UJMR, Volume 4 Number 2, December, 2019, pp 18 - 25 ISSN: 2616 – 0668

https://doi.org/10.47430/ujmr.1942.004

Received: 25/04/19 Accepted: 19/06/19

Optimization of Culture Condition in the Production of Bioenzymes by Bacteria Isolated
from Poultry Waste in Sokoto State, Nigeria
1
Hauwa, H., 2S.B. Oyeleke and 3Adamu, Shahidah Ahmed
1
Faculty of Science, Department of Microbiology. Faculty of Science. Sokoto State University,
Sokoto.
2
Faculty of Science, Department of Microbiology. Federal University of Science And
Technology Minna , Niger State.
3
Department of Microbiology. Faculty of Science.Sokoto State University, Sokoto
(08069303535) h_hauwau@yahoo.com

Abstract
Poultry wastes obtained from a poultry farm in Sokoto metropolis were analyzed for
cellulose producing bacteria. Bacillus megaterium, Bacillus laterosporus, and Bacillus
amyloliquifeciens isolated were screened for their ability to produce cellulase enzyme. All
the isolates showed cellulose activity by exhibiting a wide halo on caboxymethylcellulase
medium (CMC).The fermentation process was optimized using the following parameters,
inoculum size, pH, Substrate concentration, temperature, and incubation periods. Cellulase
activity was determined using DNS method, Banana peels was used as a substrate for the
production of the enzymes, this was analysed with atomic absorbtion spectrophotometer
(AAS).Cellulase enzyme was produced at innoculum size 1, 2, 3, 4, and 5%. pH 3, 5, 7, 9, and
11. Substrate concentration 1g, 2g, 3g, 4g, and 5g. Temperature 35, 45, 55, 65, and 75, for
1, 2, 3, 3, and 5, days respectively. Bacillus laterosporus recorded the highest cellulase
activity 0f 0.37mg/ml in 5% substrate concentration among all the isolates while Bacillus
amyloliquefeciens recorded highest cellulose production at pH3 with 45mg/ml Bacillus
laterosporus recorded highest activity of cellulose production with 0.71mg/ml Temperature
was also studied in the cellulose production and Bacillus laterosporus showed highest activity
at 75⁰C with activity of 0.66mg/ml. This study showed that Bacillus laterosporus was the
best cellulase producing bacteria among all the isolates.
Key words: poultry wastes, cellulase, Banana peels, Bacteria, Enzymes.
INTRODUCTION agricultural, urban, or industrial in origin, sewage
Cellulose is the principal constituent of the cell sludge might also be considered a source of
wall of most terrestrial plants. The source of cellulose since its cellulosic content provides the
cellulose is in plants and it is found as micro- carbon needed for methane production in the
fibrils (2-20nm in diameter and 100 – 40,000nm anaerobic digestion of sludge. Agricultural wastes
long). These form the structurally strong frame include crop residue, animal excreta and crop
work in the cell walls. Despite a worldwide and processing wastes slashing generated in logging,
enormous utilization of natural cellulosic sources, saw dust formed in timber production and wood
there are still abundant quantities of cellulose- products in forestry originated activities
containing raw materials and waste products that (Pranner, 1979).
are not exploited or which could be used more The previous negative attitude in which wastes
efficiently. Various biomass inducing residues were viewed self consciously as valueless and
including lignocellulosic material, paper waste, even offensive and for disposal only has been
pulses, cereals straw and bagasses have been replaced in large part by a positive view in which
widely used as carbon sources for commercial wastes are recognized as raw materials of
cellulose fermentation Brijwani, and Vadlani, potential value (Pranner, 1979). Currently, there
(2011), Wen, et al., (2005) Belghith, et al., are two major ways of converting cellulose to
(2001). glucose: chemical versus enzymatic. Enzymatic
The problem in this respect is however to hydrolysis of cellulose is an important reaction in
develop processes that are economically nature for it marks the first step in the decay of
profitable. Cellulose-containing wastes may be

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 UJMR, Volume 4 Number 2, December, 2019, pp 18 - 25 ISSN: 2616 – 0668

cellulose, the most abundantly occurring organic material.

In the early 1970s, the oil crisis generated on the influence of various fermentation
interest in using cellulose as a chemical and variables on cellulase production by different
energy resource. One promising approach was to bacteria and fungi isolated from various natural
hydrolyze the cellulose to glucose with fungal environments (Garcia-Martinez et al., 1980;
enzymes and then to ferment the glucose to Stewart and Parry, 1981; Coral et al., 2002;
ethanol which could be used as a liquid fuel Rajoka, 2004; Immanuel et al., 2006),
(Mandels et al., 1974). information on the optimum fermentation
conditions for cellulase production by fish gut
Sources of Cellulase bacteria is lacking.
Cellulases are easily obtained from microbial and
fungal sources, but vertebrates lack the ability to MATERIALS AND METHODS
produce endogenous cellulases, and hence are The cellulolytic activity of the isolated organism
reliant upon gastrointestinal microorganisms for was determined as described by Ghose, (1987).
cell wall degradation of ingested plants. The Loop full of each isolate was streaked aseptically
beneficial effects of microorganisms in the on carboxymethyl cellulose medium. The plate
digestive processes of terrestrial animals are well was then incubated at 37 oC for 48 hours for
established (Combe et al., 1976; McBee, 1977; bacterial isolates. After incubation period, the
Goldin, 1986; Moriarty, 1990). Some culture was flooded with 0.1% Congo red dye
investigations have also suggested that solution and later 40% Nacl solution and allowed
microorganisms have a beneficial effect in fish to stand for 15-20 minutes. A zone of clearance
digestive processes e.g. microbial breakdown of formed around the bacterial colonies. The zone
chitin (Minami et al., 1972; Goodric and Morita, of clearance was measured using a meter rule.
1977; Danulat and Kausch, 1984; Kono et al., This represents the cellulolytic activity of the
1987), p-nitrophenyl-β-N-acetylgalactosamine bacterial isolates.
and collagen (MacDonald et al., 1986), cellulose Medium composition for cellulase production
(Stickney and Shumway, 1974; Trust et al., 1979; The fermentation media used was Mary Mandels
Saha and Ray, 1998; Bairagi et al., 2002 a, b, mineral salts solution and it was used along with
2004; Saha et al., 2006), and the vitamin B12 different carbon and nitrogen sources. The
producing ability of the bacteria (Sugita et al., medium (M1) contained the following (per L)
1991). However, the specific cellulolytic activity Banana peels, 20g ; Peptone,1g ; (NH 4)2SO4, 1.4g
shown by the bacterial species is found to depend ; KH2PO4, 2g ; CaCl2, 0.3g ; MgSO4.7H20, 0.3g ;
on the source (Saxena et al., 1993). Urea, 0.3g ; trace metal solution (2.5g FeSO 4;
Microbial enzymes have the enormous advantage 0.98g MnSO4.H2O ; 1.76g ZnSO 4.H2O; 1.83g COCl
of being able to be produced in large quantities l2.6H2O dissolved in 1000ml of distilled water and
by established fermentation techniques. Enzyme heated to homogenized ; pH 6.8 (Jeffries, 1996).
production is closely controlled in micro Cellulase assay
organisms and therefore, to improve its Liquid media was used for the quantitative assay
productivity these controls can be exploited and of cellulase production from the three bacterial
modified. Cellulase yields appear to depend on a strains. Cellulase activity was measured
complex relationship involving a variety of according to the method of Denison and Koehn
factors like inoculum size (carbon source and (1977). The production of reducing sugar
cellulose quality), pH value, temperature, (glucose) from CMC substrate through cellulolytic
presence of inducers, medium additives, activity was measured at 540 nm by the
aeration, growth time, etc. (Immanuel et al., dinitrosalicyclic acid method using glucose as the
2006). Therefore, attention has been focused on standard.
studying the cellulolytic activity and cellulase One cellulase unit (U) was defined as the
enzyme production by several micro organisms in amount of enzyme per milliliter culture filtrate
various products as well as in various that released 1 microgram glucose per minute.
environments. To establish a successful Optimization of Cellulase Production Process
fermentation process it is necessary to make the Effect of optimum pH on cellulase production
environmental and nutritional conditions by Bacteria isolates
favorable for the micro organism for over- The reaction mixtures of 120 hrs containing
production of the desired metabolite. enzymes from the different bacteria isolates
Thus an elaborate investigation is therefore, were prepared; the optimum pH for enzyme
required to establish the optimum conditions to cellulase activity was examined by running the
scale up enzyme production in an individual assay activity between pH ranges of 3.0, 5.0, 7.0,
fermentation process. Although there are reports 9.0, and 11.
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 UJMR, Volume 4 Number 2, December, 2019, pp 18 - 25 ISSN: 2616 – 0668

The enzyme activity for each pH was determined of 0.10 mg/ml. Bacillus megaterium had an
by adding DNSA reagents and the absorbance activity of 0.40 mg/ml at pH of 5 this was
read at 540 nm. followed by a gradual decrease as the pH
Effect of optimum temperature on cellulase increases. Bacillus amyloliquifeciens recorded
production by Bacteria isolates maximum activity of 0.45 mg/ml at pH of 3.There
The optimal temperature for activity was was a slight decrease as the pH approaches 11
determined by assaying the activity of the with cellulase activity of 0.19 mg/ml. This shows
enzyme at different temperature ranges of 35 0C, that Bacillus amyloliquifeciens recorded the
45 0C, 55 oC, 65 oC, and 75 0C. highest cellulase activity.
Effect of incubation period on cellulase The effect of innoculum size on the activity of
production by Bacteria isolates cellulase produced by bacterial isolates. It can be
The incubation was carried out for different seen in figure 3 that Bacillus laterosporus
periods in this study including days 1, 2, 3, 4, and exhibited high cellulase activity of 0.94 mg/ml
5 for the bacteria isolates after which an assay and 0.65 mg/ml in 5% and 4% respectively,
was determined by Dinitrosalicyclic method minimum cellulase activity was recorded at 3%
(Betrand et al, 2004). with 0.46 mg/ml. Bacillus megaterium and
Effect of innoculum size on cellulase Bacillus amyloliquifeciens also recorded
production by Bacteria isolates maximum activity 1n 3%. Bacillus megaterium
This activity was determined by assaying the had cellulase activity of 0.85 mg/ml while
enzymes at different sizes of 1%, 2%, 3%, 4% and Bacillus amyloliquifeciens had 0.89 mg/ml from
5% and the enzyme solution was maintained with the result Bacillus laterosporus recorded the
different sizes of the innoculum. highest cellulase activity while Bacillus
Effect of substrate concentration on cellulase megateruim recorded the least activity. lower
production by Bacteria isolates innoculum size require longer time for the cells
The effect of substrate concentration was to multiply to sufficient number to utilize the
determined by assaying the activity of the substrate and produce enzyme, an increase in the
enzymes of each bacteria with different number of cells in the innoculums would ensure a
substrate concentration of 1, 2, 3, 4and 5%. DNSA rapid proliferation and biomass synthesis. When
reagent was added and the absorbance was taken innoculum size was increased from 1-5% there
on the spectrophotometer at 540nm as described was increases in enzyme production but after
by Betrand et a.,l (2004). that the activity was decreased (Fig 3) due to
depletion of nutrients by the enhanced biomass,
RESULTS which resulted dwindle in metabolic activity
The effect of substrate concentration on (Kashyap et al., 2002). A balance between the
cellulase activity produced by bacterial isolates is increasing biomass and accessible nutrient would
shown in Fig1. Bacillus laterosporus had yield an optimal enzyme production
maximum activity recorded in 2% substrate (Ramachandran et al., 2004).
concentration with cellulase activity of 0.35 The effect of incubation period on the activity of
mg/ml and the least activity recorded in 5% cellulase producing isolates. From figure 4 it can
substrate concentration with activity of 0.15 be seen that Bacillus laterosporus exhibited high
mg/ml Bacillus megaterium recorded maximum cellulase activity of 0.71 mg/ml after 120 hrs
cellulase activity in 2% substrate conc with (day 5) These agrees with the report of Gautam
activity of 0.37 mg/ml, which was then followed et al ., (2011) who reported optimum yield on
by a decrease in activity as the substrate the 5th day but contrary to his other report of
concentration increases. Bacillus highest cellulase activity by A.niger and
amyloliquifeciens recorded its least activity in Trichoderma sp on the 4th and 6th day which was
5% substrate concentration with activity of 0.14 suitable for commercial point of view Kang et al.,
mg/ml and the highest recorded at 1% substrate (2004) , and it might be due to the depletion of
concentration with activity of 0.36 mg/ml. nutrients in the medium which stressed the
The effect of pH on the activities of cellulase fungal physiology resulting in the inactivation on
enzyme produced by bacterial isolates is shown in secretary machinery of the enzmes (Nochur et
figure 2. From the result the highest activity al., 1993), the least activity recorded was at 48
produced by Bacillus laterosporus was at pH of 5 hrs (day 2) with 0.4 mg/ml .Bacillus megaterium
with activity of 0.36 mg/ml, these agrees with recorded its highest activity after 48 hrs (day 2)
the report of Lynd et al., 2002 and Gautam (0.56 mg/ml) this was followed by a decline in
(2011) Who reported maximum yield at pH 5 and cellulase activity at 72 hrs (day 3) and 120 hrs
6 by A. niger and Trichoderma sp while the least (day 5).
recorded at pH value of 9 with cellulase activity
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 UJMR, Volume 4 Number 2, December, 2019, pp 18 - 25 ISSN: 2616 – 0668

The least activity recorded was at 96 hrs (day 4) exhibited its high cellular activity at 55 oC with
(0.21 mg/ml) Bacillus amyloliquifeciens had its 0.58 mg/ml but the activities fluctuated by
highest activity recorded at 24 hrs (day 1) this increasing and decreasing as temperature rises
was followed by a slight decrease as the hours and fall the lowest recorded for Bacillus
increases, to120 hrs (day 5) the activity drop to megaterium was at 35oC with cellulase activity
0.18 mg/ml which was the least recorded. of 0.36 mg/ml . Bacillus amyloliquifeciens had its
The effect of temperature on cellulase highest activity at 35oC with 0.59 mg/ml, it was
production by the bacterial isolates is shown in then followed by a slight decrease as the
figure 5 from the figure Bacillus laterosporus tempreture increases with the lowest activity at
had cellulase activity of 0.04 mg/ml at 35 oC, this 75oC (0.30 mg/ml ). From the result Bacillus
was followed by a slight increase at 45 oC (0.55 laterosporus recorded the highest cellulase
mg/ml ) these disagrees with the report of Ray et activity. These agrees with the findings of Bakare
al (2007) who reported that minimum cellulose et al., (2005) who found that cellulose enzyme
yield was observed when fermentation was producded by pseudomonas fluorescence was
carried out at 45 oC by B. subtilis and B.circulans activated at 30-35 oC, also disagrees with the
it was then followed by a sharp decrease at 55 oC report of immanuel et al., 2006 who reported
and 65oC 0.23 mg/ml and 0.13 mg/ml but as the maximum endogluconase activity in cellulomonas
temperature increases to 75oC the activity also Bacillus and Micrococcus sp at 40 oC .
increases to 0.66 mg/ml. Bacillus megaterium

Figure 1; Effect of substrate concentration on cellulase activity produced by B. laterosporus, B.

megaterium, B. amyloliquifeciens

Figure2: Effect of pH on cellulase activity produced by B. laterosporus, B.megaterium, B.

amyloliquifeciens
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 UJMR, Volume 4 Number 2, December, 2019, pp 18 - 25 ISSN: 2616 – 0668

Figure 3: Effect of innoculum size on cellulase activity produced by B. laterosporus, B.megaterium, B.

amyloliquifeciens

Figure 4: Effect of incubation periods on cellulase activity produced by B. laterosporus, B.megaterium,

B. amyloliquifeciens

Figure 5: Effect of Temperature on cellulase activity produced by B. laterosporus, B. megaterium, B.

amyloliquifeciens
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 UJMR, Volume 4 Number 2, December, 2019, pp 18 - 25 ISSN: 2616 – 0668

DISCUSSION mg/ml at 55oC. Hence, further increase in

Three species of Bacillus were characterized and temperature lead to a decrease in enzyme
identified on the basis of macroscopy and activity of 0.32 mg/ml at 75oC .This is in line with
microscopy. The macroscopic and microscopic the report of Kelly et al (1997) that during
characterization revealed the presence of isomerization, temperature is preferably
Bacillus laterosporus, Bacillus megaterium, and maintained within the range of 20-90oC and the
Bacillus amyloliquifeciens. All the bacterial best activity is obtained at 50-750C.
isolates were hydrolizers of carboxymethyl Further increase in cellulose concentration
cellulose meduium, based on cellulolytic activity beyond the level that gave optimum cellulase
of the isolates following their zones of clearing enzyme production will result in proportionate
on CMC medium. Temperature and pH are the decrease in yield. This is at variance with
most important factor that influence enzyme Haapela et al., (1995) and Jeffries, (1996) who
activity .Bacillus laterosporus had an enzyme reported that maximum enzyme activity was
activity of 0.44 mg/ml at 35 oC this was followed recorded with the medium concentration of
by an increase in enzyme activity as the cellulose at 10g/l. Lynd et al., 2002), reported
temperature increase, with maximum activity of that the production of cellulase for the
0.56 mg/ml. cellulase enzymes when hydrolyzed utilization of cellulose is induced only in the
by cellulolytic microorganisms instead of being presence of specific substrate but suppressed
left alone for natural degradation, these enzymes when easily utilizable sugars such as glucose are
can be utilized effectively under optimum available.
conditions, to produce cellulase as realized as
the temperature increased beyond 45oC the CONCLUSION
enzyme activity decreased ,The minimum This study revealed that poultry wastes and
enzyme activity for Bacillus laterosporus was Banana peel, are of domestic and industrial agro-
recorded at 75oC with enzyme activity of 0.17 wastes, that can be use to produce large amounts
mg/ml while Bacillus magaterium had an of cellulase enzymes when hydrolyzed by
enzyme activity of 0.56 mg/ml at 35 oC and the cellulolytic microorganisms instead of being left
temperature increased to 45oC enzyme activity alone for natural degradation, these enzymes can
also increases to 0.66 mg/ml this was followed be utilized effectively under optimum conditions,
by a sharp increase in enzyme activity of 0.82 to produce cellulose.
Methods on Physiochemical
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