43-50.
Syed, S. and Chinthala, P. 2015.Research article: Heavy
metal detoxification by different Bacillus species
isolated from solar salterns. Scientifica.Vol 2015.
Tasic, S. Kojic, M. Obradovic, D. and Tasic, I. 2014.
Molecular and biochemical characterization of
Pseudomonas putida isolated from bottled
uncarbonated mineral drinking water. Arch. Biol. Sci.
66 (1):23-28.
Tekin, N. Cihan, A. C. Takac, Z. S. Yagci, T. C. Tunc, K.
View publication stats
and Cokmus, C. 2012. Alkaline protease production
of Bacillus cohnii APT5.Turk J Biol.36: 430-440.
Vijayadeep, C. Sastry, P. S.2014.Effect of Heavy Metal
Uptake by E. coli and Bacillussps. Journal of
Bioremediation & Biodegradation. 5:238.
Worthington.2017. http://www.worthington-biochem.com/
introbiochem/substrateconc
Zaghloul, T. I. Abdel – Aziz, A. and Mostafa, M. H.1994.
High level of expression and stability of the cloned
alkaline protease (aprA) gene in Bacillus subtilis.
Enzyme Microb. Technology.16:537.
Zaghloul, T. I. Haroun, M. A., El-Gayar, K. and Abdelal,
A. 2004. Recycling of Keratin-containing Materials
(Chicken Feather) Through Genetically Engineered
Bacteria. Polymer-Plastics Technology and Engineering.
43 (6) : 1589-1599.