isolation, identification and production of amylases from...

AASCIT Journal of Bioscience

2017; 3(6): 52-68

http://www.aascit.org/journal/bioscience

ISSN: 2381-1250 (Print); ISSN: 2381-1269 (Online)

Keywords Amylases Activity,

Starch Hydrolysis,

Bacillus spp.,

Corn Starch,

Broken Rice

Received: July 10, 2017

Accepted: August 29, 2017

Published: September 26, 2017

Isolation, Identification and Production of Amylases from Thermophilic Spore Forming Bacilli Using Starch Raw Materials Under Submerged Culture

Rawia Fathy Gamal, Khadiga Ahmed Abou-Taleb*,

Basma Talaat Abd-Elhalem

Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo,

Egypt

Email address [email protected] (K. A. Abou-Taleb) *Corresponding author

Citation Rawia Fathy Gamal, Khadiga Ahmed Abou-Taleb, Basma Talaat Abd-Elhalem. Isolation,

Identification and Production of Amylases from Thermophilic Spore Forming Bacilli Using Starch

Raw Materials Under Submerged Culture. AASCIT Journal of Bioscience.

Vol. 3, No. 6, 2017, pp. 52-68.

Abstract Out of 133 amylase producing bacterial isolates, two isolates were selected, which gave

the highest values of starch hydrolysis ratio (SHR) on agar plates ranged from 2.80 to

3.06 and α-amylase activity in broth medium ranged from 73.5 to 77.0 Uml-1

after 48 h

at 50°C were recorded by B85 and B87. These isolates were identified based on

phenotypic characteristics and confirmed by 16S rRNA gene analysis (genotypic

characterizes), the isolates B85 and B87 were identified as Bacillus megaterium and B.

licheniformis, respectively. The results also showed both strains achieved the highest

growth and amylases activity during 10 – 24 h and 18 - 24 h incubation periods,

respectively. Corn starch and broken rice proved to be the best carbon sources for the

production of the highest enzymes by B. megaterium and B. licheniformis at 2%

concentration, respectively. 0.44 gL-1

ammonium sulphate and 3.65 gL-1

corn steep

liquor were the best nitrogen concentration for amylases activity by B. megaterium and B.

licheniformis, respectively. The increase of enzymes activity for α-amylase about 2.9-

fold, β-amylase about 18.5 &15.6-fold and γ-amylase about 3.8 & 23.9-fold on modified

medium, as compared with basal medium for B. megaterium and B. licheniformis,

respectively.

1. Introduction

The most important amylases are α-amylase; β-amylase and glucoamylase [1]. Soluble

starch degradation by α-amylase into soluble malto-oligosaccharides and limit dextrins

[2]. Then, dextrins and oligosaccharides are hydrolyzed to maltose and glucose by other

enzymes (β and γ) amylase respectively, therefore α-amylase tends to be faster-acting

than ß-amylase [3, 4]. These enzymes are found in many living organisms such as

animals (saliva, pancreas), plants (malts), several microorganisms like bacteria [5] and

fungi [6]. Microbial amylases are available commercially and they have almost

completely replaced chemical hydrolysis of starch in the starch processing industry [7].

The major advantage of using microorganisms for the production of amylases is in

53 Rawia Fathy Gamal et al.: Isolation, Identification and Production of Amylases from Thermophilic Spore

Forming Bacilli Using Starch Raw Materials Under Submerged Culture

economical bulk production capacity and microbes are also

easy to manipulate to obtain enzymes of desired

characteristics [8]. The thermophilic bacteria were capable of

producing thermostable amylases with high thermostability

in production processes and they can maintain their activity

longer, such as Bacillus stearothermophilus, B. licheniformis,

B. acidocaldarius, B. macerans, B. megaterium and B.

amyloliquefaciens [3, 9, 10]. Microorganisms play an

important role in transforming agricultural waste into

important products such as enzymes [11]. a low and/or good

starchy waste or by-products used as carbon source for

biosynthesis of amylases like wheat bran, soybean, broken

rice, potato peel, potato starchy waste, sun flower, cotton

seed meal, rice bran and husk [12-14].

This study was aimed to isolate thermo-starch degrading

Bacillus strains from rhizosphere samples and utilize starch

agro-industrial materials as a carbon source for amylases

production.

2. Materials and Methods

2.1. Samples Collection

Rhizosphere samples were obtained from the fertile fields

planted with wheat (Triticum aestivum), Egyptian clover

(Trifolium alexandrinum), broad bean (Vicia faba) and sugar

cane (Saccharum officinarum), in Qalyubia and Menoufia

governorates. Soil samples were collected from 3 to 5 cm

depth after removing 5 cm from the ground surface. These

samples were collected into sterilized plastic bags and stored

in ice-boxes during their transport to the laboratory. In the

laboratory, all samples were kept refrigerated until isolation.

2.2. Media Used

Nutrient agar medium [15] was used for maintenance and

preservation of bacteria. Starch agar medium [16] was used

for isolation of starch-degrading bacteria. Its composition

was as follows (gL-1

): soluble starch, 10; KNO3, 0.5; K2HPO4,

1; MgSO4.7H2O, 0.2; CaCl2, 0.1; FeCl3, traces; agar, 15 and

adjusted to pH 7.0. Starch broth medium was the same as

starch agar medium without adding agar.

2.3. Isolation and Screening of the

Amylolytic Bacteria

Ten-gram representative soil sample was suspended in 90

ml of sterile tap water and shaken thoroughly for 10 min.

Starch-degrading microorganisms were isolated from

collected samples by the soil dilution plate technique using

starch agar medium [17]. Serial dilutions up to 10-7

of each

soil sample were prepared using sterilized water. Suitable

dilutions were plated (in triplicates) on the above solid

medium. After incubation of plates at 50°C for 48 h, the

plates were immersed with 1% Lugol’s iodine reagent for 20

min, then washed with distilled water to remove the excess

color. The clear halo-zone around colonies was measured to

calculate starch hydrolysis ratio (SHR) [18]. The selected

isolates were preserved on agar slant for further use.

2.4. Phenotypic and Genotypic Identification

Identification of selected isolates were carried out

according to their morphological including shape, size and

Gram & endospore staining were observed under light

microscope) and biochemical tests (catalase, starch

hydrolysis, gelatin hydrolysis, casein hydrolysis, indole

production and Voges–Proskauer test) based on Bergey's

Manual of Systematic Bacteriology [19]. It was then

confirmed by 16S ribosomal ribonucleic acid (rRNA)

sequencing, the genomic deoxyribonucleic acid (DNA) was

isolated from two isolates using the method suggested by

Hiney and colleagues [20]. Amplification of 16S rDNA by

polymerase chain reaction (PCR) was performed using

bacterial universal primers (27F) forward F (5`-AGA GTT

TGA TCC TGG CTC AG-3ˊ) and (1492R) reverse R (5`-

GGT TAC CTT GTT ACG ACT T-3ˊ) [21]. PCR was

carried out in a 50 µl reaction volume. The thermal cycle

(PCR) steps were applied as follows; 5 min initial

denaturation at 95°C, followed by 30 cycles of 1 min

denaturation at 95°C, 1 min primer annealing at 55°C, 1 min

extension at 72°C and a final 10 min extension at 72°C. The

PCR products were detected on 1% (w/v) agarose gel

electrophoresis, eluted and purified using the Qiaquick gel

extraction kit (Qiagen, Germany) following the

manufacturer’s protocol [22]. The purified PCR product was

sequenced by the Big-Dye terminator kit ABI 310 Genetic

Analyzer (Applied Biosystems, USA). Sequences were

further analyzed using Basic Local Alignment Search Tool

(BLAST) from the National Center of Biotechnology

Information (NCBI) website (http://www.ncbi.nlm.nih.gov)

[23] and the software package CLC Main Workbench version

5.5 (Windows 7 6.1) www.clcbio.com was used for multiple

alignments and phylogenetic analysis [24]. The phylogenetic

tree was constructed neighbor-joining algorithm. A bootstrap

analysis (100 repeats) was performed to evaluate the

topology of the phylogenetic tree.

2.5. Submerged Fermentation Process

It was carried out in 250 ml plugged Erlenmeyer flasks,

each containing 100 ml sterile starch broth medium

(medium 2) and inoculated with 1% of standard inoculum

(2.74 ×106 CFUml

-1) for the tested bacterial strains and

incubated at 50°C for 48 h on a rotary shaker at 150 rpm.

Samples (5 ml) were taken from the growing cultures

periodically every 6 hours under aseptic conditions and

centrifuged at 10,000 rpm for 10 min in order to determine

periodically the cell dry weight and amylases activity in the

precipitate and supernatant, respectively. All the

experiments were carried out at least in triplicate [25]. The

relation between time and the optical density (at 620 nm) of

growth (growth curve) was plotted using Microsoft Office

Excel (2013). The growth parameters were calculated from

the exponential phase.

AASCIT Journal of Bioscience 2017; 3(6): 52-68 54

2.6. Influence of Starchy Raw Materials as

Carbon Sources on Enzymes Activity

This experiment was performed to study the effect of

different carbon sources on amylases production by tested

strains. Therefore, The appropriate carbon source was

selected by replacing the original carbon source of the used

medium with equivalent carbon amount of each of the tested

carbon source (wheat bran, wheat straw, broken rice, rice

bran, rice straw, rice husk, corn cobs, corn starch and potato

waste) to eliminate errors which may occur as a result of

differences in carbon concentrations in each source.

Different corn starch and broken rice concentrations being

0.5, 1.0, 1.5, 2.0 and 2.5% were added to the fermented

medium to study their effect on amylases production by the

tested isolates.

2.7. Influence of Nitrogen Sources on

Enzymes Activity

The effect of various nitrogen sources on enzyme production

was evaluated by replacing KNO3 by equivalent nitrogen

amount of each of the tested organic by equivalent nitrogen

amount of each of the tested organic nitrogen source [beef

extract, corn steep liquor (CSL), malt extract, peptone, soy bean

meal, tryptone and yeast extract] and inorganic [ammonium

chloride, ammonium citrate, ammonium nitrate, ammonium

sulphate & ammonium phosphate] nitrogen sources.

Different concentrations of ammonium sulphate (0.11 -

0.55 gL-1

) or corn steep liquor (0.73 - 4.83 gL-1

) were

estimated for enzymes synthesis by the tested strains.

2.8. Analytical Procedures

Alpha amylase activity was assayed using starch–iodine

method [26]. One gram soluble starch (Sigma S-2630) was

gelatinized in 100 ml distilled water and continue mixing at

100°C for 15 min. then 0.5 ml of the mixture was mixed with

0.5 ml of 0.1 M phosphate buffer, pH 7.0 and 1 ml of crude

enzyme. The mixture was incubated at 60°C for 30 min. the

reaction was stopped by addition of 1 ml HCl (1M). The

residual starch in the supernatant was determined

colorimetrically by 1ml of iodine reagent (5mM I2 and 5mM

KI) and read at 620 nm using spectrophotometer (Unico S2100

series UV/Vis). Starch–iodine assay is defined as the

disappearance of an average of 1mg of iodine binding starch

material per min in the assay reaction. One unit (U) of alpha

amylase was defined as the amount of enzyme which the

disappearance of an average of 1mg of iodine binding starch

material per min. Uml-1

was calculated using the formula [27]:

Uml-1 = (A620nm control - A620nm sample) / (A620nm/mg starch)

/30 min / 1ml /dilution factor. (1)

Where,

A620nm control is the absorbance obtained from the starch

without the addition of enzyme, A620nm sample is the

absorbance for the starch digested with enzyme,

A620nm /mg starch is the absorbance for 1 mg of starch as

derived from the standard curve.

Beta and gamma amylase activities were estimated the

reducing sugars (expressed as glucose) liberated from starch

using glucose oxidase peroxidase kits (BIO-ADWIC)

obtained from EL NASR PHARMACEUTICAL

CHEMICALS Co., Egypt [28]. The reaction was started by

adding 0.5 ml sample of a crude enzyme into 0.5 ml of 1%

soluble starch and 0.5 ml of 0.1 M acetate buffer, pH 4.8 or

4.5 and incubated for 3 min at 45 and 55°C, respectively. The

glucose released in the supernatant was estimated

colorimetrically using spectrophotometer (Unico S2100

series UV/Vis) at 550 nm. One unit (U) of beta and gamma

amylases is that amount of enzyme which catalyzed the

formation of 1 µ mole of glucose in 1 min. Both enzyme

activities were calculated by following formula [29]:

Uml-1 = (Amount of reducing sugar × dilution factor) / (1000

× MW of glucose (180.2) × time × enzyme volume). (2)

2.9. Parameters Calculations

Specific growth rate per hour (µ) [30]=

(ln X –ln X0) (t - t0)-1 (3)

Where:

X = Amount of growth after t time (t).

X0 = Amount of growth at the beginning time (t0).

Doubling time (td) [30] = ln2 (µ)-1 (4)

Where:

µ = Specific growth rate per hour.

Multiplication rate (MR) [31] = 1(td)-1 (5)

Where:

td = Doubling time.

Number of generation (N) [32] = (t - t0) (td)-1 (6)

Where:

t = Ending time.

t0 = Beginning time.

td = Doubling time.

Productivity (Uml-1h-1) [33] = Enzyme activity / Time (h) (7)

SHR [18] = Clear halo zone diameter (mm) / Colony growth

diameter (mm) (8)

2.10. Statistical Analysis

The collected data were statistically analyzed using IBM®

SPSS® Statistics software [34] and the correlation

coefficient was analyzed with Microsoft Office Excel 2013.

3. Results and Discussion

3.1. Isolation and Screening the Most

Efficient Starch Degrading Bacteria

In the present investigation, 51 out of 133 bacterial isolates



were isolated from different plants rhizosphere on starch agar

medium at 50°C. The percentage distribution of thermophilic

starch degrading isolates (Figure 1a). The highest

thermophilic starch degrading isolates were detected from

Egyptian clover followed by wheat and broad bean being

45.0%, 27.5%, and 25.5%, respectively.

These isolates were classified into three categories

according to the diameter of clear halo zone (starch

hydrolysis) namely; high, moderate and low starch

hydrolyzing isolates which showed halo-zone diameter

ranged between & mm, & mm and &mm, respectively

(Figure 1b). The highest diameter of clear halo-zone was

recorded for B87 isolate (110.0 mm) followed B85 isolate

(90.0 mm) as illustrated in Figure 2a.

Figure 1. The percentage distribution of thermophilic starch degrading isolates, a) obtained from different plants rhizosphere, b) into three categories which

express as a diameter of zone hydrolysis (mm).

Figure 2. Screening the most efficient starch degrading bacteria by qualitative on starch agar plate floating with Lugol’s iodine reagent a) and quantitative

estimations on broth medium at 50°C for 48 h.

B = Broad bean, E= Egyptian clover, S = Sugarcane and W =Wheat. Different letters on top of bars in the same column indicate significant differences and the

same letter do not significantly differ from each other, according to Duncan’s at 5% level.


Data recorded in Figure 2b clearly shows that SHR

(starchy hydrolysis ratio) and α-amylase activity ranged from

1.05 - 3.06 and 15.0 - 77.0 Uml-1

for thermophilic isolates,

respectively. The statistical analysis (Analysis of variance by

the ANOVA and means of difference by Duncan) of the data

proved that isolates B87 and B85 (which isolated from the

broad bean) gave the highest values of SHR (3.06 and 2.80)

and α-amylase activity (77.0 and 73.5 Uml-1

), respectively.

As previously observed, the starch degrading

microorganisms from different sources and respective

amylases activity [5]. The highest ratios of starch degradation

ranged from 3.4-4.0 for tested isolates [35]. While the ratio

of starch degradation by B. licheniformis was 1.5 compared

to the other tested bacterial species [36]. From all the

previous data, it could be stated that B87 and B85 were the

best isolates for amylase activity on solid and broth media at

50°C. So, both isolates were selected as the potential starch

degrading isolates and selected for subsequent investigations.

3.2. Identification of the Most Potent Starch

Degrading Bacterial Isolates

3.2.1. Phenotypic Characteristics

The selected isolates were identified depending on their

cultural, morphological and biochemical properties based on

Bergey's Manual of Systematic Bacteriology [19]. The

results obtained show that the most potent starch degrading

isolates namely B85 and B87 were isolated from rhizosphere

broad bean plants at 50°C. All tested isolates B85 and B87

were Gram positive, rod shaped, motile, endospore-forming

bacteria, aerobic and positive reaction with catalase, starch

hydrolysis, casein hydrolysis, gelatin hydrolysis, citrate

utilization, while gave negative results for nitrate reduction

and Voges-Proskauer. These preliminary characteristics

suggested that B85 and B87 were characterized as Bacillus

species.

3.2.2. Genotypic Characteristics and the

Phylogenetic Tree

Molecular identification and classification in a base of 16S

rDNA sequence analysis is an important tool for correct

identification of microbial species then morphological,

physiological and biochemical characterization due to

cumbersome and time-consuming [37]. This method of

identification is mainly based on the conservation of 16S

rDNA sequence among bacterial species. That is to say, the

16S rDNA sequence is actually species specific. Each species

has a unique 16S rDNA sequence which reflects the validity

of the test. In this method, the total genomic DNA was

isolated, purified and used as a template for PCR reaction

(Figure 3a). The analysis of 16S rRNA gene of B.

megaterium was sequenced with R1 primer at the reverse

direction and produced 1212 and 1253 bp, respectively.

While the analysis of 16S rRNA of isolate B. licheniformis

sequenced with F1 primer at the forward direction produces

1223 bp. The results of PCR sequences were compared with

the other sequenced bacteria in National Center for

Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov)

in Gene Bank and the Ribosomal Database Project (RDP)

database showed a similarity of derived sequences with some

sequences belonging to the 16S small subunit rDNA of other

bacteria. A phylogenetic tree was conducted by taking the

sequences obtained in blast search. The sequence obtained

from BLASTN (nucleotide blast) was obtained in FASTA

format and relation between each sequence could be known

by multiple sequence alignment using a software CLUSTAL

algorithm. The tree was generated using neighbor joining (NJ)

a distance-based algorithm of phylogenetic analysis.

Bacterial isolates (B85 and B87) were clustered. Based on

16S rRNA gene analysis, isolates B85, B87 were grouped

into genus Bacillus. The sequence of B85 and B87 was most

closely related to B. licheniformis (accession number

DSM_13) and B. megaterium (accession number QMB_1551)

with a similarity of 97%, respectively (Figure 3b). Similar

results were isolated two hundred and seventy amylolytic

isolates from soil samples in Khartoum State and were

identified as Bacillus sp. which found the most potent

amylases producers isolates were identified as B.

licheniformis, B. subtilis, B. cereus and B. megatarium [36].

Also, nine isolates collected from the waste potato dumpsites,

which characterized as Bacillus species [38]. In addition to,

identified the most efficient amylolytic isolate as Bacillus sp.

[39].



Figure 3. Molecular identification based on 16S rRNA sequence, a) agarose gel electrophoresis shows PCR product of 16S rDNA sequence from the three

isolates. b) the phylogenetic tree was constructed via the bootstrap test of the neighbor-joining algorithm based on the 16S rRNA gene sequences of isolates

B85 and B87, and related species of the genus Bacillus.

Lane M represents DNA base pair marker. Bootstrap analysis was performed with 100 replicates, are shown at the branch points. GenBank sequence accession

numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in CLC Main Workbench software version 5.5.

3.3. Time Course of Biomass Production and

Amylases Activity

The samples were taken every six hours during the 48 h.

The tested bacteria were grown on starch broth medium and

incubated at 50°C for 48 h. The results revealed that B.

licheniformis and B. megaterium grew exponentially during

the first 10 - 24 h of incubation periods. The time course

analysis of exponential phase increased to reach the peak in

the 24th

hour for B. licheniformis and B. megaterium (Figure

4a). The growth was found to be more constant (stationary

phase) during 48 h, then began to decrease (decline phase).

The correlation coefficient (r) between fermentation periods

and growth was a high positive (r= 0.90) for both strains. The

illustrated data of growth parameters for the tested bacteria

(Figure 4b) pointed out that the specific growth rate (µ) of B.

megaterium and B. licheniformis were 0.20 and 0.13 h-1

,

respectively. The corresponding figures of multiplication rate

(MR) were 0.26 and 0.19, respectively. The lowest doubling

time (td) was achieved by B. licheniformis followed by B.

megaterium to be 4.0 and 5.3 h, respectively. The highest

values of the number of generations were 3.6 for B.

megaterium, while it was 2.6 for B. licheniformis.

Results illustrated in Figure 5 showed that the highest

amylases activity obtained by B. licheniformis was 90.1 Uml-

1 of α-amylase, 0.16 Uml

-1 of β-amylase and 0.18 Uml

-1 of

glucoamylase (γ- amylase) after 24, 18, 24 h of fermentation

periods. While the maximum amylases activity have been

recorded on 24 h of α-amylase (80.4 Uml-1

) and 18 h of β &

γ-amylases (0.15 & 0.20) for B. megaterium. The increasing

activity of amylases was achieved during the log phase of the

bacterial growth profile. Amylases activity were decreased

after 36 - 42 h for all the tested bacteria. a high positive

correlation coefficient (r) between fermentation periods and

each of α-amylase activity (Uml-1

), β-amylase (Uml-1

)

activity and γ-amylase (Uml-1

) activity, r values were 0.93 &

0.93, 0.86 & 0.88 and 0.90 & 0.91 for B. megaterium and B.

licheniformis, respectively. The highest amylases activity and

biomass were obtained during 24 h of incubation periods for

B. subtilis and Bacillus sp. ANT-6 [13]. With regard Karnwal

[40] reported that the highest α-amylase activity was

observed at 24 h of incubation periods for Pseudomonas

fluorescence Apk10 strain. Moreover, the level of amylase

activity increased during the exponential phase of

Lactobacillus fermentum growth [25].


Figure 4. Growth curves (a) and parameters (b) of B. megaterium and B. licheniformis grown on starch broth medium (basal medium) at 50°C using shake

flasks as a batch culture.

Figure 5. Amylases activity of B. megaterium and B. licheniformis grew on starch broth medium (basal medium) during 48 h of incubation periods at 50°C

using shake flasks as a batch culture.



3.4. Starchy Raw Materials as Carbon

Sources

Biosynthesis of amylases were made on agro-industrial

wastes and by-products as a trial to reduce pollution

problems and obtained a low-cost medium [41]. To

investigate the effect of various carbon sources such as corn

cobs, corn starch, potato starchy waste, rice bran, broken rice,

rice husk, rice straw, soluble starch (control), wheat bran and

wheat straw on amylases activity for B. licheniformis and B.

megaterium at various time intervals, the experiment were

incorporated with basal medium by replacing soluble starch

(control 1%). the amylases activity of α, β and γ for B.

megaterium were increased gradually during the fermentation

periods and reached to maximum peak after 18 & 18 and 24

h on medium supplemented with corn starch by 95.4, 0.34

and 0.65 Uml-1

, respectively followed by broken rice with

93.6 & 0.30 and 0.52 Uml-1

, at 24 h of fermentation periods,

respectively (Table 1).

In case of B. licheniformis the α, β and γ amylases activity

were increased gradually during the fermentation periods and

reached to maximum peak after 18 h on medium

supplemented with broken rice by 97.4, 0.80 & 2.10 Uml-1

,

respectively followed by potato starchy waste with 96.8, 0.45

and 0.90 Uml-1

, at 18 h of fermentation periods, respectively

(Table 2). The lowest amounts of enzymes observed in

medium supplemented with wheat bran or corn cobs for all

the tested bacteria ranged from 27.5 – 37.7 Uml-1

of α

amylase, 0.01- 0.07 Uml-1

of β amylase and 0.03-0.06 Uml-1

of γ amylase. The significant reduction of enzymes activity

may be due to thickness of the fermentation medium for

wheat bran leading to decrease culture aeration, which was

essential for the growth and amylases activity [12] also, corn

cobs weren’t suitable as carbon source, it could be due to

their content of starch is very poor, to be insufficient for

amylases activity [42]. All other carbon sources gave less

significant results as compared to corn starch or broken rice

for B. megaterium and B. licheniformis, respectively. So,

these carbon sources were selected for subsequent studies.

Table 1. Effect of different carbon sources on amylases activity of B. megaterium on basal medium incubated at 50°C during 48 h using shake flasks as a

batch culture.

Carbon

sources

Time

(h)

Enzymes activity (U/mL) Carbon

sources

Time

(h)

Enzymes activity (U/mL)

α β γ α β γ

Soluble starch

(Control)

0 0.0 0.0 0.0

Rice bran

0 0.0 0.0 0.0

6 13.3 0.0 0.05 6 5.1 0.0 0.01

12 27.3 0.1 0.1 12 12.2 0.0 0.03

18 50.4 0.2 0.2 18 27 0.0 0.05

24 80.4 0.1 0.19 24 44.2 0.0 0.08

30 75.8 0.1 0.18 30 60.1 0.0 0.08

36 69.5 0.1 0.17 36 55.8 0.0 0.07

48 60.2 0.1 0.15 48 50.7 0.0 0.07

rt 0.93 0.9 0.91

rt 0.91 0.9 0.93

Broken rice

0 0.0 0.0 0.0

Rice husk

0 0.0 0.0 0.0

6 15.2 0.1 0.06 6 9.1 0 0.03

12 32.7 0.2 0.18 12 21.5 0.1 0.04

18 50.2 0.2 0.4 18 37.2 0.3 0.05

24 93.6 0.3 0.52 24 64.9 0.4 0.08

30 88.8 0.3 0.5 30 62.5 0.3 0.13

36 80.3 0.2 0.46 36 58.3 0.3 0.11

48 70.7 0.2 0.43 48 52.1 0.3 0.09

rt 0.91 0.9 0.93 rt 0.92 0.8 0.88

Corn cobs

0 0.0 0.0 0.0

Rice straw

0 0.0 0.0 0.0

6 4.1 0 0.01 6 5.1 0 0.01

12 9 0 0.01 12 12.3 0 0.02

18 14.7 0.1 0.03 18 25.6 0 0.03

24 22.1 0.1 0.04 24 39.8 0.1 0.05

30 37.7 0.1 0.06 30 67.1 0.1 0.07

36 34.5 0.1 0.06 36 62.5 0.1 0.07

48 28.4 0.1 0.05 48 54.2 0 0.06

rt 0.86 1 0.91 rt 0.88 0.9 0.94

Corn starch

0 0.0 0.0 0.0

Wheat bran

0 0.0 0.0 0.0

6 18.6 0.1 0.08 6 4.1 0 0.01

12 52.3 0.2 0.16 12 8.2 0 0.02

18 95.4 0.3 0.39 18 13.5 0 0.02

24 93.1 0.3 0.65 24 21.9 0 0.03

30 88.5 0.3 0.63 30 37.7 0 0.03

36 80.7 0.3 0.6 36 33.5 0 0.02

48 71.9 0.2 0.57 48 27.4 0 0.02

rt 0.92 0.9 0.91 rt 0.86 0.9 0.91


Carbon

sources

Time

(h)

Enzymes activity (U/mL) Carbon

sources

Time

(h)


α β γ α β γ

Potato starchy

waste

0 0.0 0.0 0.0

Wheat straw

0 0.0 0.0 0.0

6 17.1 0.1 0.02 6 5.8 0 0.01

12 42.1 0.1 0.06 12 10 0 0.02

18 93.1 0.2 0.09 18 17.2 0 0.03

24 92.2 0.2 0.17 24 25.7 0 0.06

30 88.5 0.2 0.16 30 40.1 0 0.06

36 83.1 0.2 0.15 36 36.3 0 0.05

48 77 0.2 0.12 48 30 0 0.05

rt 0.91 0.9 0.9

rt 0.89 0.9 0.9

Basal medium = starch broth medium, rt= Correlation coefficient between time and amylases activity.

Table 2. Effect of different carbon sources on growth and amylases activity of B. licheniformis on basal medium incubated at 50°C during 48 h using shake


Carbon Time Enzymes activity (U/mL) Carbon Time Enzymes activity (U/mL)

sources (h) α β γ sources (h) α β γ

Soluble starch

(Control)

0 0.0 0.0 0.0

Rice bran

0 0.0 0.0 0.0

6 16.3 0.0 0.03 6 8.9 0.0 0.01

12 30.2 0.1 0.06 12 18.6 0.0 0.01

18 55.1 0.2 0.09 18 34.2 0.0 0.03

24 90.1 0.2 0.18 24 46.6 0.1 0.06

30 86.3 0.2 0.17 30 65.5 0.1 0.06

36 81.8 0.1 0.15 36 60.3 0.0 0.05

48 77 0.1 0.14 48 53.5 0.0 0.05

rt 0.93 0.9 0.9 rt 0.94 0.8 0.87

Broken rice

0 0.0 0.0 0.0

Rice husk

0 0.0 0.0 0.0

6 19.3 0.2 0.58 6 12.5 0.0 0.01

12 46.5 0.4 1.08 12 30.8 0.0 0.03

18 97.4 0.8 2.1 18 53.4 0.1 0.05

24 96.1 0.8 2.08 24 72.6 0.1 0.08

30 91.8 0.7 2.04 30 70.3 0.1 0.08

36 85.4 0.6 2 36 65.1 0.1 0.07

48 80.8 0.6 1.5 48 58.6 0.1 0.07

rt 0.92 0.9 0.95 rt 0.96 0.9 0.93

Corn cobs

0 0.0 0.0 0.0

Rice straw

0 0.0 0.0 0.0

6 3.63 0.0 0.004 6 13.2 0.0 0.01

12 7.2 0.0 0.01 12 26.9 0.0 0.04

18 12.2 0.0 0.02 18 39.1 0.0 0.06

24 19.3 0.0 0.03 24 55.8 0.1 0.08

30 27.6 0.0 0.05 30 74.7 0.1 0.12

36 24.8 0.0 0.05 36 69.5 0.1 0.11

48 19.6 0.0 0.04 48 61 0.1 0.09

rt 0.9 0.8 0.85 rt 0.95 0.9 0.91

Corn starch

0 0.0 0.0 0.0

Wheat bran

0 0.0 0.0 0.0

6 17.3 0.1 0.06 6 3.5 0.0 0.01

12 29.2 0.1 0.18 12 7.6 0.0 0.012

18 51.2 0.3 0.3 18 13.5 0.0 0.026

24 96.4 0.4 0.58 24 19.5 0.0 0.04

30 91 0.4 0.52 30 28.3 0.0 0.035

36 84.5 0.4 0.49 36 24.5 0.0 0.028

48 73.4 0.3 0.44 48 19.4 0.0 0.021

rt 0.9 0.9 0.9 rt 0.9 1 0.87

Potato starchy

waste

0 0.0 0.0 0.0

Wheat straw

0 0.0 0.0 0.0

6 4.1 0.0 0.25 6 8.2 0.0 0.03

12 14.2 0.2 0.54 12 14.7 0.1 0.06

18 96.8 0.5 0.9 18 26.7 0.1 0.09

24 94.1 0.5 0.87 24 40.4 0.1 0.11

30 90.5 0.4 0.8 30 59.1 0.2 0.2

36 83.6 0.4 0.75 36 53.4 0.2 0.19

48 78 0.3 0.71 48 45.6 0.2 0.15

rt 0.81 0.9 0.92

rt 0.91 0.9 0.89

Basal medium = starch broth medium, rt= Correlation coefficient between time and amylases activity.



As previously reported the potato and rice starches were

suitable substrates for amylases production by Bacillus sp.

[43, 44]. Similar results indicated that amylases activity were

high when maize starch was used as carbon source followed

by potato starch [45]. The corresponding figures of amylases

activity α, β and γ were increased by 1.19, 2.27 and 3.25 fold

and 1.08, 4.0 and 11.7 fold for B. megaterium and B.

licheniformis, respectively comparing to control (Tables 1

and 2).

From the above experiments, it could be stated that

availability of carbon source to the organism is one of the

most important factors to be considered in the production of

bacterial amylolytic enzymes.

3.5. Different Starch Substrate

Concentrations

An experiment was carried out to study the effect of

different concentrations of corn starch and rice broken which

exhibited superiority among other tested carbon sources for B.

megaterium and B. licheniformis, respectively. So, five

concentrations of tested carbon sources ranging between 0.5

and 2.5% were used for amylases activity by the tested

bacteria.

Modified medium = Basal medium– (soluble starch) + KNO3+ corn starch.

Figure 6. Effect of different concentrations of corn starch on amylases activity of B. megaterium on modified medium incubated at 50°C during 48 h using

shake flasks as a batch culture.

Modified medium = Basal medium–(soluble starch) + KNO3+ broken rice.

Figure 7. Effect of different concentrations of broken rice on amylases activity of B. licheniformis on modified medium incubated at 50°C during 48 h using

shake flasks as a batch culture.


Data present in Figures 6 and 7 clearly show that, the

highest values of α, β, and γ amylases activity in medium

supplemented with 2% corn starch and broken rice by B.

megaterium (137.3, 0.46 & 0.81 Uml-1

) and B. licheniformis

(161.9, 1.1 & 2.5 Uml-1

) were attained after 18, 18 and 24 h

of fermentation periods, respectively. The statistical analysis

demonstrated a high positive correlation coefficient (r)

between carbon source concentrations and each of α, β and γ

amylase (Uml-1

) for B. megaterium and B. licheniformis.

Correlation coefficient (r) were incorporated in high ranked

(0.82 - 0.97). In similar studies, the 2.0% starch was the best

carbon source for amylases activity by Bacillus sp. [39, 46].

Moreover, the addition of 1.5 to 2.5% starch to fermented

medium gave a high yield of the enzyme using Pseudomonas

fluorescence and decreased at 3.5% starch [40]. At high

concentrations of starch in production medium, the activity

of amylases was decreased [47].

So, it could be stated that 2% of each of corn starch or

broken rice were the best carbon sources for the growth and α,

β and γ amylase activity by B. megaterium and B.

licheniformis resulting to increase activity by 1.44, 1.66 and

1.58 fold and 1.3, 1.38 and 1.9 fold as compared to the

lowest proper carbon concentration.

It is rather generally accepted rule in enzymology that

production of the specific enzyme by the microorganism is

stimulated when the culture medium in which the organism

grows, contains the substrate to be attacked by the enzyme. It

is assumed that enzymes are produced by microorganisms for

the modification of potential nutrient substrates to bring them

into a form which can be assimilated by the organism.

3.6. Influence of Nitrogen Sources

Microorganisms behave differently in the presence of

different N sources. The efficiency of 13 different nitrogen

sources on amylases activity was investigated. Data in Table

3 show that the highest figure of enzymes activity secreted by

B. megaterium was recorded when ammonium sulphate was

used as inorganic nitrogen source being 186.0 Uml-1

of α-

amylase, 1.4 Uml-1

of β-amylase and 3.20 Uml-1

of γ-amylase

after 18, 24 and 24 h, respectively. Soybean meal was found

next nitrogen source for enzyme synthesis (154.7, 1.23 &

2.75 Uml-1

of α, β and γ amylase). Generally, it could be

stated that ammonium sulphate had a positive impact on α, β

and γ amylase activities by B. megaterium (1.36, 3.04 & 3.9

fold), comparing with potassium nitrate (KNO3) as a control.

These results are in agreement with those obtained by

Kumarai et al [45] who noticed that sodium nitrate and

ammonium sulphate were the best nitrogen sources for

biosynthesis of amylases.

On the other hand corn steep liquor was the best organic

nitrogen sources for amylases synthesis by B. licheniformis,

respectively (Table 4) could be interrupted on the basis corn

steep liquor not only as a nitrogen sources but also as a

source of growth factors and protein which play a vital role

in enhancement the amylases activities. Among the tested

nitrogen sources, corn steep liquor followed by soybean meal

were the best organic nitrogen sources for B. licheniformis

giving 212.2 & 156.6 Uml-1

, 1.6 & 1.1 Uml-1

and 3.10 & 2.60

Uml-1

of α, β and γ amylases after 18, 18 & 24 h of

incubation periods, respectively. The minimum values of

enzyme activities were recorded on medium supplemented

with malt extract or ammonium phosphate. Also, it could be

noticed that the values of enzyme activity of α, β and γ

amylase increased by 1.31, 1.45 & 1.19 fold in medium

supplemented with corn steep liquor than enzyme activity in

medium supplemented with KNO3 (control), respectively

(Table 4). On the other hand, it was noticed that the

availability of (NH4)2SO4 and corn steep liquor to B.

megaterium and B. licheniformis is one of the most important

factors to be considered in the production of amylolytic

enzymes. Similar results recorded that soybean meal

presented a positive effect and was the best nitrogen source

for α-amylase production by Bacillus sp. 1-3 strain [48, 49].

Also, soybean meal and yeast extract were the best nitrogen

sources and showed a significant effect on α-amylase [50]. In

addition, Rasooli et al [9] and Božić et al [10] revealed that

the organic nitrogen sources were stimulated amylases

synthesis from Bacillus spp., it could be contained amino

acids, growth factors and vitamins [51].

3.7. Influence of Nitrogen Sources

Concentrations

Variations in concentrations of ammonium sulphate or

corn steep liquor were effective for amylases synthesis by B.

megaterium and B. licheniformis, respectively. Therefore

different concentrations of ammonium sulphate (0.11 - 0.55

gL-1

) or corn steep liquor (0.73 - 4.83 gL-1

) were estimated

for enzyme activities by the tested bacteria (Figures 8 and 9).

The highest values of enzymes activities produced by B.

megaterium and B. licheniformis being 236.2 & 258.5 Uml-1

of α-amylase, 1.90 & 2.5 Uml-1

of β-amylase and 3.70 & 4.3

Uml-1

of γ-amylase were obtained at 0.44 gL-1

ammonium

sulphate and 3.65 CSL after 18, 18 and 24 h of fermentation

periods.

Results in Table 5 show that the highest level of enzymes

activities achieved on the modified medium by all tested

bacilli being 236.0 & 258.5 Uml-1

of α-amylase, 1.9 & 2.5 &

1.6 Uml-1

of β-amylase and 3.7 & 4.3 Uml-1

of γ-amylase for

B. megaterium and B. licheniformis, respectively. Enzymes

productivity were 13.1 & 14.4 Uml-1

h-1

of α-amylase, 0.11 &

0.14 Uml-1

h-1

of β-amylase and 0.15 & 0.18 Uml-1

h-1

of γ-

amylase for B. megaterium and B. licheniformis, respectively.



Table 3. Effect of different nitrogen sources on amylases activity of B. megaterium on modified medium incubated at 50 °C during 48 h using shake flasks as a

batch culture.

Nitrogen

sources

Time Enzymes activity (U/mL) Nitrogen Time Enzymes activity (U/mL)

(h) α β γ sources (h) α β γ

KNO3

(Control)

0 0.0 0.00 0.00

Malt

extract

0 0.0 0.00 0.00

6 25.6 0.09 0.21 6 13.6 0.06 0.11

12 61.3 0.26 0.42 12 40.8 0.13 0.31

18 137.1 0.46 0.82 18 91.4 0.53 0.71

24 131.6 0.40 0.81 24 86.6 0.80 1.50

30 124.1 0.35 0.76 30 81.4 0.75 1.43

36 117.6 0.31 0.71 36 77.2 0.70 1.35

48 109.1 0.29 0.67 48 72.1 0.61 1.28

rt 0.91 0.95 0.97

rt 0.89 0.87 0.88

Amounium

nitrate

0 0.0 0.00 0.00

Yeast

extract

0 0.0 0.00 0.00

6 26.9 0.04 0.01 6 8.8 0.04 0.07

12 75.9 0.11 0.25 12 21.2 0.16 0.26

18 140.5 0.32 0.58 18 45.3 0.36 0.51

24 136.3 0.62 1.16 24 86.5 0.61 1.20

30 130.5 0.55 1.07 30 81.5 0.51 1.13

36 124.1 0.50 0.95 36 75.4 0.47 1.06

48 112.2 0.45 0.84 48 68.1 0.33 0.91

rt 0.92 0.85 0.85

rt 0.89 0.86 0.85

Amounium

phosphate

0 0.0 0.00 0.00

Beef

extract

0 0.0 0.00 0.00

6 9.6 0.01 0.02 6 5.3 0.03 0.07

12 20.2 0.06 0.07 12 14.1 0.07 0.13

18 44.8 0.17 0.27 18 30.2 0.15 0.28

24 76.4 0.30 0.65 24 62.8 0.21 0.56

30 70.3 0.28 0.64 30 57.3 0.20 0.54

36 63.7 0.21 0.55 36 50.1 0.18 0.46

48 50.7 0.17 0.40 48 43.1 0.17 0.43

rt 0.89 0.82 0.81

rt 0.85 0.93 0.87

Amounium

sulphate

0 0.0 0.00 0.00

Peptone

0 0.0 0.00 0.00

6 30.9 0.35 0.57 6 11.5 0.03 0.21

12 95.4 0.69 1.59 12 29.1 0.25 0.59

18 186.0 1.00 2.16 18 66.5 0.44 1.11

24 180.2 1.40 3.20 24 95.2 0.80 2.00

30 166.2 1.33 2.88 30 90.5 0.76 1.89

36 161.1 1.23 2.77 36 86.8 0.72 1.73

48 150.7 1.15 2.62 48 81.4 0.62 1.51

rt 0.95 0.97 0.95

rt 0.93 0.91 0.9

Amounium

chloride

0 0.0 0.00 0.00

Tryptone

0 0.0 0.00 0.00

6 15.2 0.06 0.15 6 24.9 0.27 0.48

12 44.4 0.19 0.58 12 70.0 0.57 1.34

18 91.5 0.42 1.00 18 123.8 1.00 2.50

24 85.6 0.75 1.70 24 119.5 0.99 2.43

30 77.5 0.66 1.64 30 113.1 0.87 2.34

36 68.1 0.58 1.57 36 109.1 0.81 2.25

48 63.3 0.49 1.33 48 101.8 0.77 2.15

rt 0.87 0.87 0.92

rt 0.92 0.90 0.92

Amounium

citrate

0 0.0 0.00 0.00

Corn steep

liquor

0 0.0 0.00 0.00

6 2.8 0.03 0.02 6 6.6 0.05 0.12

12 10.7 0.05 0.03 12 21.3 0.11 0.30

18 27.3 0.09 0.07 18 43.9 0.34 0.67

24 50.6 0.16 0.13 24 85.1 0.57 1.10

30 45.8 0.16 0.11 30 81.0 0.51 1.05

36 40.6 0.13 0.09 36 76.6 0.51 0.93

48 36.7 0.12 0.09 48 71.1 0.45 0.84

rt 0.86 0.91 0.85

rt 0.92 0.9 0.92

Soybean

meal

0 0.0 0.00 0.00

6 38.5 0.14 0.41

12 84.1 0.44 0.84

18 154.7 0.77 1.72

24 150.5 1.23 2.75

30 144.9 1.13 2.53


Nitrogen

sources

Time Enzymes activity (U/mL) Nitrogen Time Enzymes activity (U/mL)

(h) α β γ sources (h) α β γ

36 137.1 1.11 2.39

48 128.5 1.08 1.95

rt 0.93 0.94 0.94

N= nitrogen source, Modified medium = basal medium – (soluble starch + KNO3) + 2% corn starch + nitrogen source, rt= Correlation coefficient between

time and amylases activity.

Table 4. Effect of different nitrogen sources on amylases activity of B. licheniformis on modified medium incubated at 50 °C during 48 h using shake flasks as

batch culture.

Nitrogen

sources

Time

(h)

Enzymes activity (U/mL) Nitrogen

sources

Time

(h)


α β γ α β γ

KNO3

(Control)

0 0.0 0 0

Malt extract

0 0.0 0.00 0.00

6 29.4 0.11 0.33 6 9.7 0.05 0.05

12 61.2 0.24 0.77 12 20.7 0.10 0.12

18 161.9 0.56 1.44 18 41.9 0.15 0.16

24 155.9 1.1 2.6 24 73.3 0.23 0.24

30 150.7 1.07 2.49 30 70.3 0.21 0.23

36 144.6 1.02 2.31 36 64.2 0.21 0.22

48 134.5 0.94 0.22 48 54.3 0.19 0.18

rt 0.92 0.95 0.96

rt 0.90 0.91 0.92

Amounium

nitrate

0 0.0 0 0

Yeast extract

0 0.0 0.00 0.00

6 35.7 0.17 0.5 6 3.5 0.07 0.07

12 87.2 0.54 1.02 12 16.1 0.13 0.15

18 184.9 1.3 1.72 18 45.7 0.22 0.23

24 173.7 1.27 2.9 24 76.4 0.30 0.46

30 163.7 1.21 2.8 30 70.2 0.27 0.45

36 152.6 1.14 2.58 36 63.7 0.24 0.42

48 142.5 1.05 2.43 48 53.1 0.21 0.35

rt 0.9 0.89 0.93

rt 0.9 0.91 0.9

Amounium

phosphate

0 0.0 0 0

Beef extract

0 0.0 0.00 0.00

6 7.5 0.02 0.04 6 19.8 0.08 0.30

12 15.3 0.05 0.06 12 47.8 0.19 0.50

18 26.5 0.09 0.1 18 86.1 0.35 0.90

24 66.7 0.19 0.2 24 81.1 0.50 1.20

30 61.2 0.18 0.2 30 75.2 0.48 1.90

36 56.3 0.16 0.16 36 68.8 0.47 1.71

48 48.4 0.12 0.15 48 60.6 0.41 1.54

rt 0.84 0.85 0.87

rt 0.9 0.92 0.92

Amounium

sulphate

0 0.0 0 0

Peptone

0 0.0 0.00 0.00

6 5.7 0.03 0.04 6 4.7 0.09 0.14

12 18.2 0.05 0.06 12 17.8 0.19 0.23

18 41.8 0.08 0.1 18 42.9 0.30 0.45

24 80.4 0.19 0.2 24 82.1 0.48 0.70

30 75.2 0.17 0.2 30 74.2 0.47 0.66

36 69.5 0.14 0.15 36 66.3 0.43 0.63

48 61.2 0.12 0.12 48 58.0 0.39 0.59

rt 0.9 0.84 0.84

rt 0.9 0.94 0.93

Amounium

chloride

0 0.0 0 0

Tryptone

0 0.0 0.00 0.00

6 21.5 0.13 0.19 6 17.0 0.14 0.26

12 51.8 0.26 0.49 12 47.0 0.23 0.51

18 93.3 0.48 0.93 18 87.2 0.47 0.80

24 87.8 0.8 1.73 24 83.3 0.70 1.32

30 83.3 0.77 1.72 30 76.7 0.69 1.30

36 79.8 0.73 1.7 36 72.6 0.66 1.24

48 72.1 0.65 1.64 48 67.3 0.61 1.20

rt 0.9 0.93 0.92

rt 0.9 0.94 0.95

Amounium

citrate

0 0.0 0.00 0.00

Corn steep

liquor

0 0.0 0.00 0.00

6 11.0 0.06 0.11 6 47.5 0.49 0.43

12 20.0 0.12 0.24 12 109.2 0.84 0.91

18 40.9 0.25 0.37 18 212.2 1.60 2.08

24 78.8 0.37 0.60 24 208.2 1.59 3.10

30 71.2 0.36 0.55 30 200.2 1.56 3.05

36 65.5 0.33 0.52 36 185.2 1.52 3.01

48 58.9 0.28 0.46 48 175.3 1.47 2.94

rt 0.9 0.93 0.94

rt 0.93 1.0 0.96



Nitrogen

sources

Time

(h)

Enzymes activity (U/mL) Nitrogen

sources

Time

(h)


α β γ α β γ

Soybean meal

0 0.0 0.0 0.0

6 35.5 0.3 0.6

12 81.2 0.5 1.1

18 156.6 0.8 1.6

24 150.3 1.1 2.6

30 141.0 1.07 2.53

36 131.3 1.06 2.42

48 120.7 1.0 2.31

rt 0.9 0.94 0.94

N= nitrogen source, Modified medium = basal medium – (soluble starch + KNO3) + 2% broken rice + nitrogen source, rt= Correlation coefficient between

time and amylases activity.

Modified medium = basal medium – (soluble starch + KNO3) + 2% corn starch + ammonium sulphate.

Figure 8. Effect of ammonium sulphate concentrations on amylases activity of B. megaterium on modified medium incubated at 50°C during 48 h using shake


Modified medium= basal medium – (soluble starch + KNO3) + 2% broken rice + C.S.L.

Figure 9. Effect of corn steep liquor concentrations on amylases activity of B. licheniformis on modified medium incubated at 50°C during 48 h using shake



Moreover, data clearly show that increased of enzymes

activity and productivity for α-amylase about 2.9 & 3.8 fold,

β-amylase about 18.5 & 3.9 fold and γ-amylase about 3.8 and

13.9 fold, as compared with basal medium (control) for B.

megaterium. While B. licheniformis gave an increase of

enzymes activity and productivity (α-amylase 2.9 & 3.8, β-

amylase 15.6 & 15.6 and γ-amylase 23.9 & 23.9 fold. so, the

modified media was the best in enzyme production, which

gave the highest production and reduction of cost and time.

these results are accordance with Haq et al [52] who pointed

that the ingredients of synthetic media (nutrient broth and

soluble starch) are very expensive and could be replaced by

economically agro-industrial residues with low costs.

From the previous results it could be summarized the

ingredients of the most suitable fermented medium for

amylases production which namely modified medium

containing 2% corn starch or broken rice, 0.44 gL-1

ammonium sulphate or 3.65 gL-1

corn steep liquor, 0.5

K2HPO4, 1.0 MgSO4.7H2O, 0.2 CaCl2, 0.1 and FeCl3 traces

for B. megaterium and/or B. licheniformis. So, the modified

basal medium 2-2 was more favorable than the basal medium

for amylases production by all the tested bacteria. From all

previous data, it could be stated that B. licheniformis was the

pioneer organism since it gave the highest amylases

comparing to B. megaterium. Therefore B. licheniformis was

selected for further studies as thermo-amylolytic bacteria.

Table 5. Comparative data for enzymes activity and productivity of the tested bacteria as influenced by fermentation media.

Strains Media Enzymes activity (Uml-1) Productivity (Uml-1h-1)

α β γ α β γ

B. megaterium

Basal medium (Control) 80.40 0.50 0.20 3.40 0.03 0.01

Modified medium 236.0 1.90 3.70 13.1 0.11 0.15

Fold increase 2.90 3.80 18.5 3.90 3.8 13.9

B. licheniformis

Basal medium (Control) 90.10 0.16 0.18 3.80 0.009 0.008

Modified medium 258.5 2.50 4.30 14.4 0.14 0.18

Fold increase 2.87 15.63 23.9 3.79 15.56 22.5

Productivity (Uml-1h-1) = Enzyme activity / Time (h).

4. Conclusions

One hundred and thirty-three starch degrading bacterial

isolates were isolated from different plants rhizosphere. Only

51 out of 133 isolates were degraded starch at 50°C. The

most efficient two isolates were selected based on the highest

starch hydrolysis ratio (SHR) and amylases production in

liquid starch medium and identified based phenotypic

characters and further confirmation by sequencing the 16S

rRNA gene. The isolates were identified as B. megaterium

and B. licheniformis. Amylases were favored in the presence

of corn starch and/or broken rice as carbon substrates and

ammonium sulphate and/or corn steep liquor as a sole

nitrogen source for B. megaterium and B. licheniformis,

respectively.

Acknowledgements

The authors would like to express their sincere

appreciation to Prof. Mohamed El-Sawy Mubarak, (Allah

have mercy on him), Depart. of Agric. Microbiology, Fac. of

Agriculture, Ain Shams Univ., for his advice.

References

[1] Akkaya B., Yenidünya A., Akkaya R., (2012), Production and immobilization of a novel thermo alkalophilic extracellular amylase from bacilli isolate. International journal of biological macromolecules, 50 (4), 991-995. doi: 10.1016/j.ijbiomac.2012.02.011

[2] Demirkan ES., Mikami B., Adachi M., Higasa T., (2005), Alpha amylase from B. amyloliquefaciens: Purification, characterization, raw starch degradation and expression in E. coli. Proc. Biochem.,

40, 2629-2646. doi: 10.1016/j.procbio.2004.08.015

[3] Shivaramakrishnan S., Gangadharan D., Nampoothiri KM., Socool CR., Pandey A., (2006), Alpha amylases from microbial sources an overview on recent developments. Food Technology and Biotechnology, 44 (2), 173-184.

[4] Liu Y., Lu F., Chen G., Snyder CL., (2010), High-level expression, purification and characterization of a recombinant medium-temperature α-amylase from Bacillus subtilis. Biotechnology letters, 32 (1), 119-124.

[5] Kathiresan K., Manivannan S., (2006), α-amylase production by Penicillium fellutanum isolated from mangrove rhizospheric soil. African Journal of Biotechnolog, 5 (10), 829-832.

[6] Kammoun R., Naili B., Bejar S., (2008), Application of a statistical design to the optimization of parameters and culture medium for α-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product). Bioresource Technology, 99 (13), 5602-5609. doi: 10.1016/j.biortech.2007.10.045.

[7] Pandya PH., Jarsa RV., Newalkar B., Bhalt PN., (2005), Studies on the activity and stability of immobilized α- amylase in ordered mesoporous silicas. Microporous and Mesoporous Materials, 77, 67-77.

[8] Aiyer PVD., (2005), Amylases and their applications. African Journal of Biotechnology, 4 (13), 1525-1529.

[9] Rasooli I., Astaneh SDA., Borna H., Barchini KA., (2008), A thermostable α-amylase producing natural variant of Bacillus spp. isolated from soil in Iran. American Journal of Agricultural and Biological Sciences, 3 (3), 591-596.

[10] Božić N., Ruiz J., López-Santín J., Vujčić Z., (2011), Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a. Biochemical Engineering Journal, 53 (2), 203- 209. doi: 10.1016/j.bej.2010.10.04



[11] Pandey A., Soccol CR., Rodriguez LJ., Nigam P., (2001), Solid State Fermentation in Biotechnology: Fundamentals and Applications. Asiatech Publishers Inc, New Delhi, pp. 1-221.

[12] Satyanarayana T., Noorwez SM., Kumar S., Rao JL., Ezhilvannan M., Kaur, P., (2004), Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles. Biochemical Society Transactions, 32, 276-278. Doi: 10.1042/.

[13] Asgher M., Asad M J., Rahman SU., Legge RL., (2007), A thermostable α-amylase from a moderately thermophilic Bacillus subtilis strain for starch processing. Journal of Food Engineering, 79 (3), 950-955. doi: 10.1016/j.jfoodeng.2005.12.053

[14] Vijayabaskar P., Jayalakshmi D., Shankar T., (2012), Amylase production by moderately halophilic Bacillus cereus in solid state fermentation. African Journal of Microbiology Research, 6 (23), 4918-4926.

[15] Difco Manual, (1984), Difco manual of dehydrated culture media and reagents for microbiological and clinical laboratory procedures. (9th Ed), Difco Laboratories, Detroit, M. (ed.) USA. DOI: http://dx.doi.org/10.5962/bhl.title.7276

[16] Madigan MT., Martinko JM., Stahl DA., Clarck DP., (2011), Brock biology of microorganisms. (13th Ed), Prentice Hall, New jersey, USA, pp. 642-656.

[17] Clark HE., Bordner GEF., Kabler PW., Huff CB., (1958), Applied Microbiology. International Book Company, New York, USA, pp. 27-53.

[18] Bahadure, RB., Agnihotri US., Akarte SR., (2010), Assay of population density of amylase producing bacteria from different soil samples contaminated with flowing effluents. International Journal of Parasitology Research, 2, 9-13. doi: http://dx.doi.org/10.9735/0975-3702.2.1.9-13.

[19] Niall AL., Paul DV., (2009), Genus Bacillus. In: Paul, D. V., George, M. G., Dorothy, J., Noel, R. K., Wolfgang, L., Fred, A. R., Karl-Heinz, S. & William, B. W. (eds), Bergey’s manual of systematic bacteriology. Vol. 3 (2nd Ed.), Springer, New York, USA, pp. 21- 128.

[20] Yadav V., Prakash S., Srivastava S., Verma PC., Gupta V., Basu V., Rawat, AK., (2009), Identification of common as species using 16S rRNA gene sequence. Bioinformation, 3 (9), 381-383.

[21] Raji AI., Möller C., Litthauer D., Van Heerden E., Piater, LA., (2008), Bacterial diversity of biofilm samples from deep mines in South Africa. Biokemistri, 20 (2), 53-62.

[22] Nimnoi P., Pongsilp N., Lumyong, S., (2010), Genetic diversity and community of endophytic actinomycetes within the roots of Aquilaria crassna Pierre ex Lec assessed by actinomycetes-specific PCR and PCR- DGGE of 16S rRNA gene. Biochemical Systematics and Ecology, 38, 595-601.

[23] NCBI, (2014), http://www.ncbi.nlm.nih.gov/BLAST/

[24] Knudsen B., Knudsen T., Flensborg M., Sandmann H., Heltzen M., Andersen A., Dickenson M., Bardram J., Steffensen PJ., Mønsted S., Lauritzen T., Forsberg R., Thanbichler A., Bendtsen JD., Görlitz L., Rasmussen J., Tordrup D., Værum M., Ravn M. N., Hachenberg C., Fisker E., Dekker P., Schultz J., Hein AK., Sinding JB., (2009), CLC Main Workbench software version 5.5 (Windows 7 6.1). CLC bio A/S, Denmark. (http://www.clcbio.com).

[25] Fossi BT., Tavea F., Jiwoua C., Ndjouenkeu R., (2011),

Simultaneous production of raw starch degrading highly thermostable α-amylase and lactic acid by Lactobacillus fermentum 04BBA19. African Journal of Biotechnology, 10: 6564-6574.

[26] Sudharhsan S., Senthilkumar S., Ranjith, K., (2007), Physical and nutritional factors affecting the production of amylase from species of Bacillus isolated from spoiled food waste. African Journal of Biotechnology, 6 (4), 430-435.

[27] Xiao ZZ., Storms R., Tsang A., (2005), Microplate-based carboxymethyl-cellulose assay for endoglucanase activity. Analytical Biochemistry, 342 (1), 176-178. doi: 10.1016/j.ab.2005.01.052

[28] Bryjak J., (2003), Glucoamylase, α-amylase and β-amylase immobilization on acrylic carriers. Biochemical Engineering Journal, 16 (3), 347-355. doi: 10.1016/S1369-703X(03)00114-1

[29] Karnwal A., Nigam, V., (2013), Production of amylase enzyme by isolated microorganisms and its application. International Journal of Pharmacy and Biological Sciences, 3 (4), 354-360.

[30] Painter PR., Marr AG., (1968), Mathematics of microbial populations. Annual Review of Microbiology, 22, 519-548. doi: 10.1146/annurev.mi.22.100168.002511.

[31] Stanier R., Doudoroff M., Adelberg EA., (1970), General Microbiology, (3rd Ed), Macmillan & Co., Ltd., London, pp. 302-306.

[32] Doelle, H. W. (1975). 2 - Enzymes, coenzymes and bacterial growth kinetics. In Bacterial Metabolism. (2nd Ed), Academic Press, New York, USA, pp. 38-83.

[33] Pirt SJ., (1975), Principles of cell cultivation. Blackwell Scientific, London, pp. 115-117.

[34] IBM® SPSS® Statistics, (2011), Version 19.0, SPSS Inc., Chicago, Illinois.

[35] Vaseekaran S., Balakumar S., Arasaratnam V., (2010), Isolation and identification of a bacterial strain producing thermostable α- amylase. Tropical Agriculture Research, 22, 1-11.

[36] Alkando AA., Ibrahim HM., (2011), A potential new isolate for the production of a thermostable extracellular α- amylase. Journal of Bacteriology Research, 3 (8), 129-137.

[37] Poorani E., Saseetharan M., Dhevagi P., (2009), L-asparaginase production and molecular identification of marine Streptomyces sp. strain EPD 27. International Journal of Integrative Biology, 7, 150-155.

[38] Verma V., Avasthi MS., Gupta AR., Singh M., Kushwaha A., (2011), Amylase production & purification from bacteria isolated from a waste potato dumpsite in district farrukhabad U. P state India. European Journal of Experimental Biology, 1 (3), 107-113.

[39] Vishnu TS., Soniyamby AR., Praveesh BV., Hema, TA., (2014), Production and optimization of extracellular amylase from soil receiving kitchen waste isolate Bacillus sp. VS 04. World Applied Sciences Journal, 29 (7), 961-967.

[40] Karnwal A, (2011), Screening and optimization of extracellular amylase production from plant growth promoting rhizobacteria. Annals Food Science and Technology, 12 (2), 135-141.


[41] Mukherjee AK., Borah M., Rai SK., (2009), To study the influence of different components of fermentable substrates on induction of extracellular α-amylase synthesis by Bacillus subtilis DM03 in solid-state fermentation and exploration of feasibility for inclusion of α-amylase in laundry detergent formulations. Biochemical Engineering Journal, 43 (2), 149-156. doi: 10.1016/j.bej.2008.09.011.

[42] Moreira FG., Lenartovicz VL., Peralta RM., (2004), A thermostable maltose-tolerant α-amylase from Aspergillus tamari. Journal of Basic Microbiology, 44 (1), 29-35. doi: 10.1002/jobm.200310302.

[43] Kamm B., Kamm M., (2004), Biorefinery-systems. Chemical and Biochemical Engineering Quarterly, 18 (1), 1-6.

[44] Kunamneni A., Permaul K., Singh, S., (2005), Amylase production in solid state fermentation by the thermophilic fungus Thermomyces funginosus. Journal of Bioscience and Bioengineering, 100 (2), 168-171. doi: 10.1263/jbb.100.168.

[45] Kumarai BL., Sairam CVS., Kumar TS., Sudhakar P., Vijetha P., (2011), Screening and isolation of thermostable α-amylase producing bacteria and optimization of physico-chemical parameters for increasing the yield. International Journal of Pharmacy and Technology, 3 (1), 1570-1583.

[46] Kanimozhi M., Johny M., Gayathri N., Subashkumar R., (2014), Optimization and production of α -amylase from halophilic Bacillus species isolated from mangrove soil sources. Journal of Applied & Environmental Microbiology, 3 (3), 70-73. doi: 10.12691/jaem-2-3-2.

[47] Santos EO., Martins MLL., (2003), Effect of the medium composition on formation of amylase by Bacillus sp. Brazilian Archives of Biology and Technology, 46 (1), 129-134.

[48] Goyal N., Gupta JK., Soni SK., (2005), A novel raw starch digesting thermostable alpha amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme and Microbial Technology, 37, 723-734. doi: 10.1016/j.enzmictec.2005.04.017

[49] Sodhi HK., Sharma K., Gupta JK., Soni SK., (2005), Production of a thermostable α-amylase from Bacillus sp. PS-7 by solid- state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production. Process Biochemistry, 40 (2), 525–534. doi.org/10.1016/j.procbio.2003.10.008.

[50] Fossi BT., Tavea F., (2013), Simultaneous production of raw starch degrading highly thermostable α-amylase and lactic acid by Lactobacillus fermentum 04BBA19. African Journal of Biotechnology, 10, 6564-6574.

[51] Gao L., Yang H., Wang X., Huang Z., Ishii M., Igarashi Y., Cui, Z., (2008), Raw straw fermentation using lactic acid bacteria. Bioresource Technology, 99 (8), 2742-2748. Doi: 10.1016/j.biortech.2007.07.001

[52] Haq I., Ashraf H., Iqbal J., Qadeer MA., (2003), Production of alpha amylase by Bacillus licheniformis using an economical medium. Bioresource Technology, 87 (1), 57-61.

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