Optimization of Production and Extraction Parameters of Bacillus megaterium

Journal of Applied Sciences Research, 4(10): 1199-1204, 2008
© 2008, INSInet Publication
Optimization of Production and Extraction Parameters of
Bacillus megaterium Levansucrase Using Solid-state Fermentation
Samia A. Ahmed
Department of Chemistry of Natural and Microbial
Products National Research Center, Cairo, Dokki, Egypt.
Abstract: The use of waste as a raw material is important for government and natural balance.
The purpose of this work was to study the production of levansucrase by Bacillus megaterium in solid
state fermentation (SSF) using different agricultural residues (orange peels, lemon waste, sawdust, banana
waste and wheat bran) as substrates. Maximum enzyme production (140.54 U/g dry solid substrate) was
obtained on moistened 5 g sawdust with 55 ml production media (initial pH 6.0) after 72 h incubation
at 30 î C under static condition. The extraction of enzyme was found to depend on different parameters like
nature of extractant, soaking time, temperature, etc. Among various organic, inorganic solvents and
salt solutions tested, maximum extraction was achieved when 10 ml/ g biomass distilled water was
adding. The optimum conditions of extraction were 90 min soaking time at 40 î C under agitated condition
(160 rpm).
Key words: Bacillus megaterium, levansucrase, sawdust, solid state fermentation
INTRODUCTION
Bacterial levansucrase (EC2.4.1.10) is an
extracellular protein found in gram-negative and grampositive bacteria. This protein catalyzes the synthesis
of levan (B-2, 6-linked fructan) from sucrose [1 ,2 ].
Levan has potential application in the fields of food as
low-caloric sweetener, cosmetic, pharmaceuticals, as
antitumor agent based on its physical properties [3 ] and
hypocholesterolemic agent[4 ] and increase bifidobacteria
population in the intestinal track [5 ]. In the last decades,
there are an increasing number of reports about
utilization of solid state fermentation (SSF) for
production of value added products such as secondary
metabolites, organic acid, fuel and enzymes, etc.[6 ].
SSF can be defined as the growth of microorganism on
wet solid materials in the absence or near absence of
free water and secrete the necessary enzyme for
degradation of the available substrate molecules in
order to meet their nutritional requirement[7 ]. SSF has
gained renewed attention from industry because it
becomes a more alternative to submerged fermentation
with simpler cultivation equipments, lower capital
in v e stm e n t, h ig h e r p ro d u ctiv ity, lo w e ne rgy
requirement, less water output, better product recovery,
lack of foam build up and reduced bacterial
contamination [6 ,8 ]. SSF appears as an interesting lowcost alternative for the production of biomoleculs
because agro-industrial residues can be employed as
culture media which reduce production costs [9 ]. [8 ]used
bran for alpha-amylase production, [ 2 ] produced
fructosyltransferase using agricultural by-products.[1 0 ]
using wheat bran for producing milk-clotting enzyme,
[1 1 ]
used melon wastes for lipase production and [1 2 ] used
orange bagasse for pectinase production. On the other
hand, the application of using agro-industrial waste for
SSF solves the pollution problems. Productivity was
affected by the nature of solid substrate (SS), SS
concentration, level of moisture content, pH of
medium, incubation temperature and incubation period.
In SSF the products are formed at or near the surfaces
of the SS with low moisture content, so it is necessary
to select suitable solvents or solution for leaching out
the product from the bulky solid mass [8 ]. A common
extractant is distilled or deionized water, other
extractants have also been used for extract other
enzymes such as using sodium chloride solution for
protease extraction [1 3 ]. [1 4 ]extracted pectinases from
wheat bran by acetate buffer (pH 4.4). This paper
reports the optimization of fermentation parameters for
levansucrase production by Bacillus megaterium
through SSF. Studies on the extraction of levansucrase
from sawdust include the effect of some factors which
influence the efficiency of leaching out of the enzyme
and its efficacy in the leaching technique.
M ATERIALS AND M ETHODS
M icroorganism: T he bacterial strain Ba cillus
megaterium was obtained from the Culture Collection
Corresponding Author: Samia A. Ahmed, Department of Chemistry of Natural and Microbial Products National Research
Center, Cairo, Dokki, Egypt.
Telephone: 002 02 6856074 Fax : +33 4 91 82 85 70 E-mail: [email protected]
1199
J. Appl. Sci. Res., 4(10): 1199-1204, 2008
of the National Research Centre, Dokki, Cairo, Egypt.
M aintenance Culture M edium: B. megaterium was
maintained on nutrient agar medium, pH 7.0 at 4 î C and
was transferred to new slant every week. The following
medium was used for production (g/l distilled water):
sucrose 80.0; MgSO 4 .7H 2 O 0.3; (NH4) 2 SO 4 2.0; yeast
extract 2.0; peptone 2.0 and K 2 HPO 4 2.0. The pH was
adjusted to 7.0 before autoclaving.
Production Process: To optimize the production
process different experiments were carried out. Five
grams of different solid substrates (orange peels, lemon
waste, banana waste and wheat bran) and 3.0 gm in
case of sawdust moistened with 25 ml of production
media (pH 7.0) in 250 ml Erlenmeyer flask and
incubated at 30 î C for 1 h and 160 rpm. Then the
flasks autoclaved for 15 min at 121 î C and each flask
inoculated with 2 ml inoculum from 24 h old
culture and incubated at 30 î C for 48 h at static
condition. The Effect of solid substrate concentration
on the enzyme production was measured at different
concentrations of sawdust (1, 2, 3, 4, 5, 6 and 7 g).
The effect of culture conditions in the present study
was carried out at different incubation periods (12, 24,
48, 72 and 96 h), effect of ratio between sawdust and
water (1:2, 1:5, 1:8, 1:11 and1:14 w/v) and initial pH
(5.0, 6.0, 7.0, 8.0 and 9.0).
Extraction Process: The extraction was conducted in
50 ml conical flask containing one gram of fermented
biomass and 5 ml of solvent solution and kept for 1 h
on a rotary shaker at 160 rpm and 30 î C. The crude
extract was centrifuged at 10000xg for 20 min at 4 î C
and the clear supernatant obtained was used in the
enzyme assays. To optimize the extraction process
different experiments were carried out.
Effect of Different Solvents: The extraction of the
enzyme from the fermented biomass was carried out
with distilled water, tap water, and inorganic salt
solutions (potassium chloride, magnesium chloride,
calcium chloride, and sodium chloride) at different
concentrations (0.025, 0.05, 0.075, 0.1 and 0.125 %)
and organic solvents (glycerol, methanol, ethanol and
acetone) at concentration of 5 %.
Effect of Distilled W ater Volume: The investigation
was carried out to see the effect of solvent level (1, 5,
10, 15, 20 and 25 ml/g fermented biomass).
Effect of Soaking Temperature: To study the effect
of temperature on the extraction process the
temperature was varied from 20 î C to 60 î C each at
10 î C intervals.
Effect of Physical State: Two different extraction
conditions were studied stationary and agitation
(160 rpm).
Effect of Soaking Time: Extraction time was tested
(30, 60, 90, 120, 150 and 180 min) keeping all other
conditions at optimum levels.
Enzyme Assay: Levansucrase assay was performed
according to the method of [7 ]. 0.5ml culture filtrate was
incubated with 1 ml sucrose solution (20 %) and 0.5
ml acetate buffer (0.1 M , pH 5.2) and incubated at
30 î C for 15 min. The concentration of glucose released
by the sucrose hydrolysis activity of levansucrase was
measured by the glucose oxidase method with a GODPAP Kit (Sigma). One unit of enzyme activity was
defined as the amount of enzyme that produced 1µmol
glucose per min. All values given are averages of three
determinations.
Paper Chromatography: The polysaccharide produced
(levan) was precipitated with 2 volumes ethanol and
indicated after acid hydrolysis (with 0.1 N HCl in a
boiling water bath for 1 h) by paper chromatography.
The descending technique was adopted using W hatman
No.1 paper and the solvent mixture n-butanol: acetone:
water (4: 5: 1). The chromatograms were sprayed with
aniline phthalate.
RESULTS AND DISCUSSION
Due to potential usefulness of levan (produced by
levansucrase enzyme) the development of methods for
cheaper production of enzyme is very important. One
alternative low cost production method is SSF [5 ].
There are several factors, which affect SSF processes
and the nature of SS is the most important factor.
This not only supplies the nutrient to the culture but
also serves as an anchorage for the microbial cells[1 5 ].
The selection of a substrate for SSF depends upon
several factors mainly related with cost, availability,
consistency, stability, ease of handling and of course
the effect on the productivity process. An ideal SS
provides all necessary nutrients to the microorganism.
However, some of the nutrients may be not present in
the SS, so it would be necessary to supplement them
externally [5 ]. Several substrates including agricultural
crop residues or industrial waste have been used by
various workers for SSF. Sawdust is a cheap SS and
very little workers used it in SSF. From the various SS
used in the present study, sawdust proved to be most
suitable for the colonization of B. megaterium, as
indicated by the maximum visible growth on the
surface of substrate and highest enzyme yield (Fig.1)
which is possibly due to its most suitable particle size
and consistency required for anchorage. However, it is
interesting to know that sawdust is a cheap SS and
very little workers used it. [2 ] studied the production of
levansucrase by A. oryzae employing a great variety of
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J. Appl. Sci. Res., 4(10): 1199-1204, 2008
Fig. 4: Effect of moisture content on levansucrase
production.
Fig. 1: Effect of different solid
levansucrase production.
Fig. 2: Effect of sawdust
production.
level
substrate on
on levansucrase
Fig. 3: Effect of incubation period on levansucrase
production.
agricultural by-products. They found that, the best
results were obtained when rice bran, wheat bran, spent
coffee and spent tea used supplemented with yeast
extract and complete synthetic media.
The effect of sawdust concentration on enzyme
production is shown in figure 2. It was observed that
5 g sawdust in the fermentation medium yielded
maximum enzyme activity (71.62 U/g dry solid
substrate). A further increase in substrate did not
increase the enzyme yield significantly because 2 ml
inoculume was added to each flask and increase in
sawdust level only could not effect the growth of
organism [1 6 ].
Fermentation time had a profound effect on
enzyme production. Figure (3) showed that the
maximum activity (88.57 U/g dry SS) obtained after 72
h of SSF at pH 7.0 and 30 î C. Enzyme level declined
with prolonged incubation, this could be due to loss of
moisture or denaturation of the enzyme resulting from
variation in pH during fermentation. This result is on
line with [1 7 ] when obtained the highest level of
levansucrase after 72 h. On the other hand, [2 ] found
that maximum production of levansucrase by A. oryzae
in SSF was 8 h.
Moisture content of the substrate is one of the
critical factors influencing the outcome of SSF, and is
governed by the water-holding-capacity of the substrate,
the type of end-product, and the requirement of the
microorganism. The inter particle mass transfer within
the solid phase to the growing microorganism depends
on the substrate characteristics and the moisture
content. The results (Fig. 4) pointed to a marked
improvement (97.91 U/g dry SS) was achieved by
optimizing moisture content. Lower moisture content
cause a reduction in solubility of nutrients provided to
organism by SS, a lower degree of swelling and higher
water tension [5 ]. On the other side, reduction in enzyme
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J. Appl. Sci. Res., 4(10): 1199-1204, 2008
Fig. 5: Effect of solvents type on levansucrase extraction from SSF.
Fig. 6: Effect of inorganic salt solutions on
levansucrase extraction from SSF.
Fig. 7: Effect of distilled
water
volum e on
levansucrase extraction from SSF.
production at high moisture content may be due to the
reduction in substrate porosity, changes in the structure
of substrate particles, reduction of gas volume and
decreasing in bacterial growth [1 8 ] .
pH is among most important factors for any
fermentation process and depended upon microorganism
because each microorganism possesses a pH range for
its growth and activity with an optimum value in
between the range [5 ]. The optimal levansucrase
production was attained at an initial pH of 6.0
(data not shown). Increasing or decreasing in pH on
either side of the optimum value resulted in poor
m ic r o b ia l a n d d e c r e a s e in gro w th p ro d u c t
fermentation [1 6 ].
SSF is fermentation in the absence of free
liquid, and recovery of the fermentation product
requires its extraction from the solid fermented
medium. The extraction efficiency is critical to the
recovery of enzyme from the fermented biomass, hence
selection of a suitable solvent is necessary. Organic and
inorganic solvents beside tap and distilled water were
used in this study. From the results (Fig. 5) it is clear
that among all the solvents, distilled water gave the
best extraction of levansucrase from the fermented
solids. This might be due to dissolution of the all
media broth by distilled water which then becomes salt
solution and hence able to extract enzyme protein from
fermented biomass. Distilled water is a common
extractant (available, save and low cost) used by other
workers,[1 9 ] used distilled water for extraction of
alkaline protease from solid wheat bran fermentation. [2 0 ]
extracted xylanase and CMCase enzymes from different
solid substrates with distilled water. However, among
all salt solutions tested (Fig. 6), sodium chloride
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J. Appl. Sci. Res., 4(10): 1199-1204, 2008
Fig. 8: Effect of tem p erature
extraction from SSF.
on levansucrase
less. This may be due to the inhibitory effect of
temperature on enzyme activity and make it less stable.
Agitation of the fermented biomass with distilled
water at 40 î C and 160 rpm gave appreciable amount of
levansucrase enzyme compared with stationary
condition (data not shown). Agitation was effective for
the leaching process due to reduce enzyme adhesion to
cell biomass and also disperses the fermented sawdust
uniformly in the continuous phase of the solvent [8 ].
Extraction of other enzymes from fermented biomass
by agitation process [8 ,1 0 ].
Soaking time was optimized for maximum enzyme
recovery from the fermented sawdust. The time period
was varied from 30 min to 180 min as shown in figure
9. It was found that 90 min soaking was optimum and
beyond that it did not have any additional effect on
enzyme extraction indicating that 90 min is the
minimum time for total penetration of distilled water
through the fermented biomass. Periods of 30 or 60
min seem not be enough for total solubilization of
levansucrase enzyme present in sawdust medium.[1 4 ]
investigated
the effect of incubation time on
protease extraction in SSF and found that 30 min
provided the best time. On the other side, [8 ] found that
150 min was optimum for extraction of á-amylase from
fermented bran.
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Fig. 9: Effect of soaking time
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