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EVALUATION OF WELL-ADJUSTED SOLID SUBSTRATE MEDIUM FOR

ENHANCED PROTEASE PRODUCTION BY BACILLUS SUBTILIS DKMNR: MIXTURE
DESIGN A CASE STUDY

Article · January 2011

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 IJIB INTERNATIONAL JOURNAL OF INDUSTRIAL BIOTECHNOLOGY
Volume 1 • Number 1 • January-June 2011, pp. 41-53,   International Science Press (India), ISSN:

Regular Articles

EVALUATION OF WELL-ADJUSTED SOLID SUBSTRATE

MEDIUM FOR ENHANCED PROTEASE PRODUCTION BY
BACILLUS SUBTILIS DKMNR: MIXTURE DESIGN A CASE
STUDY
Kezia Devarapalli 1,2,*, Prasanthi Velaga3, Narasimha Rao Medicherla. 1 and
Sureddy Visweswara Naidu1
1
Centre for Biotechnology, Department of Chemical Engineering, Andhra University,
Visakhapatnam, India.
2
Department of Biotechnology, St. Martins Engineering College, Dhulapally, Secundrabad, India.
3
Department of Quality Control, Startek Laboratories, Hyderabad, India.

Abstract: Alkaline protease production under solid state fermentation was investigated using the isolated
strain of Bacillus subtilis DKMNR. Among all agro-industrial waste material evaluated, green gram husk,
soya bean meal and bengal gram husk supported maximum protease production. The simplex lattice design
was used to improve the enzyme production and to observe the effect of the three substrates. Maximum
enzyme production (1045 U/gds) was observed when the substrate mixture was of 0.8 g of green gram husk,
3.4 g of soybean meal and 0.8 g of bengal gram husk. The yield was improved with the supplementation of
carbon and nitrogen sources to the solid medium. Optimum enzyme production was achieved with 1.25 g of
glucose and 0.5 g of casein. Glucose did not repress the enzyme production but the inorganic nitrogen
sources except urea, all showed little negative impact. The physiological fermentation factors such as pH of
the medium (pH 9.0), moisture content (120 %), inoculum concentration (2 ml), incubation temperature
(32 °C) and incubation time (48 hr) played a vital role in alkaline protease production.
Keywords: Alkaline protease, Bacillus subtilis DKMNR, Solid state fermentation, Mixed design, Optimization.

1. INTRODUCTION conditions. Further, it has advantages in downstream

Among the industrial enzymes, proteases make up the processing in spite of the cost-intensiveness for medium
greatest portion of worldwide sales with a steadily components [1, 4-5]. However, solid-state fermentation
increasing demand for applications in the leather, has gained renewed interest and fresh attention from
cosmetic, pharmaceutical, detergent, brewing and food researchers because of its edge in biomass energy
industries[1-2]. However, the largest share of the enzyme conservation, solid waste treatment and its application to
market has been held by detergent alkaline proteases produce secondary metabolites over submerged
active and stable in the alkaline pH range as they play a fermentation [6-9]. Production of biocatalysts using agro-
specific catalytic role in the hydrolysis of proteins [3]. biotech substrates under solid-state fermentation
This has created increasing attention in exploitation of conditions provide several advantages in productivity, cost-
exotic microbial strains for production of alkaline effectiveness in labour, time and medium components
proteases. further the effluent production is less and thus it is eco-
friendly [6-10]. However, these production characteristics
Most of the microbial products at industrial scale are have to offer a competitive advantage over existing
generally produced using submerged fermentation due to products.
its apparent advantages in consistent enzyme production
characteristics with defined medium and process There are a large number of techniques available to
design culture media. They can vary from the traditional
* Corresponding Author: [email protected], Telephone: one-variable at- a-time method to more complex statistical
+91 9703507043, 040 27230536. and mathematical techniques [2] involving experimental
 42 International Journal of Industrial Biotechnology (IJIB)

designs such as full and partial factorials, Plackett- 2.2 Collection and Processing of the Substrates
Burman design, followed by optimization techniques such Various agro industrial materials such as rice bran, wheat
as response surface methodology (RSM) [9], artificial bran, coconut oil cake, zuzubi oil cake, peanut press cake,
neural networks (ANNs) [3,4], fuzzy logic and genetic rice husk, green gram husk, red gram husk, bengal gram
algorithms (GA)[3,4] among others. Regrettably, no husk, black gram husk, soya bean meal, corn cobs, corn
multipurpose technique is known to be applicable to all stover, corn leaves, sweet sorghum pulp, sugarcane
situations. Mixture designs were found to be effective baggase and sugarcane leaves are obtained from the local
tool for screening and evaluation of the various solid agricultural market. Whereas applepomac, pine apple
substrates [8]. In a mixture experiment, the independent waste, orange waste, banana peels and orange peels were
factors are proportions of different components of a blend. obtained from the local fruit market and juice shops. The
The interpretation of data in mixture experiments where potato peels, mashed potatoes, processed tea powder and
the components represent proportionate amounts of the processed coffee waste was collected from the home. All
factors differs from classical factorial experiments where the materials were dried at 80°C for 2 hours and the leaf
the response varies depending on the amounts of each materials and fruit pulp were powdered. All the materials
input variable. The key to mixture experiments is that the were sieved to avoid the fine powder which causes the
mixture components are subject to a constraint requiring clumps formation upon addition of water and decrease the
that the proportions sum to one. In mixture experiments, substrate availability to the microorganism [6].
the measured response is assumed to depend only on the
relative proportions of the ingredients or components in 2.3 Solid State Fermentation
the mixture, and not on the amount of the mixture. Selected substrates materials were used as a solid medium
However, one can overcome this limitation by adding the for protease production. Five grams each of the substrate
amount of mixture as an additional factor in the experiment, was taken separately in 250 ml Erlenmeyer flasks and
thereby allowing mixture and process variables to being moisturized with 5ml of distilled water. These flasks were
treated together. The advantage of mixture experiments sterilized and inoculated with 2 ml of inoculum solution.
over factorial design is that one can more efficiently study The flasks were mixed thoroughly and incubated at 30°C
the interaction influence amongst factors on the production, in an incubator for 48 hours.
and subsequently eliminate both neutral- and negative-
factors. Mixture experiments have been the subject of 2.4 Mixture Design
many studies and have enjoyed extensive application in
Mixture design was chosen to study the effect of mixed
pharmaceuticals, geology, petroleum, food, and tobacco
substrates on protease production. The best three
industries.
substrates which could yield highest protease at individual
The present study was aimed to exploit the locally level were chosen. A simplex lattice design was employed
available, inexpensive agro-substrate for alkaline protease to optimize the substrate mixture. All the experiments
production using Bacillus subtilis DKMNR under solid- were conducted according to the Sathish et al [8].
state fermentation and optimization of the various
In a mixture design, two models have to be taken into
parameters for improvement of the yield.
account, one for each mixture being considered. In this
2. MATERIALS AND METHODS case, a product model can be used with the two groups
of components x and z. This model is represented by
2.1 Microorganism and Culture Conditions Eq. (1):
The Bacillus subtilis DKMNR (MTCC No: 10551 y = f ( x) ∗ g ( z ) + e (1)
Genebank no: FR717670) culture used in this study was In Eq. (1) y is the response, the functions f (x) and
isolated from garden soil samples around the department g (z) are separated polynomial models that represent the
of Chemical Engineering, Andhra University, two mixtures, and e is a zero-mean random variable with
Visakhapatnam, A.P., India. The culture was periodically variance independent of x and z.
sub cultured on the nutrient agar slants. The inoculation The polynomial models used in Eq. (1) was modified
media was composed of (g/L) of Glucose, 18.8; peptone, some terms from the complete polynomial expression in
11.24; K2HPO4, 0.50, MgSO4, 0.05 and CaCl2, 0.02 and order to eliminate the constraint originated in the correlated
pH 9 after inoculation of the media, incubated at 31°C on variables. Eq.(2) shows the canonical form of the
rotary shaker at 225 rpm for 24 hrs. quadratic model:
Evaluation of Well-Adjusted Solid Substrate Medium for Enhanced Protease Production by Bacillus Subtilis… 43

q q enzyme production in solid-state fermentation process

y = ∑ βi xi + ∑ ∑ βij xi x j (2) depends upon several factors, mainly related with cost
i =1 i< j and availability of the substrate material. Thus it involves
Geometrically, in Eq. (2) the parameter β i represents screening of several agro-industrial residues.Agro industrial
the expected response to the pure mixture xi = 1, xj = 0, materials such as rice bran (RB), wheat bran (WB),
j ≠ i. The first term in the Eq. (2) represents the response coconut oil cake (COC), zuzubi oil cake (ZOC), peanut
when blending is strictly additive and there are no press cake (PPC), rice husk (RH), green gram husk (GH),
interactions between the components of the mixture. The red gram husk (RH), bengal gram husk (BGH), black
quadratic term β ij xixj represents the excess response gram husk (BLH), soya bean meal (SM), corn cobs (CC),
over the linear model due to the interaction between two corn stover (CS), corn leaves (CL), sweet sorghum pulp
components, and this effect is often called synergism or (SSP), sugarcane baggase (SB) and sugarcane leaves
antagonism. (SL) (Fig.1) and household waste materials like apple
pomac (AP), pine apple waste (PW), orange waste (OW),
2.5 Enzyme Extraction banana peels (BP), orange peels(OP), potato peels(PP),
mashed potatoes (MP), processed tea powder (PTP) and
The enzyme was extracted according to the method
processed coffee waste (PCW) (Fig.1) were used as
described by Prakasham et al [6]. Fermented medium
solid support/substrate matrices for production of enzyme
was mixed thoroughly with 50 mM glycine–NaOH buffer,
by B. subtilis DKMNR. The data indicated that protease
pH 11 for 30 min and the extract was separated by
production pattern varied with the type of agro-waste.
squeezing through a cloth. This process was repeated
This phenomenon might be attributed to the dual role of
three times and extracts were pooled together and then
solid materials on supply of nutrients to the growing
centrifuged. The supernatant was used as enzyme source
microbial culture and providing anchorage for the growing
for protease assay.
cells.
2.6 Estimation of Protease Activity
The protease was assayed according to the method of
modified Auson-Hagihara method [12]. One unit of
alkaline protease activity was defined as 1 µg of tyrosine
liberated per ml under the assay conditions.

2.7 Optimization of the Various Process

Parameters
In order to increase the protease production in the solid
state fermentation various process parameters such as
pH (5 to 12), moisture content (20 to 200 % w/w), inoculum
size (0.5 to 3.5) and temperature (26 to 40 °C) were
optimized.

2.8 Effect of Carbon and Nitrogen Additives on Figure 1: Protease Production by Isolated Bacillus Subtilis
Protease Production DKMNR Using Market and Household Waste
The optimum SSF medium was supplemented with Maximum protease production (914U/gds) was
different carbon and nitrogen sources initially at 0.5g. observed with green gram husk while minimum protease
Further the best suitable additional carbon and nitrogen production (145 U/gds) was noticed with rice husk as
source was studied at various concentrations in order to substrate/support material. Soya bean meal and bengal
improve the protease production. gram husk were found to be best substrates for protease
production next to the green gram husk. This data further
3. RESULTS AND DISCUSSION supported that, the composition of the substrate was one
of the important parameters for evaluation of extracellular
3.1 Evaluation of Different Agro-Industrial microbial enzymes production. The results were in
Material for Alkaline Protease Production accordance with the observations made with alkalophilic
The selection of an ideal agro-biotech waste for economic and thermophilic Bacillus species JB-99[13] and others
 44 International Journal of Industrial Biotechnology (IJIB)

reported about bacterial strains [6, 14]. However, the for analysis of the mixture design using following
results varied in the production values suggesting that the equations.
present investigated bacterial strain was different in its
Y = ∑ i =1 βi xi
v
metabolic and biochemical aspects to that of Bacillus Linear model (3)
species JB-99[13]. Evaluation of protease production
Y = ∑ i =1βi xi + ∑ ∑ i < j βij xi x j
v p
values of the strain with different organisms reported in Quadratic model (4)
the literature did not indicate that, this strain could be a
Y = ∑ i =1βi xi + ∑∑ i < j βij xi x j + ∑∑∑ i < j <k βijk xi x j xk
v p p
potential organism after optimization and scale up studies
(Table 1). To obtain the maximum protease production
the fermentation media is modified by mixing the different Special cubic (5)
agro-industrial wastes. Three different wastes such as
Y = ∑ i =1 βi xi + ∑∑ i < j βij xi x j + ∑∑ i < j δij xi x j ( xi – x j )
v p p
soybean meal, bengal gram husk and green gram husk
were selected as they exhibited the maximum protease + ∑∑∑ i < j < k βijk xi x j xk
p

activity when used as individual substrates.

Full cubic (6)
Table 1
Protease Production by Different Microorganisms Where Y is a response, β i is a linear, β ij is a quadratic
and β ijk cubic coefficients, βij is a parameter of the model.
Microorganism Solid substrate Protease
used production The β ix i represents linear blending portion and the
(U/gds parameters β ij represents either synergic or antagonistic
material) blending.
Bacillus sp [6] Green gram husk 35,000 Table 2 presents the varying concentrations of solid
Bacillus species JB99 [13] Pigeon pea 12,430 substrate used during fermentation process and the
Bacillus species [15] Wheat bran 429 protease production data for each experiment. The
Bacillus species [15] Lentil husk 168 protease production values varied from 685 to 1045 U/
Penicillium species [17] Soyabean 1950 gds. This variation of protease yield under similar
Aspergillus oryzae[18] Wheat bran 1500 fermentation conditions but with different substrates
Rhizopus oryzae [19] Wheat bran 358 suggested the importance of substrate composition on
Rhizopu soryzae [20] Wheat bran 58.7 fermentative protease production. Analysis of individual
substrate impact on protease production pattern indicated
3.2 Mixture Designs for Substrate Optimization that green gram husk is the best substrate with 53% higher
yield compared to other two selected materials. Further
An augmented simplex lattice design was employed for
analysis of the data (Table 2) revealed that mixed substrate
the present investigation. In order to observe the mixed improved protease yield. This can be evidenced based on
substrate effect on protease production, three solid higher protease yield from experiment 5 compared to 1
substrates soybean meal (SM), green gram husk (GH) and 2 (Table 2). However, presence of GH in mixed
and bengal gram husk (BGH) were used in these designs, substrate fermentations, supported better production
the total proportions of the different substrates made to compared to SM and BGH which can be evidenced from
100% i.e., 5g material. The design consists of total 10 runs experiments 4,6, 7 and 9 where a higher protease
where three with pure mixtures (one for each component), production was observed than individual or in other
other three with binary blends for each possible two- combination substrates as sole substratum. These results
component blend, another three with complete blends (all suggest that green gram husk is playing the vital role in
three components are included but not in equal proportions) the mixed substrate fermentation.
and last one as centroid (where equal proportions of all In view of the variation in protease yield with
three components are included in this blend). Table 2 different substrates and its production by fermentation is
shows all the experimental runs with the composition of associated with availability of nitrogen source, the nutrient
substrate as per mixture design model and the protease release pattern during sterilization of substrate material
output values. Assuming that, the measured response of was investigated by extracting with known amount of
protease production was dependent on the relative distilled water and their concentration was analyzed. It
proportions of the components in the mixture, linear was noticed that more nutrients were release to
through to cubic models (STATISTICA 6.0) were used fermentation medium by the GH was observed compared
Evaluation of Well-Adjusted Solid Substrate Medium for Enhanced Protease Production by Bacillus Subtilis… 45

Table 2
Mixed Design Experimental Layout and Protease Production
S. No Soybean Meal Green Gram Husk Bengal Gram Husk Protease Activity
(g)(SM) (g)(GH) (g)(BGH) (U/gds)
Coded Real Coded Real Coded Real Observed Predicted Error
1 1 5 0 0 0 0 685.00 685.74 -0.74
2 0 0 1 5 0 0 904.00 910.38 -6.38
3 0 0 0 0 1 5 823.00 808.83 14.16
4 0.5 2.5 0.5 2.5 0 0 966.00 969.17 -3.17
5 0.5 2.5 0 0 0.5 2.5 998.00 986.26 11.73
6 0 0 0.5 2.5 0.5 2.5 967.00 949.62 17.37
7 0.666667 3.4 0.166667 0.8 0.166667 0.8 1045.00 1023.91 21.08
8 0.166667 0.8 0.666667 3.4 0.166667 0.8 915.00 923.74 -8.74
9 0.166667 0.8 0.166667 0.8 0.666667 3.4 1019.00 1010.83 8.16
10 0.333333 1.6 0.333333 1.7 0.333333 1.7 917.00 970.47 -53.47

Table 3
ANOVA
S. No Model SS MS F-Value p-Value R2 Adjusted R2
1 Linear 73184.79 13711.06 1.3114 0.328304 0.2725 0.0647
2 Quadratic 4138.62 23015.39 22.2445 0.005882 0.9588 0.9074
3 Special Cubic 4088.73 49.89 0.0366 0.860492 0.9593 0.8780
4 Cubic 1609.46 1239.64 0.7702 0.627401 0.9840 0.8560

to BGH and SM denoting that the higher protease yield only 4.12% of the variability remaining unexplained. In
by GH may be correlated with availability of nutrients in the present study, a good co-relation was identified
the composition of substrate. between predicted and experimental protease production
Data from the experimental design was further with a variation of 5.8 %. The empirical relationship
analyzed by employing a multiple linear regression using between protease production (Y) and substrate variables
protease yield as the response. Sequential F-tests, on the in coded units is obtained by the application of second
linear to full cubic solutions were performed for order model as per Eq.7.
appropriate model selection (based on highest F-statistics
Y = 685.75 × SM + 910.38 × GH + 808.84 × BGH
significance) suitable for protease production. ANOVA
+ 684.41 × SM × GH + 809.32 × SM × BGH
results of the all four models are presented in Table 3.
+ 506.6 × GH × BGH
The quadratic model showed a high F value (22.24) and
low p value (0.00588). The analysis of R2 value revealed (7)
that the special cubic and cubic model have higher fit
(R2 special cubic = 0.9593 & R2 Cubic = 0.984) than the Table 4
Quadratic Model Terms
quadratic model (R2quadratic = 0.9588). Inspite of the
greater R2 values than the quadratic model they have Coefficients t-Value p-Value
smaller F-value (F special cubic = 0.0366 & F cubic = SM 685.7483 22.10519 0.000025
0.7702) and higher p-values (P special cubic = 0.8605 & GH 910.3847 29.34638 0.000008
P cubic = 0.6274). Such data suggest that the quadratic
BGH 808.8392 26.07304 0.000013
model is the most significant. Therefore further data
analysis was performed using only quadratic model. The SM*GH 684.4141 4.78690 0.008731
noticed R2 value of the quadratic model is 0.9588 indicating SM*BGH 809.3232 5.66054 0.004801
that 3 substrate components altogether would explain GH*BGH 506.5960 3.54321 0.023944
about 95.88% of the variability in the response leaving
 46 International Journal of Industrial Biotechnology (IJIB)

Table 5 graphical representation of a combination of raw materials,

Optimum Temperature Values for rather than a predictive illustration of the product yield.
Maximum Protease Production
Figure 2 depicts the triangular graphs showing the level
Optimum Organism curves of protease yield (obtained from Eq .7) as a
Temperature (°C) function of the substrate type. From the graph it is
30 Bacillus species B21-2 [28] observed that the highest yield was nearer to the GH
35 Bacillus species Y [29] having equal distance to the GH – SM and GH-BGH
Bacillus species.MH5-6 [30] axis. The SM- BGH axis demonstrated the least protease
36 Bacillus licheniformis [31] production.
Bacillus species strain GX6638 [32] Further, a numerical method given by Myers and
Bacillus species no. AH-101 [33] Montgomery [21] was used to solve the regression Eq. 7
37 Bacillus firmus [34] to optimize the substrate mixture ratio. The results
39.5 Bacillus licheniformis [35] indicated that the optimum mixture for higher protease
production is 44.4% of GH, 25% SM and 30.6% BGH by
Where Y is the response of protease production in dry weight. For this substrate combination the predicted
U/gds and GH, SM and BGH were the substrates with protease yield was 1029.864 U/gds. Our results validated
the respective coded experimental values testing the these fermentation conditions with a protease production
experiments as per the Table 2. The significance of each rate of 1054 U/gds. A similar optimization approach was
coefficient in Eq. 7 was determined by Student’s t-test performed by Sathish et al [8] for L-glutaminase
and p-values and listed in Table 4. The larger magnitude production in solid state fermentation. Rispoli and Shah
of the t-value and smaller p-value denote the corresponding [22], in cutinase production in submerged fermentation.
coefficient significance. The observed lower p-value The role of each substrate material was optimized for the
(<0.05) in the present experiment suggested that all linear production of protease in mixture design fermentation using
and interactive terms were significant. For the substrates, natural agro-wastes such as SM, BGH and GH. Among
GH has one the highest – test value with a magnitude of all materials, GH presence is essential and SM is the least
910.38 and the p-value (8×10–6) was one of the least important component for maximizing protease production
which indicates that the largest influence in the mixture among selected substrates. An optimum protease
for protease production. Even BGH has highest magnitude production of 1054 U/gds could be obtained with a 44: 31:
of 808.84 and p-value of 13×10–6. The interaction of GH 25 ratio of GH: BGH: SM respectively without any
with BGH indicated the lowest magnitude (3.54) and pretreatment of the material. Further work was preceded
higher p-value (23.9×10–3). Similarly, the interaction of with respect to optimization of the fermentation parameters
SM with the BGH has the highest magnitude (809.32) to increase the protease production. The parameters such
and lowest p-value (4.8×10–3) suggesting a large influence as pH, moisture content, inoculum concentration, incubation
on protease production which can be seen from the 5th time, and incubation temperature, concentration of the
experiment in Table 2. optimized carbon and nitrogen sources from the selected
sources were investigated. Keeping the potentiality of this
microbial strain in protease production further evaluation
was continued using the mixture of soybean meal, bengal
gram husk and green gram husk as solid support/substrate
for solid state fermentation.

3.3 Role of pH on Protease Production

Enzyme production by microbial strains strongly depends
on the extracellular pH because culture pH strongly
influences many enzymatic processes and transport of
various components across the cell membranes which in
Figure 2: Triaxial Diagrams of Protease Production as a turn support the cell growth and product production [3]
Function of Substrate Concentration
pH dependent alkaline protease production studies by B.
The task of optimizing mixtures of different substrates subtilis DKMNR in solid state fermentation using the
for protease production can be predicted using a triangular mixture of SM, BGH and GH suggested that, the enzyme
surface response methodology as triaxial diagrams are production was influenced by the pH of the medium.
Evaluation of Well-Adjusted Solid Substrate Medium for Enhanced Protease Production by Bacillus Subtilis… 47

Maximum protease production (1246 U/gds) was fermentation using a particular substrate, moisture level
observed at pH 9.0 (Fig. 3). The synthesis of the enzyme (content)/water activity was one of the most critical
increased with increase of the pH of the medium towards factors [8, 23]. Solid-state fermentation processes are
alkaline range from neutrality up to 9.0 and was less different from submerged fermentation culturing, since
constant in the pH range 9.0–12.0 by B. subtilis DKMNR. microbial growth and product formation occurs at or near
The enzyme production pattern suggested that, the isolated the surface of the solid substrate particle having low
bacterial strain was alkalophilic in nature and produces moisture contents [10]. Thus, it is crucial to provide
maximum quantity of enzyme at alkaline pH conditions. optimized water level that controls the water activity (aw)
of the fermenting substrate for achieving maximum
product production. Reports on enzyme production by
microbial species under solid-state fermentation indicated
that the availability of water in lower or higher
concentrations affected microbial activity adversely [15].
Moreover, water is known to have profound impact on
the physico -chemical properties of the solids and this, in
turn, affects the overall process productivity [10].

Figure 3: Effect of pH on the Production of Protease by

Isolated B.subtilis DKMNR

Further evaluation of enzyme data in the studied pH

range indicated a linear increase in the biocatalyst
production up to pH 9.0. The observed variation in
protease production under solid state fermentation with
mixed substrate attributed to higher enzyme production
in the pH range of 9.0 to 10.0. In the literature the authors
Figure 4: Effect of Moisture Content on the Production
reported that protease from solid state cultures of of Protease by B. Subtilis DKMNR
Aspergillus parasiticus showed optimum activity at pH
8.0 and 80 % less activity at pH 5.0. Johnvesly et al[13] The data indicated that, characteristic nature of
working on alkaline thermo stable protease found that, enzyme production along with studied moisture level and
the enzyme produced by thermoalkalophilic Bacillus moisture content played a critical role in alkaline protease
species JB-99 showed catalytic activity in a broad pH production in B.subtilis DKMNR. Maximum enzyme
range (6.0 to 12.0) with 11.0 as optimum pH. The data production was observed with 140 % moisture content,
generated in the present investigation suggested that, the which was noticed as 9824 U/gds matrix biomass (Fig 4).
influence of pH on alkaline protease produced by isolated Linearity between moisture content and enzyme
B. subtilis DKMNR might be related with synthesis level production was observed up to 140 % and thereafter
because, the extraction of the enzyme after solid state further increase in moisture level in the fermentation
fermentation was performed using alkaline pH buffer. medium resulted in reduction of protease production. The
The adapted extraction procedure eliminated the inhibition percent reduction in enzyme production from either side
of protease at cellular transport and at activity level and of the optimum moisture level (120 %) varied (Fig.4).
the observed growth associated nature of this enzyme This was evidenced from the fact that, an increase in 40
production in this bacterial strain. % moisture level reduced the production to the tune of
only 17 % to that of optimum production while decrease
3.4 Role of Moisture Content on in same quantity of moisture level caused 30 % reduction
Protease Production indicating the severity of damage to cell metabolism and
Among the several factors that are important for microbial subsequent enzyme production will be more with low
growth and enzyme production under solid-state water activity in solid state medium than higher water
 48 International Journal of Industrial Biotechnology (IJIB)

level to that of critical requirement. The decrease in studied inoculum range (Fig.5). Maximum protease
production with increase in moisture level in the solid synthesis (1391U/gds) was noticed in 2 % inoculum
medium might be attributed to decrease in mass transfer supplemented fermentation conditions. The reduction
of heat and gases caused by water logging among the pattern of enzyme production with increase or decrease
inter-particulate area which in turn adversely affects in inoculum supplementation from the optimum level also
cellular and biosynthetic activities associated with showed difference. Increase of inoculum level from 2 to
microbial growth [24]. The observed reduction of enzyme 3.5 % adversely caused 33 and 48 % reduction in the
production at reduced moisture level might be associated enzyme production. Decrease of the same from 2 % to
with reduced availability of water content for microbial 1.5 and 1 % resulted in 36% and 69 % reduced protease
growth. The results further indicated that, moisture production, respectively depicting the importance of
content during solid-state fermentation played a major inoculum level optimization for efficient protease
role in regulating alkaline protease production in the production under solid-state fermentation conditions.
isolated B. subtilis DKMNR. Though the pattern of
protease production with the function of moisture level in 3.6 Role of Incubation Temperature on
the bacterial strain and comparison of protease production Protease Production
values of strains reported in the literature were observed Temperature is another critical parameter that has to be
to be similar. However, optimum requirement of moisture controlled and varied from organism to organism. The
content during solid state fermentation process varied with mechanism of temperature control of enzyme production
the type of organism and agro industrial material [6]. is not well understood [26]. However, studies by Frankena
et al [7] showed that a link existed between enzyme
3.5 Role of Inoculum Concentration on synthesis and energy metabolism in Bacilli, which was
Protease Production controlled by temperature and oxygen uptake. The
Initial inoculum level influenced the cellular metabolic optimum temperature values reported for maximum
activity, growth of the microorganism and metabolite protease production are given in the table 4.
production [25]. The role of initial inoculum concentration
on alkaline protease production under solid-state
fermentation environment with soybean meal, bengal gram
husk and green gram husk as medium was investigated
to determine the optimum inoculum requirement. Inoculum
level selected for this study ranged from 0.5 % to 3.5 %
from 24 hr grown bacterial cell suspension having an
absorbance of 0.8 at 600 nm.

Figure 6: Effect of Incubation Temperature on the Production

of Protease by Isolated B. Subtilis DKMNR

Alkaline protease production by B. subtilis DKMNR

in solid state fermentation was increased by optimizing
the temperature of the environment. Maximum protease
production was 1505 U/gds at 32 °C. The incubation
temperature showed parabolic nature in this study (Fig.6).
Figure 5: Effect of Inoculum Concentration on the The increase and decrease of temperature showed a lot
Production of Protease by B. Subtilis DKMNR of difference in the protease production. An increment
Alkaline protease production by B. subtilis DKMNR of 4 °C in the temperature reduced the enzyme production
strain under solid-state fermentation conditions varied with to 39% and decrease of 4 °C in the incubation temperature
initial inoculum level and showed parabolic nature in the also decreased the protease production to 30%. Therefore,
Evaluation of Well-Adjusted Solid Substrate Medium for Enhanced Protease Production by Bacillus Subtilis… 49

the shifting of temperature from 26 to 40 °C has enhanced Protease production was observed to be 12 % increase
the protease production in the fermentation media to two- over control condition, with glucose as external carbon
fold when compared to the control experiment. source. This data suggested that, glucose was not a
repressor of protease enzyme in the bacterial strain under
3.7 Role of Different Carbon Sources on investigation unlike the observed catabolic repression by
Protease Production glucose in Bacillus subtilis and Bacillus licheniformis
The selection of an ideal substrate for enzyme production [27, 38].
in solid-state fermentation process is one of the critical One interesting phenomenon in this investigation was
factors to be considered [1, 4]. This is because, some of maltose, which is a disaccharide with two monomers of
the nutrients may be available in sub-optimal concentrations, glucose units supported protease production better from
or even absent in the substrate and therefore no substrate this bacterial species when compared to carbon source
may be suitable. In such cases, it would be necessary to (Fig.7). This might be attributed to the fact that, maltose
supplement substrate externally with deficit nutrients to as such might enter inside the cell before conversion into
have optimal yields. Several carbon sources such as glucose units. In fact, maltose metabolism in bacterial
galactose, arabinose, fructose, maltose, soluble starch, cells begins at maltose-6 phosphate and subsequently gets
glucose, xylose, mannose and ribose were selected and converted to glucose-6 phosphate and is metabolized
supplemented to the solid medium at 1 % level in order to further. This apparently eliminates the glucose associated
enhance the microbial growth. physiological changes in the medium such as lowering of
solution pH especially the medium is alkaline in nature.
Such glucose-mediated reduction in protease production
associated with lowering of pH was noticed by Zamost
et al [39] while working on production and characterization
of a thermostable protease by an asporogenous mutant of
Bacillus stearothermophilus. This was further evidenced
from the fact that reduced alkaline protease production
in lower pH medium environment.

Figure 7: Effect of Different Carbon Sources on the

Production of Protease by Isolated B. subtilis DKMNR
The data suggested that, supplementation of external
carbon source influenced alkaline protease production in
this bacterial strain and all the selected carbon sources
showed positive impact on cellular metabolism leading to
the production of the enzyme (Fig.7). The results indicated
Figure 8: Influence of Glucose Concentration on the
that the selected media (mixture of soybean meal, bengal Production of Protease by Isolated B. subtilis DKMNR
gram husk and green gram husk) was not the ideal
substrate for alkaline protease production by this isolated In order to know optimum requirement of carbon
Bacillus subtilis DKMNR because of the deficiency in source for better alkaline protease production by the
carbon source. Improvement of cell growth and subsequent bacterial strain is under investigation in solid-state
metabolite synthesis, in several microorganisms, was fermentation conditions, the enzyme production pattern
noticed upon supplementation of external carbon sources was investigated by supplementation of different
[36-37]. However, enhancement in enzyme production concentrations of glucose (0 to 2 %) (Fig.8). The data
levels varied with the type of carbon source (Fig.7). revealed that alkaline protease production in this bacterial
Glucose supplemented conditions supported maximum strain was regulated by availability of glucose in the
production with an increase of 112 % over control (no medium and maximum production (1971 U/gds) occurred
external carbon source supplementation). with 1.25 % glucose concentration under experimental
 50 International Journal of Industrial Biotechnology (IJIB)

conditions. Approximately 15 % improvement of enzyme as nitrogen source showed maximum influence by

yield was noticed under 1.25% glucose supplemented enhancing the enzyme production to that of other organic
conditions when compared to 0.5 %. Further increase in and inorganic nitrogen sources. The increase in protease
glucose concentration adversely affected protease yield was observed to be approximately 2-fold over control.
production in B. subtilis DKMNR under solid-state Similar observations were noticed in the case of protease
fermentation condition. The results obtained were in production by different microbial species [13,41].
accordance with reported alkaline protease production in Alkaline protease production data in different inorganic
the presence of different sugar [40]. nitrogen sources supplemented condition indicated that
enzyme production was having negative influence in the
3.8 Role of Different Nitrogen Sources on presence of ammonium-based and nitrate-based nitrogen
Protease Production sources. However, very little impact was observed when
Nitrogen source is one of the essential requirements for compared to control (Fig.9). Sinha and Satyanarayana
healthy microbial growth and is required to produce [42] noticed ammonium nitrogen associated regulation in
several cellular organic compounds such as amino acids, protease production in thermophilic Bacillus licheniformis.
nucleic acids, proteins and cell wall components. Though These observations clearly suggested that complex nitrogen
most of microorganisms metabolize inorganic and organic compounds had an edge over inorganic nitrogen sources
nitrogen sources, the preference varies with the genetic in alkaline protease production. This might be due to the
nature of microbe and type of product produced [13]. It lowering of pH, by ammonium ion caused reduction in
was reported that alkaline protease comprised 15.6 per the enzyme yields.
cent nitrogen and its production was regulated by the However, genetic nature of microbial species especially
availability of nitrogen in the medium [10, 38,40]. with respect to ammonium ion concentration in the medium
Therefore, influence of different nitrogen sources on and enzyme production could not be ruled out because,
alkaline protease yield by isolated B. subtilis DKMNR protease synthesis was known to be repressed by rapidly
was investigated by supplementing 0.5 % selected nitrogen metabolizable nitrogen sources [23]. These observations
compound to solid medium under optimal fermentation were in accordance with the noticed protease production
environment and measuring enzyme production. in the presence of complex nitrogen sources in Bacillus
licheniformis [41] and alkalophilic Bacillus species [28].
Experiments were conducted to optimize the casein
concentration as nitrogen source for production of alkaline
protease production, by varying the concentration of yeast
extract in the medium. The results indicated that the
protease production decreased with increase in casein
concentration in the medium. This data suggested that an
economic production of alkaline protease by this bacterium
can be obtained with 0.5 % casein supplementation to

Figure 9: Effect of Different Nitrogen Sources on the

Production of Protease by Isolated B. subtilis DKMNR

Experimental data revealed that complex nitrogen

sources yield maximum alkaline protease production in
the bacterial strain. The data indicated the importance of
nitrogen requirement for production of the protease by
Figure. 9. A media with the mixture of SM, BGH and GH
was insufficient nutrient medium for protease production.
So, different nitrogen sources on protease production by
B .subtilis DKMNR under solid state fermentation
Figure 10: Effect of Casein Concentration on the Production
conditions with the above media was investigated. Casein of Protease by Isolated B.subtilis DKMNR
Evaluation of Well-Adjusted Solid Substrate Medium for Enhanced Protease Production by Bacillus Subtilis… 51

the mixture of SM, BGH and GH based solid-state 4. CONCLUSION

fermentation medium though enzyme yield was 3267 Alkaline protease production by isolated Bacillus subtilis
U/gds. The results are depicted in (Fig.10). DKMNR under solid state fermentation was influenced
by the chemical nature of the mixture of the substrates
3.9 Role of Incubation Time on Protease Production (soybean meal, green gram husk and bengal gram husk).
Incubation time is one of the essential physiological Overall, the enzyme production was 117 % higher
fermentation parameters to be evaluated for optimal compared to initial level (i.e., without any nutrients
production of any microbial product/metabolite. To addition). The results obtained further supported that;
determine the optimum incubation time required for nitrogen and carbon sources were the major limiting
alkaline protease production by isolated B. subtilis factors in extracellular protease production by the
DKMNR, the enzyme production pattern was investigated microbial strain. Enhanced protease production was
during solid-state fermentation process using soybean yielded by the supplementation of glucose and casein to
meal, bengal gram husk and green gram husk as solid the solid medium.
matrix. It was observed that protease production increased
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