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Production, Optimization and Characterization of Alpha Amylase
Isolated from Wastewater
*1Sulaimon Adebisi Musbau., 1Fasiku Oluwafemi Omoniyi., 2Aishat A., 3Hafsat A.L., 4Nafisat S.T
1Science Technology Department, Waziri Umaru Federal Polytechnic, Birnin-Kebbi, Kebbi, Nigeria
2,3Science Eductaion Department, Waziri Umaru Federal Polytechnic, Birnin-Kebbi, Kebbi State,
Nigeria
4Kebbi State University of Science and Technology Aliero, Kebbi, Kebbi State, Nigeria
Corresponding Author*
DOI: https://doi.org/10.51244/IJRSI.2025.120800183
Received: 07 Aug 2025; Accepted: 13 Aug 2025; Published: 18 September 2025
ABSTRACT
The study produced, optimized and characterized α-amylase from a bacterium isolated from waste water with a
view to obtaining best optimized conditions required for growth of the organism for the production of the
enzyme for industrial uses. The waste water collected from Daula restaurant located in Birnin Kebbi were
taken to laboratory and analysed. Isolates from the plates were screened for amylase activity using starch agar
and are detected on potato starch solution. The bacterium with the highest amylase activity was selected for
enzyme production. Optimal conditions for enzyme production by the bacterium were determined. The best
isolate from the waste that showed better ability for amylase production was identified molecularly as
Lysinibacillus sphaericus C4-31. The peak amylase activity was observed at day 4 of incubation
(3.44mM/min). The optimum pH and temperature for the production of Lysinibacillus sphaericus C4-31 α-
amylase was 8 and 30°C respectively. The result also revealed that ammonium phosphate supported higher
enzyme activity of 5.86mM/min among the nitrogen source. Glucose as a carbon source gave the highest
activity of 6.89mM/min. The study concluded that α-amylase can be synthesize by Lysinibacillus sphaericus
C4-31which is moderately thermostable and able to degrade many cheap raw starches and can therefore find
applications in the food industry.
Keywords: Enzymes; restaurant waste; bacteria; amylase.
INTRODUCTION
Microbial enzymes have a great number of applications in food, pharmaceutical, textile, paper, leather and
other industries (Hasan et al., 2006). Their applications have been increasing rapidly. Among industrially
important enzymes, hydrolases come in the first place and include enzymes with a wide substrate specificity.
Carbohydrases, proteases, pectinases and lipases are classified into hydrolases. They catalyze the hydrolysis of
natural organic compounds (Sundarram and Krishna, 2020; Underkofler et al., 2007; Rajan, 2001). A special
focus on amylase from a warm-adapted bacterium in this particular study. The α-amylases are enzymes that
hydrolyze starch molecules to generate progressively smaller polymers composed of glucose units (Windish et
al., 2005). The enzyme accounts for 65% of enzyme market in the world. To meet the growing demands in the
industry, it is necessary to improve the performance of the system and thus increase the yield without
increasing the cost of production (Gangadharan et al., 2008).
Today, a large number of microbial amylases have almost completely replaced the chemical hydrolysis of
starch. The main advantage of using microorganisms for the production of amylase is the ability to bulk
produces the enzyme and the easy manipulation of microbes to achieve enzymes with desired characteristics.
Moreover, the stability of microbial amylases are higher than those from plant and animal sources (Tanyildizi
et al., 2005).
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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While studies exist on enzyme production from various sources and optimization of parameters like pH,
temperature, and carbon/nitrogen sources, a lack of focus on wastewater-derived amylase, especially in the
context of industrial-scale applications, presents a key area for further investigation. Therefore there is need in
translating findings from laboratory-scale amylase production to real-world industrial applications. This study
focused on demonstrating the characterization of wastewater-derived amylase and the type of microbe that
could tolerate, and produce amylase so a sto be used in specific industrial processes.
METHODOLOGY
Microbial and innoculum preparation
Lysinibacillus sphaericus C3-41 was isolated from dietary oil rich waste water as characterized and identified
molecularly. The isolate was and re-cultured at laboratory of the Department of Microbiology, Landmark
University, Omu-Aran, Kwara State, Nigeria for further analysis. Lysinibacillus sphaericus C3-41 cell free
supernatant was obtained by growing the organism in a sterile PBS buffer, pH 7.2 and centrifuging the broth
culture. One hundred (100 μl) of the sixth 10-fold serial dilutions were plated on De Man Rogosa and Sharpe
(MRS) agar, followed by picking some colonies at random and growing in MRS broth at 300C for 24hrs
(Sundarram and Krishna, 2020). The broth culture was then centrifuged at 10,000 rpm for 5mins. Cell pellets
harvested from the MRS broth cultures were re-suspended in MRS broth containing 15% glycerol and aliquots
were frozen for use when needed. Cell free supernatant was used as the crude enzyme for assay and further
analysis (Das et al., 2004).
Preparation of Wastewater and medium for Enzyme production
Wastewater treatment was done by distributing sterile wastewater into five portions of 100 mL each contained
in 250 mL Erlenmeyer flasks followed by inoculating 1 mL Lysinibacillus sphaericus C3-41 culture (1.86 x
106 CFU/ml) into the wastewater sample contained in each flask and the absorbance was measured at 600nm
(Gupta et al., 2004). The flasks were kept in shaking incubator with 150 r.p.m at 37℃. Samples were drawn
from each of the flasks at intervals of 6 h for a period of 24 h and later centrifuged at 5000 x g for 1 minute at
4℃. The medium for amylase production contains (g/L): soluble starch (10), peptone (20), MgSO4.7H2O (1.0),
Na2HPO4 (3), FeSO4 (0.3) and NaCl (0.1), which was sterilised then left to cool until 27 °C. One mL of 24 h
culture of Lysinibacillus sphaericus C3-41 (0.5 McFarland) was inoculated to 99 mL of cultivation medium
containing (soluble starch (10), (NH4)2SO4 (2), MgSO4.7H2O (1.0), Na2HPO4 (3), FeSO4 (0.3) and NaCl (0.1))
in a 250 mL volumetric flask and allowed to incubate for 48 h at 45 °C with agitation (150 rpm) (Msarah et al.,
2020). Culture samples (1mL) were removed at 6h intervals and centrifuged at 10,000g for 10min. Growth was
measured using turbidity of harvested samples at 600 nm. The supernatant obtained after centrifugation was
used as crude enzyme solution. The enzyme was usually stored at 4℃ until when needed.
Assay for Amylase Activity of Lysinibacillus sphaericus C3-41
In the assay for Amylase activity, 0.5 mL of the supernatant was added into a tube containing 1.5 mL of 2 %
(w/v) of potato starch solution and 1 mL of 0.05 M acetate buffer, pH 5.0. The reaction mixture was incubated
at 400C for 15 min in water bath. Then, 1 mL of the mixture was transferred to a new tube containing 1 mL of
3, 5-dinitrosalicylic acid (DNS) reagent and kept in boiled water for 10 min (Mawadza et al., 2000). The
colour density was determined spectrophotometrically at 540 nm. One international unit (IU) of amylase
activity was considered as the required amount of the enzyme to release 1 μmol of reducing sugar from the
sugar source within one minute under standard experimental conditions (Rao et al., 2011).
Purification of amylase enzyme
Purification of amylase enzyme was achieved by ammonium sulphate precipitation followed by dialysis. One
hundred milli litre (100 ml) of cell-free extract was saturated with centrifuged at 7000 rpm for 15 min. The
supernatant was collected and saturated up to 0–30 and 30–80% with ammonium sulphate. Then, the content
was centrifuged at 7000 rpm for 15 min and the pellet was collected for further analysis (Rao et al., 2011). The
enzyme mixture was transferred in a dialysis bag with space size 70 cm and immersed in phosphate buffer pH
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7 at 4°C for 24 hr. The buffer was continuously stirred using a magnetic stirrer throughout the process. The
buffer was changed several times during the process in order to obtain proper purification.
Optimization of conditions for Microbial growth and Amylase production
Various process parameters affecting enzyme production were optimized. Such different growth conditions
were optimized independent of each other. The parameters investigated included (i) incubation time (24h-
12days), (ii) incubation temperature (20-80℃), (iii) pH of medium (4-8), (iv) nitrogen source (ammonium
nitrate, ammonium nitrite, ammonium sulphate, ammonium chloride, ammonium carbonate and ammonium
phosphate; 1%w/v), (v) supplementary carbon source (glucose, fructose, galactose, sucrose, maltose, lactose;
1%w/v), and (vi) salt ions (Na, K, Mg, Ca, Fe PO4, SO4, Cl-). Inoculation of the organism into media without
supplement serves as control (Gupta et al., 2004).
Growth and amylase production of Lysinibacillus sphaericus C3-41at different incubation periods
Amylase production was carried out for 12 days and the samples were collected after every 24hours to amylase
production and microbial growth. Enzyme assay was carried out using standard assay procedure as described
by Mawadza et al. (2000).
Effect of temperature on growth and amylase production by Lysinibacillus sphaericusC3-41
The optimum temperature for amylase activity was determined by incubating the assay mixture described
above at different temperatures between 20oC and 80oC (at 10oC intervals) for 24hours while keeping other
parameters constant and the resulting enzyme activity using standard assay procedure was determined as
described by Mawadza et al. (2000).
Effect of pH on growth and amylase production by Lysinibacillus sphaericusC3-41
Enzyme production and the growth of Lysinibacillus sp were observed on the media with varying pH ranging
between pH 4 to pH 8, while keeping other parameters constant and the resulting enzyme activity using
standard assay procedure was determined as described by Mawadza et al. (2000).
Effect of various nitrogen sources on growth and amylase production by Lysinibacillus sphaericusC3-41
The influence of ammonium nitrate, ammonium nitrite, ammonium sulphate, ammonium chloride, ammonium
carbonate and ammonium phosphate (1%w/v) each was examined on the growth and amylase production of
Lysinibacillus sp. in the basal medium while keeping other parameters constant. Enzyme assay was carried out
using standard assay procedure (Francisco et al., 2004).
Effect of various carbon sources on growth and amylase production
Starch present as carbon source in the production medium was replaced with different carbon sources like
glucose, fructose, galactose, sucrose, maltose and lactose at 1% v/v. The control contains the media without
carbon supplement. Enzyme assay was carried out using standard assay procedure.
Effect of Metal and non-metals on the growth and amylase production
Effect of metals and non metals were examined on the growth of the organism. Metals such as sodium (Na),
potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), and non-metals such as phosphate (PO4), sulphate
(SO4) and chloride (Cl) each at (1mg/100 vol of water) were used. They resulting enzyme activity was
determined using spectrophotometry as described by Mawadza et al. (2000) using standard assay procedure
RESULTS AND DISCUSSION
Many amylase producing organisms were reported previously from the source of soil marine (Mohsen et al.,
2018). Cost of fermentation medium is one of the important factors in microbial enzyme production and
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utilization of several waste sources can play a vital role in the trimming down these costs. Microorganisms
isolated from local wastes, when combined with cheap substrates can be used to reduce amylase production
cost.
Growth and α-amylase production by Lysinibacillus sphaericus at different incubation periods
Findings from this study showed that optimal amylase production occurred on day 4 with a value of
3.44mM/min while the optimal growth was observed on day 5 of incubation for the organism, following which
a decline in growth and amylase production was observed (Figure 1). This results closely agrees with Vishnu
et al. (2014) who reported that Bacillus sphaericus had optimal amylase production after 72h of incubation.
This suggests that amylase production was growth dependent. This contrasts with findings by Simair et al.
(2017) who reported optimal amylase production at 36h during optimization. Qureshi et al. (2013) suggested
that further decline could be due to decrease in cell growth, a deficiency of nutrients, and a change in the final
pH.
Figure 1: Growth of Lysinibacillus sphaericus and amylase enzyme productionat different incubation periods.
Effect of temperature on α-amylase production by L. sphaericus
The effect of temperature on α-amylase production by L. sphaericus C3-41 in the sample revealed that enzyme
activity peaked at 30°C (Figure 2). The enzyme activity decreased beyond this temperature till 70°C and later
dropped sharply at 80°C. The organism grew in the medium at the rate of 0.02 mg/day while it produced the
amylase enzyme at the rate of 0.29 mM/min every day. This closely agrees with findings by Akzan et al.
(2011) who reported maximum amylase production from Bacillus licheniformis at 37 °C, further increase in
the temperature yielded reduced amylase activity, this could be due to the decreasing microbial growth and
denaturation of the enzyme at higher temperatures.
Figure 2: Effect of different temperatures on amylase enzyme production by Lysinibacillus sphaericus C3-41
Effect of pH on α-amylase production by L. sphaericus C3-41
Findings also showed that optimal amylase production occurred at pH 8. Beyond this optimal level, lower pH
also resulted in reduction in α-amylase production such that at pH 8, the enzyme produced was 2.89mM/min
while pH 7 it produced 2.11mM/min (Figure 3). This finding agrees with Simair et al. (2017) who reported
optimal amylase production by Thermophilic Bacillus sp. BCC 021-50at pH 8. The organism grew in the
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medium at the rate of 0.06 mg/day while it produces the amylase enzyme at the rate of 0.78mM/min every day.
Microbial stains possessing thermo-alkaliphilic features offer promises as industrially-important strains owing
to their commercial potential, especially alkaline pH-stable amylase, which could be used in detergent
formulations.
Figure 3: Effect of different pHs on amylase enzyme production by Lysinibacillus sphaericus
Growth and production of amylase by L. sphaericusC3-41 in different Nitrogen sources
Growth and production of amylase by L. sphaericus C3-41 in media supplemented with different nitrogen
sources showed that ammonium phosphate was a better nitrogen source for L. sphaericus C3-41(5.86mM/min)
and optimal growth of the organism with 0.281mg. On the other hand, ammonium carbonate was the least
favoured nitrogen sources that supported the growth and synthesis of α-amylase by L. sphaericus C3-41
(Figure 4).
Figure 4: Growth of Lysinibacillus sphaericus and amylase production with different nitrogen sources
Growth and production of amylase by L. sphaericusC3-41 in different carbon sources
Supplementation of carbon sources in the form of monosaccharides, disaccharides and polysaccharides
resulted in marginal increase in growth and α-amylase production by L. sphaericus C3-41 in domestic waste
water. The highest amylase production was observed in medium supplemented with glucose with optimal
growth of 0.326mg and amylase production of 6.89mM/min (Figure 5). Higher growth of L. sphaericus
resulted in increased amylase activity. This is in contrast with the work of Gurudeeban et al. (2011) and
Sivakumar et al. (2011) who reported maltose as best carbon source for amylase production from Bacillus sp.
Higher glucose production could be due to short incubation time and simple transport mechanism (Hassan and
Abd, 2019).. Haq (2003) stated that maltose synthesis may undergo lengthy period due to their characteristic as
disaccharide and various mechanisms of maltose transport system which consist of cytoplasmic membrane
proteins, a perisplasmic binding protein and a specific outer-membrane porin.
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Figure 5: Growth of Lysinibacillus sphaericus and amylase production with different carbon sources.
Effect of Metals and non-metals on the growth of Lysinibacillus sp C3-41 and amylase production
Among the different metals and non-metals, the assay mixture supplemented with sodium yielded optimal
amylase production. Amylase secretion by the organism also corresponded significantly to the growth of the
organism. The maximum amylase produced was 6.55mM/min at a growth of 0.292 mg/L. Reduced growth of
Lysinibacillus sphaericus and amylase production was observed in the medium supplemented with iron. Also,
the sulphate supplemented medium gave the maximum secretion of the enzyme among the non-metallic ions
used (Figure 6). This finding differs from reports by Sudha et al. (2012) who reported higher amylase
production by Bacillus amyloliquefaciens Ca2+ (0.439) IU/ml/min at 7g/l concentration in comparison to other
metal ions. This result shows that metal ions may stimulate the enzyme activity by acting as a binding link
between enzyme and substrate combining with both and so holding the substrate and the active site of the
enzyme.
Figure 6: Effect of various metals and non metals on Amylase enzyme production by L. sphaericus C3-41
CONCLUSIONS
This work have revealed that the use of liquid state fermentation for production of α-amylase by Lysinibacillus
sphaericus C3-41using wastewater as a substrate is an economical process and is very simple to apply.
Optimization of the fermentation parameters and the use of suitable carbon and nitrogen supplements resulted
in 3 folds increase in the enzyme yield. The enzyme was significantly active at room temperatures and the
optimum temperature for the activity was found to be 30°C. The enzyme was found to be active over a wide
range of pH and showed the optimum activity at pH 8. These isolates can thus be industrially exploited for the
synthesis of α- amylase which can have several industrial applications.
REFERENCES
1. Baysal Z, Uyar F, Aytekin C (2003). Solid state fermentation for Production of α- amylase by a
thermotolerantB. subtilisfrom hot-spring water. Process Biochem. 38:1665-1668.
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue VIII August 2025
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2. BhaskaraRao KV, Ashwini K, Gaurav K, Karthik L (2011). Optimization, production and partial
purification of extracellular α-amylase from Bacillus sp. marini. Arch. Appl. Science Res. 3(1):33-42.
3. Das K, Doley R, Mukherjee AK (2004). Purification and biochemical characterization of thermostable,
alkaliphilic, extracellular α-amylase from B. subtilisDM-03, a strain isolated from the traditional food
of India. Biotechnol. Appl. Biochem. 40:291-298.
4. Francis F, Sabu A, Nampoothiri KM, Ramachandran S, Ghosh S, Szakacs G, Pandey A (2003). Use of
response surface methodology for optimizing process parameters for the production of α-amylase by
Aspergillusoryzae.Biochem. Eng. J. 15:107-115.
5. Franscisco, J., Ustariz, L.A., Luis, A.G., and Diaz, M. (2004).Fermentation of individual proteins for
protease production by Serratiamarcescens.BiochemEngineering Journal, 19: 147-153.
6. Gangadharan D, Sivaramakrishnan S, Nampoothiri KM, Sukumaran RK, Pandey A (2008). Response
surface methodology for the optimization of alpha amylase production by Bacillus
amyloliquefaciens.Bioresour. Technol. 99: 4597-4602.
7. Gangadharan, D., Sivaramakrishnan, S., Nampoothiri, K.M, and Pandey, A. (2008).Solid culturing of
Bacillus amyloliquifaciensfor amylase production.Food Technology Biotechnology, 44:269-274.
8. Gupta, R., Gupta, N. and Rathi, P. (2004). Bacterial lipases: an overviewof production, purification and
biotechnological properties. Application MicrobiolBiotechnol, 64: 763-781.
9. Gurudeeban, S., Satyavaniand, K. and Ramanathan, T. (2011). Production of extra cellular-amylase
using Bacillus megateriumisolated from White Mangrove (Avicennia marina). Asian Journal
Biotechnology, 3:310-316.
10. Haq I, Ashraf H, Iqbal J, Qadeer MA (2003). Production of alpha amylase by Bacillus
licheniformisusing an economical of medium.Biores. Technol. 87: 57-61.
11. Hassan H, and Abd K K.(2019). Optimization of alpha amylase production from rice straw using solid-
state fermentation of Bacillus subtilis. Int. J. Sc. Environ. Technol: 4 (1), 1-16.
12. Hasan, F., Shah, A. A. and Hameed, A. (2006).Industrial applications of microbiallipases.Enzyme and
Microbial Technology, 39: 235-251.
13. Mawadza, C., Hatti-Kaul, R., Zvauya, R. and Mattiasson, B. (2000).Purification and characterization of
cellulases produced by two Bacillus strains.Journal of Biotechnology, 83(3): 177-187.
14. Mohsen, H., Ali m., and Uygut,. M. (2018). Optimization of alpha-amylase production by Bacillus
amyloliquefaciens grown on orange peels. Iranian J. Sci. Technol. Transactions A: Science
15. Panneerselvam T, and Elavarasi S.(2015). Isolation of α-amylase producing Bacillus subtilis from Soil.
Int. J. Curr. Microbiol. Appl. Sci: 4 (2), 543-552.
16. Rao JLUM, Sathyanarayana T (2003). Enhanced secertion and low temperature stabilization of a
hyperthermostable and Ca2+ dependent α-amylase of Geobacillusthermoleovoransby surfactants.Lett.
Appl. Microbiol. 36:191-196.
17. Sivakumar, T., Ramasubramanian, V., Shankar, T., Vijayabaskar, P. and Anandapandian, K.T.K.
(2011).Screening of keratinolytic bacteria Bacillus cereus from the feather dumping soil of
sivakasi.Journal Basic & Applied Biology, 5: 305-314.
18. Suhaimi SN, Phang LY, Maeda T, Abd-Aziz S, Wakisaka M, Shirai Y, Hassan MA (2012).
Bioconversion of glycerol for bioethanol production using isolated Escherichia coli SS1. Braz. J.
Microbiol. 506-516.
19. Sundarram K, and Krishna T P. (2020). α-Amylase Production and Applications: A Review. J. Appl.
Environ. Microbiol.: 2 (4), 166-175.
20. Tanyildizi, M.S., Ozer, D. and Elibol, M. (2005).Optimization of amylase production by Bacillus sp.
using response surface methodology. Process Biochemistry, 40, 2291-2296.
21. Underkofler, L.A., Barton, R.R. and Rennert, S.S. (2007). Production of Microbial Enzymes and Their
Applications.Applied Microbiology, 6(3): 212-221.
22. Vishnu, T.S., Soniyamby, A.R., Praveesh, B.V and Hema, T.A. (2014). Production and optimization of
extracellular amylase from soil receiving kitchen waste isolate Bacillus sp. VS 04. World Applied
Scientific Journal, 29(7):961-967.
23. Windish, W.W. and Mhatre, N.S. (2005).Microbial amylases. In: Wu, W., Ed., Advances in Applied
Microbiology, Elsevier Inc., Houston, 273-304.