Isolation and Characterization of Alpha-Amylase Producing Bacteria from Bishoftu

Lakes, Ethiopia

Misgana Anbessa1, Asnake Desalegn1, Feng-Yan Bai2, Fitsum Tigu1,*

1
Department of Microbial Sciences and Genetics, Addis Ababa University, Addis Ababa,
Ethiopia
2
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Science,
Beijing 100101, PR, China
* Corresponding Author: [Link]@[Link]
ORCID: 0000-0001-9741-0062
Abstract

Alpha amylase is an enzyme that hydrolyze starch into simple sugar and has wide applications
in detergent, food, paper, textile, and pharmaceuticals industries. This study involved in
isolation, characterization and optimization of alpha-amylase-producing bacteria (APB) from
soil and water samples collected from Bishoftu lakes, Ethiopia. A total of 60 APB isolates were
obtained among them 3 potential isolates were selected and identified as Bacillus cereus
(B1W1), Aeromonas rivipollensis (G2W1) and Klebsiella pasteurii (G3W1) by 16S rRNA gene
sequence analysis. Based on growth optimization, glucose was identified as suitable carbon
source whereas beef extract, tryptone and ammonium chloride were identified as suitable
nitrogen sources. The highest crude enzyme activity of 4.5 ± 0.1, 4.3 ± 0.4 and 4.1 ± 0.1 U/mL
was obtained by Bacillus cereus, A. rivipollensis, and K. pasteurii, respectively. The strain B.
cereus was produced maximum crude enzyme activity of 5 ± 0.4 U/mL at 45℃ and pH 7. The
partially purified alpha amylase from B. cereus has a molecular weight of 50 kDa, and 10.1
U/mg specific activity, which is 2.3-fold of the crude enzyme activity. Soap made by this
enzyme successfully cleaned the cloth stained with potato waste.

Keywords: Amylase; Bacteria; Bishoftu lakes; Enzyme activity; Optimization

1
1. Introduction

Today, more than half of the carbohydrates that consumed by humans is come from starch,
which is the most prevalent source of diet (Benalaya et al., 2024). There are two types of
polysaccharides in starch: amylopectin, which is branched, and amylose, which is linear
(Schirmer et al., 2013). Amylose is made up of 1-4 glycosidic linkages and up to 6000 glucose
units. It consists of alpha-D-glucose molecules linked by α -1, 4- glycosidic link to form
covalent bonds (Doublier et al., 2007). Amylopectin is made up of short linear chains with α-
1,4 links that contain 10-60 glucose units and side chains with α-1,6 links that have about 1545
glucose units (Thompson, 2000).

Amylose and amylopectin proportions are influenced by the starch supply, typically, 20-30%
amylose is present in most common starches, such as barley, corn, and potatoes (Forssell et al.,
2002). To be used in soluble form, this starch must be break down into its smaller molecules
by process called liquefaction and saccharification. The efficient breakdown of starch into its
constituent glucose molecules requires the action of amylase (De Souza & de Oliveira
Magalhães, 2010). Plants, animals and microorganisms are capable of synthesizing amylase in
nature [1]. Among them, amylase produced from microorganisms is more advantageous due to
least cost, bulk production capability and the ease of manipulation of microbes to produce
enzymes with desired properties (Burhan et al., 2003).

Despite the extraction of alpha amylase-producing microbe by many researchers all over the
world, it is useful to find more effective amylase for the practical applications in Ethiopia
(Guta et al., 2024). The current enzyme employed in industry is suffered by thermal instability
and optimization challenges Therefore, it is important to identify and technologically
characterize amylase-producing bacteria to overcome the challenges faced in starch breakdown
processes and explore their potential for industrial applications. Five Bishoftu lakes were
selected as a sample collection site to achieve this goal in this study.

2. Materials and Methods

2.1. Description of the study area and sample collection

Bishoftu is located approximately 47 Km from the capital city, Addis Ababa, Ethiopia. The city
has an altitude of 1900 meters above sea level with an average temperature range from 20 to
25ºC. Among several lakes found in Bishoftu, five lakes; Hora, Babogaya, Kuriftu, Cheleleka,

2
and Arenguadie were purposively selected for the isolation and characterization of amylase-
producing bacteria. The physicochemical characteristics including temperature, pH and salinity
of each lake was measured. The temperature is 28 ºC (Hora and Babogaya), 30 ºC (Kuriftu), 31
ºC (Cheleleka) and 35 ºC (Arenguadie) lakes. The pH is 7.3 in Hora, 7.2 in Babogaya, 6.8 in
Kuriftu, 6.9 in Cheleleka, and 7.9 in Arenguadie. Salinity levels also exhibited noticeable
differences among the lakes: 10.8 ppt (Hora), 7.5 ppt (Babogaya), 8.7 ppt (Kuriftu), 3.9 ppt
(Cheleleka) and 14.1 ppt (Arenguadie), and. A total of 15 soil and 15 water samples were
collected from the five lakes. The samples were aseptically transported to laboratory and kept in
refrigerator at 4ºC for further analysis.

2.2. Isolation and screening

One gram of soil or 1 mL of water sample was blended with 9 mL of distilled water to make
the first 10-1 dilution, then further diluted up to 10-7 (soil) or 10-4 (water). A volume of 0.1 mL
aliquots transferred onto nutrient agar plates and incubated at incubated at 37ºC for 24 h. From
the plates having 30 to 300 colonies, morphologically distinct colonies were aseptically
transferred into nutrient broth and streak plating was performed to obtain a pure colony. Each
pure colony was grown on 1% starch media at 37 ºC for 24 h. After incubation, the plates were
flooded with 1 % iodine solution; a clear zone around the colonies indicated amylase
production (Yassin et al., 2021). The amylolytic index (AI) was used to screen the amylolytic
isolates using the formula:

Clear zone diameter (mm)

Amylolytic index (AI) =
Colony diameter

2.3. Submerged fermentation and Crude enzyme activity

To identify the best amylase producers among 60 amylase positive isolates, a modified
submerged fermentation method was conducted (Al-Johani et al., 2017). The fermentation
media contained 10 g/L of starch, 10 g/L of peptone and 20 g/L along with essential minerals:
0.02 g/L of MgSO4, 0.01 g/L of FeSO4, 0.05 g/L of CaSO4, and 0.05 g/L of K2HPO4. After 24
h of incubation period, the culture broth was centrifugated for 10 min in 4000 rpm, and the
supernatant was collected as crude enzyme.

3
2.4. Enzyme assay

Enzyme activity was measured by the DNSA method using maltose as standard curve. The
reaction mixture contained, 1 mL of crude amylase, 1 mL of starch (1%) and incubated for 15
min at 37ºC. After the reaction, l mL of DNSA reagent was added and the reaction was stopped
by boiling for 10 min in a water bath. The absorbance was measured at 540 nm using a
spectrophotometer (Hussein et al., 2020). One unit (U) of enzyme activity was defined as the
amount of amylase that liberates 1 mole of reducing sugar equivalent to maltose per minute
under the test conditions.

Release of maltose× Reaction volume × Dilution factor

Amylase activity = × 1000
Molecular mass of maltose × Incubation time

2.5. Identification and characterization of amylolytic isolates

2.5.1. Morphological and cultural characterization

The morphological features such as shape, color, and texture on agar plates was observed using
colony counter. These isolates were further described by their microscopic features under
compound microscope. The bacterial cells were stained and observed under oil immersion
(100×) objective lens.

2.5.2. Biochemical characterization

Biochemical tests such as gram reaction, methyl red, Voges-Proskauer, citrate, urease and
catalase tests were done using a 24 h bacterial culture inoculated to the appropriate media.

2.5.3. Molecular identification

Molecular identification was conducted by sequencing 16S rRNA gene. The full length of 16S
rRNA gene was amplified with primers pair 27 F: 5’-AGAGTTTGATCCTGGCTCAG-3′ and
1492 R: 5’ GGTTACCTTGTTACGACTT-3’. The PCR was conditioned at 94 °C for 10 min,
followed by 32 cycles at 94 °C for 30 sec, 55°C for 20 sec, and 72 °C for 55 sec; and a final
extension at 72 °C for 5 min with a total reaction volume of 25 μL. The amplified PCR products
were sequenced, and analyzed using the BLAST algorithm at NCBI GenBank database for
identification. Sequences alignment was conducted by ClustalW on MEGA 12. The
phylogenetic tree was constructed under the criteria: statistical method, neighbor-joining;
phylogeny test, bootstrap method with 1000 replications; model/method, Kimura 2-parameter
model as indicated elsewhere [2].
4
2.6. Growth optimization for amylase production

Different growth parameters for the production of amylase were optimized by one-factor-at-
time approach. The optimal temperature for amylase production was tested by incubating the
isolate at 20, 30, 40, and 50ºC, and produced reducing sugar was measured by DNSA
method.

The effect of pH on amylase production was determined by culturing the microbes in the
production media by adjusting the pH to 5, 6, 7, 8, and 9. The enzyme assay was conducted
after 24 h of incubation at 40ºC. Various incubation periods of 12, 24, 48, and 72 h was
conducted. The optimum incubation time for amylase production for the isolate was
determined by measuring the amylase activity at each incubation of 24-72 h (Laiz et al.,
2003).

Different carbon source: glucose, maltose, sucrose, lactose and fructose were tested by
adding 1% w/v to fermentation media to identify the best carbon source (Vijayabaskar et al.,
2012). For nitrogen sources (urea, tryptone, beef extract) and inorganic source (ammonium
chloride, ammonia) were checked after 24 h, at 40ºC and pH 7.

Inoculation concentration of bacterial isolates was done by adding bacterium at different

inoculum concentrations such as 0.5, 1, 1.5 and 2 mL. The enzyme activity was measured
after 24 h of incubation at 40ºC and pH 7 using the enzyme assay. The effect of metal ions
(0.002% w/v) on amylase production was checked using potassium chloride, sodium
chloride, zinc sulphate, and manganese sulphate added to basal media. The concentration of
starch and NaCl was checked by addition of 0.5 to 2% and the best activity was measured
after 24 h of incubation at 40ºC and pH 7 (Jha et al., 2013).

2.7. Characterization of crude enzyme

To determine the optimal temperature of the alpha-amylase activity, the enzymatic activity
was measured at different temperatures extending from 35, 40, 45 and 50ºC. For pH, the
substrate solutions were prepared by dissolving 1% soluble starch in different pH solutions
starting from 5 to 9 using phosphate buffer adjusted by NaOH and HCL (Simair et al., 2017).
The effect of divalent and monovalent ions, Ca2+, Mg2+, Mn2+, Zn2+, Na+ and K+ at a
concentration of 5 mM were used to determine their effects on the activity of amylase. The
effect of the reaction time was checked by incubating the enzyme with substrate for 15, 30,

5
45 and 60 min (Ahmed et al., 2020). Finally, the thermostability of the alpha-amylase was
determined by pre-incubated the enzyme at temperatures ranging from 45, 50, 60 and 65ºC
for 30 and 60 min. The activity of the non-treated enzyme was considered as 100% enzyme
activity.

2.8. Purification and SDS-PAGE analysis

Bacillus cereus (B1W1) which had the highest enzyme activity was selected for partial
purification and conducted using ammonium sulphate salt precipitation at saturation of 20-
40%, 40-60%, and 60-80% along with 0.1 M phosphate buffer at 4ºC and pH 7. The
precipitated protein was collected from each range of ammonium sulphate concentration and
centrifuged for 10 min at 4000 rpm. Then the supernatant was discarded and the pellet was
saved. Then, tightly sealed with membrane filter bag and dialyzed against 0.1 M potassium
phosphate buffer for 24 h. The assay of each concentration of collected enzyme was checked
and the best activity was selected for sodium dodecyl-sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) (Sani et al., 2014).

2.9. Specific activity

The specific activity of crude and partially purified enzyme was employed using egg
albumin standard by Bradford method. An equal volume of Coomassie Brilliant Blue reagent
was added to each standard sample. The samples were incubated at room temperature for 10
min, and optical density measured at 595 nm and standard curve was created using the
absorbance measurements to calculate protein concentration. Crude and partially purified
enzyme samples were then made and the absorbance was checked at 595nm as standard.
Then, the specific activity was calculated by the formula (Simpson, 2006 and Manns, 2011).

Specific activity (U/mg) =¿

2.10. Application of enzyme

The effectiveness of the collected enzyme was checked by detergent application. Two types of
soap were prepared at laboratory condition using sodium hydroxide, coconut oil, sodium
chloride and distilled water. One soap was without enzyme while the other was with 3 mL of
B1W1 (B. cereus) enzyme. Prepared equal pieces of white clothes contaminated with potatoes
waste; one was treated with soap without enzyme (20g), another was soap containing enzyme

6
(20g), and compared with clean cloth as a control. After that, rinsed with distilled water and
compared the cleanliness of each cloth against control (Roy et al., 2012).

2.11. Statistical analysis

The analysis of variance (ANOVA) at a 95% significance level (p < 0.05) was used. Origin
Lab 2024 software was used for graphic representation. To ensure reliability, all analyses
were conducted in duplicate. The data are displayed as mean ± standard deviation to clearly
illustrate variance.

3. Results

3.1. Microbial isolation and selected for amylase production

The results showed that 60 of isolates tested positive for amylase activity (Supplementary
Table 1). Clear zones forming around colonies in starch media indicated starch breakdown
due to amylase activity. These 60 isolates were subjected to submerged fermentation, after
fermentation, the enzyme activity was assayed, the best 3 isolates, B1W1, G2W1, and G3W1
which had, 1.28 ± 0.05, 1.96 ± 0.04, and 1.40 ± 0.02 ≥ 1.25 U/mL, respectively were
selected further analysis.

3.2. Conventional and molecular identification

The morphology of B1W1 was transparent colored colony, G3W1 was white while G2W1
yellowish in color. B1W1 isolate was dry in texture while G2W1 and G3W1, which were
rounded, and mucoid, respectively. Microscopically, the 3 isolates were rod in shape. B1W1
was gram positive while G2W1 and G3W1 were gram-negative bacteria. In terms of
motility, only G3W1is non-motile and B1W1 and G2W1 could not. Based on the 16S rRNA
gene sequences analysis, the three isolates were identified as identified as Bacillus cereus
(B1W1), as A. rivipollensis (G3W1) and K. pateurii (G2W1) with accession number of
……...

3.3. Optimization of amylase production by one factor at a time (OFAT) approach

The optimum temperature for the three isolates was identified at 40ºC, in which the highest
alpha amylase was produced by B. cereus, 2.3 ± 0.02 U/mL (Fig.1a). The best pH for amylase
production was observed at pH 7 (2.3 ± 0.03 U/mL) also by B. cereus (Fig. 1b). For incubation
time, the highest alpha amylase activity by all strains was seen at 24 h of incubation and the

7
highest was recorded 2.3 ± 0.02 U/mL by B. cereus (Fig.1c). There was a significant difference
within and between all strains at p < 0.05.

2.5
Temperature 30oc 5
40oc 2.5 pH 6
7
2.0 50oc
Enzyme Activity(U/mL)

Enzyme Activity(U/mL)
60oc 2.0
9

1.5
1.5

1.0
1.0

0.5
0.5

0.0 0.0
[Link] [Link] K. pasteurii [Link] A. rivipollesnsis [Link]
Strain
b Strain

12 hr
2.5 Incubation Time 24 hr
48 hr
72 hr
(U/mL)

2.0
Enzyme Activity

1.5

1.0

0.5

0.0

c
[Link] [Link] [Link]
Strain

Figure 1: The effect of temperature (a), pH (b), and incubation time (c) on strains.

All the 3 strains produced high amylase by glucose among carbon source and maximum activity
was scored by A. rivipollensis (3.4 ± 0.1 U/mL) followed by B. cereus () and K. pasteurii (3.4 ±
0.03 U/mL) (Fig. 2a). B. cereus showed the highest amylase activity with beef extract and
ammonium chloride as organic nitrogen and inorganic nitrogen sources, respectively. A.
rivipollensis and K. pasteurii scored high alpha amylase activity with tryptone as organic
nitrogen source (Fig. 2b & ?). Different isolates used different nitrogen sources. Overall, the
best amylase activity was observed upon utilization of tryptone as a nitrogen source (3.4 ± 0.03
U/mL) with A. rivipollensis followed by B. cereus (3.3 ± 0.1 U/mL) (Fig. 2b).

Up on utilization of metal ions, B. cereus scored high activity with Zn, and A. rivipollensis
scored with K and, K. pasteurii was performed best activity with Mn (Fig. 2c). It was observed
that different strains used different metal ions for alpha amylase production. Maximum activity
was scored by A. rivipollensis (3.3 ± 0.1 U/mL) with K followed by K. pasteurii (3.2 ± 0.04
U/mL). In most strains, Na ion source scored lowest production of amylase (Fig. 2c).

8
At 1.5 mL of inoculum concentration, the highest (3.3 ± 0.1 U/mL) amount of amylase activity
was observed in A. rivipollensis, followed by B. cereus (3.3 ± 0.02 U/mL). The more cell
suspension may decrease the activity of enzymes due to nutrient depilation. The optimum
inoculum size for all isolates was 1.5 mL (Fig. 2d). All strains scored maximum enzyme activity
at 1.5% starch saturation. Further increase in enzyme activity was not observed when the
medium saturated above 1.5% starch concentration (Fig. 2e). The 3 strains scored high activity
with 1.5% of NaCl (Fig. 2f).

4.0
Lactose
Carbon Source Glucose K
Metal ions
Sucrose
3.5
Nitrogen Source CO(NH2)2
[Link] 3.5 Na
3.5 Maltose NH4Cl Zn
Fructose NH3 Mn
Tryptone
3.0
3.0 3.0

2.5 2.5
2.5
Enzyme Activity(U/mL)

Enzyme Activity(U/mL)

Enzyme Activity(U/mL)
2.0 2.0
2.0

1.5 1.5 1.5

1.0 1.0 1.0

0.5 0.5 0.5

0.0 0.0 0.0

[Link] [Link] [Link] [Link] [Link] [Link] B,cereus A,rivipollensis [Link]

a
strain
b Strain

c
Strain

Inoculum Size
500 µL
1000 µL 3.5
Starch Concentration 0.5%
1% 3.5
NaCl Concentration 0.5%
3.5 1%
1500 µL 1.5%
2000 µL 2% 1.5%
2%
3.0 3.0 3.0

2.5 2.5 2.5

Enzyme Activity(U/mL)

Enzyme Activity(U/mL)

Enzyme Activity(U/mL)

2.0 2.0 2.0

1.5 1.5
1.5

1.0 1.0
1.0

0.5 0.5
0.5

0.0 0.0
0.0 [Link] [Link] [Link] [Link] [Link] [Link]
B,cereus [Link]
Strain
[Link]

e
Strain f Strain

Figure 2: Carbon source (a), nitrogen source (b), metal ions (c), inoculum size (d), starch
concentration (e), NaCl concentration (f) on isolates.

3.4. Characterization of crude enzyme

The alpha amylase extracted from the strains showed high enzymes activity at 45ºC which is
slightly greater than the strains optimum temperature for the enzyme production (Fig. 4a). The
optimum pH of alpha amylase was the same with that of strains at pH 7 (Fig. 4b). The metal

9
ions showed high activity with Ca2+, Mg2+, Mn2+, Na+, K+, and Zn2+ in respective orders.
Among metal ions, Ca2+ is the most active ion that enhanced the enzyme activity for all isolates
(Fig. 4c). In the case of reaction time, the amylase activity was decreased with the increasing
reaction time and the best reaction time was at 15 min (Fig. 4d).

In terms of thermal stability, the residual activity of alpha amylase was decreased as an
increase of temperature and time. At 45ºC for 30 min, the residual activity was retained above
90% of activity while for 60 min, all were retained the activity ≥ 86% (Fig. 4e). At 55ºC for 30
min ≥ 80% and after 60 min, it decreased to below 50% (Fig. 4f). The residual activity of alpha
amylase was still retained greater than 50% of activity at 65ºC when treated for 30 min, but
after 30 min of heat treatment, only < 40% was retained compared to the original activity.
These findings suggest that the enzymes have good thermal stability at moderate temperatures,
but are significantly compromised at higher temperatures.

10
 5 5
35
40
Temerature in oC pH
5
6
45 7
50 8
4 4
Enzyme Activity(U/mL) 9

Enzyme Activity(U/mL)
3 3

2 2

1 1

0 0
[Link] [Link] [Link] [Link] [Link] [Link]
Strain Strain

a b

5 Ca²⁺ 5
Effect of metal ions Mn²⁺ Reaction time 15min
Mg²⁺ 30min
Na⁺
45min
Zn²⁺
K⁺ 60min
4 4
Enzyme Activity(U/mL)

Enzyme Activity(U/mL)
3 3

2 2

1 1

0 0
[Link] [Link] [Link] [Link] [Link] [Link]
Strain
d Strain

11
 102
At 45ºC [Link] 100 At 55ºC [Link]
100 [Link] [Link]
[Link] [Link]
98 90

Residual Activity (%)

Residual Activity (%)

96
80

94
70
92

60
90

88 50

86
40
84
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (min) f Time (min)
e

110
At 65ºC [Link]
100 [Link]
[Link]
90
Residual Activity (%)

80

70

60

50

40

30

0 10 20 30 40 50 60

g Time (min)

Figure 4 showed enzymological characterization of the crude enzymes extracted from nine
isolates. The effect of temperature (a), the effect of pH (b), the effect of metal ions (c), the
effect of reaction time (d), thermal stability of treated enzyme for 30 min (e), and thermal
stability of treated enzyme for 45ºC for 30 and 60 min, (f) thermal stability of treated enzyme
for 55ºC for 30 and 60 min, (g) thermal stability of treated enzyme for 65ºC for 30 and 60 min
on amylase activity.

3.5. Partial purification and specific activity

The strain B. cereus (B1W1) was employed to SDS-PAGE for partial purification after
ammonium sulphate precipitation and dialyzed in 0.1 M phosphate buffer for 24 h. The 60-
80% fraction and crude extract were loaded to the gel along with protein ladder for
estimation of amylase molecular weight. Based on the molecular weight marker, the amylase
purified to 60-80% was observed around 50 kDa as shown in the Fig. 5.

12
 As shown in Table 1 initially, the total protein concentration was 46.9 mg/mL with a total
activity of 201.5 U/mL for crude enzyme. However, after precipitation of the crude enzyme
using ammonium sulphate at 40-60%, the volume was reduced to 6 mL with a total protein
concentration of 5.9 mg/mL. At 60-80% of ammonium sulphate, the volume was further
decreased to 3 mL with a total protein of 3.2 mg/mL. The maximum specific activity of 4.3
U/mg of protein was obtained from crude enzyme extract. While the partially purified
enzyme scored 8.0 and 10.1 U/mg of specific activity at 1st and 2nd steps of ammonium
sulphate precipitation, respectively. Overall, the ammonium sulphate purification technique
was slightly improved the enzyme purity and specific activity of amylase by 2.3-fold
compared to the crude enzyme.

M 60-80% Crude
180
150
100
80
60
50 50kDa
40

30
20
10

Figure 5: SDA PAGE result

Table 1: Crude and partial purified specific activity of alpha amylase enzyme

Step Volum Total Activit Total Specif Fold Yield

e of protei y activit ic (%)
fractio n (U/mL y activit
n (mg/m ) (U/mL y
(mL) L) ) (U/mg
)

Crude 50 46.9 4.0 201.5 4.3 1 100

enzym
e

40- 6 5.9 7.9 47.3 8.0 1.9 23.5

60%

60- 4 3.2 8.2 32.6 10.1 2.3 16.2

80%

13
3.6. Biotechnological application of enzyme

The effectiveness of the extracted alpha amylase was checked by detergent application and it
was properly worked. The results showed that soaps containing alpha amylase performed
better than soaps without enzyme. After washing, the piece of cloth treated with enzyme-
containing soap had more clarity, indicating that the enzyme successfully broke down the
starch in the potato waste.

4. Discussion

Bacillus cereus are gram-positive, rod shape, spore forming, and motile bacteria and known
to produce several industrially important enzymes, including alpha amylase (Bottone, 2010).
It is commonly known as a soil-dwelling bacterium, but a study demonstrated its presence in
aquatic environments, including lakes and other water bodies (Glasset et al., 2016). During
rainfall, B. cereus spores can be washed from soil into lakes and rivers, where they persist in
water and sediments (Jensen et al., 2003). Additionally, groundwater movement plays a role
in the dispersal of B. cereus into aquatic systems, as spores can leach into underground water
sources and eventually mix with surface water (Vilain et al., 2006).

The ability of B. cereus to form biofilms on surfaces such as sediments, aquatic plants, and
even industrial water pipes further supports its adaptation to water environments (Houry et
al., 2010). The presence of B. cereus in lakes and other water bodies can also be linked to
human activities such as agricultural runoff, wastewater discharge, and contamination from
food processing facilities, further supporting its role as both a soil-associated and waterborne
microorganism.

Aeromonas species are known as indigenous to aquatic environments and causes many
opportunistic infections in humans. A. rivipollensis is opportunistic pathogen with gram
negative, rod shaped, non-spore forming, and motile bacteria (Fono-Tamo et al., 2023). It was
not reported as alpha amylase producing bacteria while it has highest amylase activity in this
study.

K. pasteurii are also considered as an opportunistic pathogen to humans, gram negative, non-
motile, non-spore-forming, straight, rod-shaped types of bacteria (Merla et al., 2019).
Although the alpha amylase production of K. pasteurii is not well studied, K. oxytoca has been
shown to produce many enzymes including alpha amylase while it was the second in this study

14
(Yang et al., 2022). According to Yang et al., 2022, K. grimontii, K. huaxiensis, K.
michiganensis, K. pasteurii, K. spallanzanii, and three unidentified novel species make up the
nine species that named as Klebsiella oxytoca. This group could produce different enzyme
including amylase and it may include K. pasteurii.

Research demonstrated that different carbon sources have different effects on the production of
amylase (Vijayabaskar et al., 2012), who found that glucose increases amylase production. In
contrast, sucrose was used best for amylase production.

Ammonium chloride was the best nitrogen supplement for the isolates identified as B. cereus
for alpha amylase production, and beef extract as organic. This report is the same as the study
reported by (Sivakumar et al., 2012). A. tryptone as organic nitrogen and ammonium chloride
as inorganic nitrogen source. This is related with the study of (van Bel et al., 2021), but not
directly those strains in alpha amylase production. Metal ions for the current findings, Mn for
K. pasteurii, K for A. rivipollensis and Zn for B. cereus raises the production of the alpha
amylase production (Vijayabaskar et al., 2012).The metal ions checked in this were also
needed for the isolates and not as inhibitors.

This study found that a concentration of 1.5% NaCl was appropriate for amylase production.
The enzyme activity was gradually reduced above this concentration. This is because the cell
gets into hypertonic condition if the environment is a saltier condition leading to plasmolysis
(where cells lose water) and inhibiting the growth and enzyme production (Mahdavi et al.,
2010). Increasing the concentration of starch was increased the growth of isolates and 1.5%
was shown maximum yield of alpha-amylase and decreased above. This was because toxic
metabolic wastes that inhibit bacterial growth and alpha-amylase production may be the cause
of the decrease in enzyme production at high starch concentrations. In addition, elevated starch
concentrations made the broth culture more viscous, which hindered O 2 transport and limited
the amount of dissolved O2 needed for microbial development (Mishra & Behera, 2008).

The three species of the alpha amylase produced in this study efficiently work at 45ºC and pH
7 and can be used in industrial application, which match with this temperature, but it needs
highest level of purification of this enzyme in order to apply for the food industry. This is
mainly because the isolate is considered as an opportunistic pathogen to humans. Although
alpha amylase itself is not toxic, the presence of this pathogen may lead to a serious risk if the
enzyme is not properly purified. Partial purification of the enzyme from B. cereus (H3S1)
15
using ammonium sulphate precipitation was conducted and showed 2.3-fold increase in
activity from 4.3 U/mg of crude to 10.1 U/mg. SDS-PAGE conducted and a band of
approximately 50 kDa was detected after sample loading which showed significant evidence of
alpha amylase. This band is the expected molecular weight of alpha amylase, typically 40 to 60
kDa showing the presence of alpha amylase in the protein sample. The finding confirms that
the purification procedure was performed correctly, indicating that the proteins in the samples
are compatible with alpha amylase (Marco et al., 1996). Biotechnological application, soap
containing alpha amylase was tested for cleaning efficiency on clothing contaminated with
potato waste with the comparison with soap without enzyme against control. The enzyme
containing soap successfully broke down the starch in the potato waste. This increase in
cleaning efficiency in particular shows the potential use of microbial enzymes in laundry to
remove starch stains as the current study.

5. Conclusion

In general, the current research indicates that it is possible to isolate bacteria that produce
amylase from the soil and water of Bishoftu lakes. The isolate produced efficient yield of
amylase during submerged fermentation. The study confirmed that Bacillus cereus, A.
rivipollensis, and K. pasteurii exhibited significant enzyme activity under optimized
conditions. The enzyme could effectiveness in breaking down starch and its successful
application in detergent formulation highlight its industrial potential. The findings
demonstrate the value of locally isolated microbes in enzyme production, reducing reliance
on imported enzymes. Further research should focus on enhancing enzyme stability and
scalability for commercial applications.

Acknowledgment

This study was supported by the Addis Ababa University 10th round Thematic Research
Fund (Grant no. RD/LT-308/2022). Thus, the authors sincerely acknowledged Addis Ababa
University in bold for their financial support and extend our sincere gratitude to the
Department of Microbial, Cellular and Molecular Biology, Addis Ababa University.

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