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 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., *Corresponding author’s email: [email protected]
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 nonsporeforming rods are considered to be of relatively low virulence (Brook and Frazier, 1993). An
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, Grampositive 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 timeconsuming. 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,
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 coppercontaining 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
KHALED E. EL-GAYAR
by stabbing on the tube containing nutrient gelatin medium. After that, incubation the inoculated tube along with an uninoculated medium at 37°C for 14 days. Removing the tubes daily from the incubator and placing in ice bath or refrigerator (4°C) for 1530 minutes (until control is gelled) every day to check for gelatin liquefaction (Acharya, 2014). Screening of Bacillus subtilis for amylase production was done. Starch agar media plates were streaked with Bacillus subtilis colony using sterile tooth pick. After incubation at 37oC for 48 hours, the Petri dishes were flooded with 5.0 mL of iodine solution. The clear zone surrounding the colony was measured in mm from the edge of the colony to the limit of clearing and also diameter of colony was recorded (Qadeer et al, 1989). Semi-quantitative estimation was possible using the following formula: EA = D-d; were D is the diameter of the clearing zone; and d is the microbial colony diameter (Sicuia et al., 2015). Factors affecting on protease and amylase activity Effect of temperature on both of protease and amylase enzymes activity was studied at different temperatures (30, 37 and 50oC). Also, the effect of substrate concentration on enzymes activity was studied. For protease activity; the skim milk substrate concentrations were 5%, 10% and 20%. For amylase activity; the starch substrate concentrations 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.
millimeter (Selim et al., 2017). RESULTS In the current study, the bacterial total count of all samples of the commercial water was zero CFU/ml. The bacterial count of tap water samples was averaged; 1700, 5600 and 410, CFU/ml. MacConkey agar was used to isolate Gram – negative bacteria in parallel with isolation on nutrient agar. There wasn’t any growth on MacConkey agar, indicated that; the samples were free from Gram – ve bacteria or sewage wastes. After purification of selected several colonies using streaking techniques, the morphology graph of isolated bacteria showed that; The isolates were short chains forming, Gram-positive and rod shaped bacteria. According to the biochemical identification tests (Table 1), they were determined as Bacillus sp. To confirm the biochemical identification result, the sequencing of the gene for 16S rRNA was performed (Song et al., 2005; Tasic et al., 2014). The sequence of 16S rRNA of isolate from tap water was compared with the sequences in the NCBI gene bank data for 16S rRNA, with maximum homology (99% identity) of Bacillus subtilis BS-1 strain (accession number AY172514.1; Figures 1 and 2). From the results of biochemical identification tests and DNA alignments of the 16S rRNA sequence of the isolate
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 dual-inoculated plates, with the fungus alone as a control were incubated at 28oC for 7 days. Then the diameters of inhibition zones were measured in
Fig. 1. Partial DNA sequences of the 16S rRNA gene of the bacterial strain isolated from Tap water, Jazan, KSA and the corresponding gene of Bacillus subtilis BS-1 (accession number AY172514).
+ve +ve +/-ve -ve -ve +ve +ve +ve +ve
N-Acetyl Neuraminic Acid
8% Nacl α-D-Glucose
Stachyose pH5 +ve
Sucrose Gentiobiose D-
+ve +ve +ve
D-Glucose- 6-PO4 D-Fructose- 6-PO4
1% sodium lactate Fusidic acid
3-Methyl Glucose D-Fucose
Table 1. Biochemical analysis of bacterial strain isolated from tap water.
p-Hydroxy- Phenylacetic Acid Methyl Pyruvate
Mucic Acid Quinic Acid
L-Galactonic Acid Lactone
Niaproof 4 Guanidine HCl
L-Pyroglutamic Acid L-Serine
L-Glutamic Acid L-Histidine
Positive control Negative Control
Propionic Acid Acetic Acid
β-Hydroxy-D,L-Butyric Acid -ve
+ve +ve γ-Amino-Butryric Acid
Lithium chloride Potassium tellurite
L-Malic Acid Bromo-Succinic Acid
α-Keto-Glutaric Acid D-Malic Acid
Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water 821
KHALED E. EL-GAYAR
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
Fig. 3. The effect of some antibiotics on Bacillus subtilis 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.
1 2 3 4 5 6 7 8 9 10 11 12 13
Gentamicin (GM) Amikacin (AK) Colistin (CL) Rifampicin (RIF) Piperacillin (PRL) Ticarcillin (TC) Imipenem (IMI) Cefepime (CPM) Clindamycin (CD) Cephalothin (KF) Cotrimoxazole (TS) Erythromycin (E) Penicillin G (PG)
R = Resistance, S= sensitive
conc. (μg/ disc)
Status of susceptibility
halo zone diameter/mm
10µg 30µg 10µg 5µg 100µg 75µg 10µg 30µg 2µg 30µg 25µg 15µg 10 µg
S+++ S++++ S+ R S++++ S++ S+++ S++++ S++ S++++ S++++ S++++ S++++
21 25 12 0 25 20 22 25 20 30 30 30 30
Isolation, Identification and Characterization of Bacillus Subtilis from Tap Water
Fig. 4B. Monitoring the effect of Salinity (NaCl%) on the growth of Bacillus subtilis at 2%, 5% and 10% , O/N at 37oC.
Fig. 4A. Monitoring the effect of addition of yeast extract(with concentration 0.1%, 0.5% and 1%) on the growth of Bacillus subtilis, O/N at 37oC.
Fig. 4D. Monitoring the effect of temperature on the growth of Bacillus subtilis O/N at room temperature, 30oC, 37oC and at 48oC.
Fig. 4C. Monitoring the effect of pH on the growth of Bacillus subtilis at pH4, pH7 and pH10 O/N at 37oC.
Table 3. The heavy metals sensitivity test for Bacillus subtilis isolated from tap water.
Heavy metals concentration
50 mg/L 100 mg/L
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
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 worldwide, mainly through infectious diarrhea (Ashbolt, 2004). The United States Environmental Protection
Agency (EPA) has determined that the presence of 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. Temperature
Effect of temperature Halozone diameter for protease production
30oC 37oC 50oC Substrate % 5%skim milk 10%skim milk 20%skim milk Trace elements concentration 20 mg/l CaCl2 40 mg/l CaCl2 80 mg/l CaCl2 20 mg/l MgSO4 40 mg/l MgSO4 80 mg/l MgSO4 20 mg/l MnCl2 40 mg/l MnCl2 80 mg/l MnCl2
14 16 22
Halozone diameter for amylase production 12 20 10
Effect of substrate Substrate %
Halozone diameter for protease production 16 20 24
Halozone diameter for amylase production
0.2%starch 0.4%starch 0.6%starch
20 10 10
Effect of trace elements concentration Halozone diameter for Halozone diameter for protease production amylase production 24 24 24 26 27 24 23 25 27
25 25 25 20 25 25 29 29 29
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 rodshaped 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). Aamylase production from Bacillus cereus using solidstate 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 halfcentury, 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
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-testprinciple-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 : 21222126.
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 áAmylase 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 solidstate 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 5aminolevulinic 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.1621.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
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): 95103. 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
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.
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.