76

CHAPTER 3

OPTIMIZATION OF PROCESS PARAMETERS

FOR PROTEASE PRODUCTION IN SOLID

STATE FERMENTATION

3.1 INTRODUCTION

Solid State Fermentation (SSF) is defined as the cultivation of

microorganisms on moist solid supports, either on inert carriers or on

insoluble substrates that can, in addition, be used as carbon and energy

source. In SSF, fermentation takes place in the absence or near absence of free

water, thus being close to the natural environment to which microorganisms

are adapted (Renteria et al 2012). More generally, it can be understood as any

process in which substrates in a solid particulate state are utilized (Mitchell et

al 2000). Free water does not appear to be the natural abode for the majority

of microorganisms. Not even marine microorganisms prefer swimming in free

seawater since more than 98% of isolates from the marine environment have

been sourced from the underwater surfaces of solid substrates (Kelecom 2002).

Growth and product formation occurs on the surface and/or the inside of the

solid. In SSF a four phase system (insoluble support, water, biomass and air)

makes non-destructive on-line monitoring more difficult than in the liquid

fermentation. This constraint reduces the ability to effect control of the

fermentation. Nevertheless, in SSF, some parameters that affect growth or

product formation, such as temperature, agitation, aeration rates and gas

composition can be controlled throughout the fermentation (Villegas et al 1993).

77

Unfortunately, SSF is usually slower because of the diffusion

barriers imposed by the solid nature of the fermented mass. However, the

metabolic processes of the microorganisms are influenced to a great extent by

the change of pH, temperature, substrate, water content, inoculum

concentration, etc. These conditions vary widely from species to species for

each organism. Therefore, it becomes very important to know the

environmental conditions of the microorganism for maximum production

(Elibol and Moreira 2005). Nevertheless, research about SSF had been

neglected for a long time not only because of the popularity of the submerged

culture process but also for the difficulties associated with the measurement

of parameters in SSF, such as microbial biomass, substrate consumption,

concentration of products formed as well as the measurement of the physical

properties of the system (Hesseltine 1972). Alkaline proteases are important

enzymes and can be used for a variety of processes such as in detergents,

leather processing, silver recovery, medical purposes, food processing, feeds

and chemical industrial, as well as waste treatment (Zamost et al 1991;

Wiseman 1993).

Although there are many microbial sources available for producing

proteases, only a few are recognized as commercial producers. Major

industrial companies are continuously trying to identify enzymes that have

potential industrial applications, either to use them directly or to create

notified enzymes that have enhanced catalytic activity for well adapted large

scale industrial processes (Elibol and Moreira 2005).

SSF processes are usually simpler and can use wastes or agro-

industrial substrates, such as defatted soybean cake (Soares et al 2005), wheat

bran (Rajkumar etal 2011; Srividya et al 2012), Lentil husk (Akcan and Uyar

2011), Potato peels (Mukherjee et al 2008), Red gram husk (Rathakrishnan

78

and Nagrajan 2011) and rice bran (Karatas et al 2012; Saxena et al 2010) for

protease production.

This chapter deals with the physiochemical parameters optimization

and scale up of the process up to tray level in order to get significant amounts

of alkaline protease from Bacillus pumilus MTCC 7514 utilizing agro-

industrial wastes as nutrient source.

3.2 MATERIALS AND METHODS

3.2.1 Materials

Wheat bran and other agro-industrial residues were purchased from

the local market in Chennai, India. Fish meal (FM) was procured from Raj

Fish Meal & Oil Company, Maple, Karnataka state, India. Tryptone, Agar-

agar, maltodextrin (MD), yeast extract (YE), skim milk (SM) were purchased

from Hi-media and Sisco research laboratory (SRL). All other chemicals used

were of analytical grade.

3.2.2 Inoculum Development and Fermentation

A new strain of Bacillus pumilus MTCC 7514 isolated from beach

soil earlier in our laboratory by Prabhawathi et al (2010) was used as a

protease producer in this study. It was maintained on nutrient agar slants at 4

°C and sub-cultured every month. Inoculum was prepared by transferring a

loopful of culture from slant to 250 mL flask containing 50 mL of LB media,

followed by incubation at 30 °C at 120 rpm in shaker (Orbitek, Scigenics

Biotech, India) for 20 h. The above grown culture at the concentration of 5%

(v/w) was used to inoculate the production medium.

10 g of substrate was taken in 250 mL Erlenmeyer flask and 10 mL

of distilled water was added, mixed and sterilized at 121 C for 20 min. The

79

flasks were inoculated as mentioned above, mixed well and incubated in a

BOD incubator at 30 °C for 120 h. Samples were collected every 24 h and

checked for protease activity. All experiments were carried out in duplicates

and average of the duplicates was presented in the results.

3.2.3 Extraction and Assay of Enzyme

Protease from the fermented substrate was extracted by simple

contact method of extraction using Tris buffer (pH 9.0) as solvent. Ten

volumes of Tris buffer per gram fermented substrate (based on initial wet

weight of the substrate) were added to the fermented media and the extraction

was performed by triturating it using mortar and pestle. The slurry was then

squeezed through cheese cloth and clarified by centrifugation at 10,000 rpm

and 4 °C for 10 min. The clear supernatant was used as crude enzyme for

protease assay. Moisture was estimated in the solid residue using a moisture

analyzer (HG 63 Halogen moisture analyzer, Mettler Toledo). The protease

activity was determined by the method of Kunitz (1947) using casein as

substrate as mentioned in Chapter 1. The protease activity obtained was

converted to per gram dry substrate using the conversion factor of wet weight

to dry weight.

3.2.4 Optimization of Physico Chemical Parameters

The effect of various physico-chemical and nutritional parameters

(substrate, combination of substrates, incubation time, moisture level,

inoculum concentration, initial pH, temperature, additional carbon and

nitrogen sources and extraction of the protease) on protease production in 250

mL flasks was studied.

80

3.2.4.1 Effect of different substrates

Different agro-industrial residues such as wheat bran, rice bran,

green gram husk, black gram husk, red gram husk, and pigeon pea husk were

tried for protease production. All the substrates were subjected to sieving

employing sieve mesh size of 14 (1.41 mm). Substrate particles that passed

through the sieve were named fine particles whereas retained substrate

particles were named as coarse particles. 10 g of substrate were used for

screening studies. Samples were collected every 24 h and analyzed for

protease activity.

3.2.4.2 Effect of different substrate combinations

The effect of combination of substrates such as wheat bran and rice

bran in the ratio of 1:1 and 3:1; wheat bran and green gram husk in the ratio

of 1:1 and 3:1 on protease production was investigated. The samples were

collected at different time intervals and analyzed for protease activity.

3.2.4.3 Effect of incubation time on protease production

The flasks were inoculated with 20 h old culture grown in LB

media and incubated at 30 C in a BOD incubator for 120 h. At every 24th h,

one gram of sample from the fermented substrate was collected and checked

for activity to find the optimum time for protease production.

3.2.4.4 Optimization of initial moisture level

Optimum initial moisture content required for the growth of

bacteria as well as protease production was determined. Different experiments

by changing the substrate: water ratio viz., 1:1, 1:1.5, 1:2, 1:2.5 and 1:3 were

carried out.

81

3.2.4.5 Effect of inoculum concentration

The inoculum was developed in LB media as stated above and used

for inoculating the flask containing medium. Inoculum concentration of 2.5,

5.0, 7.5 and 10.0 (v/w) was used in order to find the optimum concentration.

The flasks were incubated at 30 °C for 96 h and samples were collected every

24 h, and analyzed for protease activity.

3.2.4.6 Effect of initial medium pH on protease production

The effect of pH on growth and protease production was

determined by varying the initial pH of medium from 6.0 to 10.0 which was

maintained by using different phosphate buffers (0.1 M) for pH 6.0-8.0 and

carbonate buffer (0.1 M) for pH 9.0-10.0 was used.

3.2.4.7 Effect of temperature

Optimum temperature for protease production was determined

incubating the production flask at different temperatures viz. 25 °C, 30 °C, 33

°C, 37 °C and RT (30±3 °C). The samples were collected at different time

intervals and analyzed for protease activity.

3.2.4.8 Effect of additional carbon and nitrogen sources

The effect of additional carbon and nitrogen sources on protease

production with wheat bran substrate was studied. Glucose, fructose, maltose,

malto-dextrin, starch and sucrose at concentration of 5% (w/w) were used as

carbon source whereas yeast extract, casein, commercial casein, soya flour,

fish meal, urea and tryptone at a concentration of 5% (w/w) were used as

nitrogen source. The effect of selected carbon and nitrogen source at different

concentrations (2, 5, 8 and 10%, w/w) on protease production was also

evaluated to find the optimum concentration.

82

3.2.5 Scale Up of Protease Production

Scale up of protease production from 10 g flask level to 200 g tray

levels were carried out. The wheat bran substrate in flasks (250 mL, 1 L and

Fern flask containing 10, 40 and 100 g of WB, respectively) and sterilized

steel trays (43 x 22 cm) each containing 200 g of WB, were inoculated with

5% (v/w) of 20 h grown culture and incubated at 30 °C for 96 h in a BOD

incubator. Samples were collected every 24 h from flasks as well as trays and

tested for protease activity. All the experiments were carried out in triplicates.

3.2.6 Effect of Different Buffers/Solution for Enzyme Extraction

Extraction of the fermented substrate was carried out with different

buffer/solution to find out the best extracting solvent. Different extraction

buffers/solution were tried out such as tap water, distilled water, carbonate

buffer (pH 9.0), Tris buffer (pH 9.0), NaOH solution (pH 9.0), Tris buffer

containing 5 mM CaCl2 and 10 mM CaCl2.

3.3 RESULTS AND DISCUSSION

Approximately 90% of all industrial enzymes are produced in

submerged fermentation (SmF), most often using specifically optimized and

genetically manipulated microorganisms. In this respect SmF processing

offers a far superior advantage over SSF. On the other hand, almost all these

enzymes could also be produced in SSF using wild-type microorganisms

(Holker et al 2004). In this study, various experiments were designed and

carried out in order to produce optimum amount of alkaline protease from

Bacillus pumillus MTCC 7514 by SSF.

83

3.3.1 Effect of Different Substrates on the Protease Production

SSF processes are significantly influenced by the nature of solid

substrates and their size. Different agro-industrial residues were tried as

substrates for protease production under solid state fermentation and the effect

of their sizes on protease production was also studied. The selection of an

ideal agro-biotech waste for enzyme production in a solid-state fermentation

process depends upon several factors, mainly related with cost and

availability of the substrate material, and thus may involve screening of

several agro-industrial residues (Pandey et al 2000). From Figure 3.1, we can

observe that wheat bran was the best substrate followed by green gram husk,

rice bran and pigeon pea husk. Red gram husk did not support any protease

production as there was no protease production with all three types of RGB.

In contrast, Rathakrishnan and Nagarajan (2011) have reported red gram husk

as a good substrate for protease production from Bacillus cereus in SSF.

Fine particles of substrates inhibited the protease production while

in the presence of coarse particle; protease production was more or less

similar to the one obtained with substrates without sieving. Green gram

course particles supported better production as compared to green gram husk

whereas , protease production was completely inhibited with Bengal gram

coarse particles while a reduced protease production was observed with

Pigeon pea coarse particles (PPCP).

84

Figure 3.1 Effect of different substrates on protease production

Abbreviations shown in Fig 3.1 are expanded in the below list:

WB (Wheat Bran) WBCP (Wheat Bran Coarse Particles)

WBFP (Wheat Bran Fine Particles) RB (Rice Bran)

RBCP (Rice Bran Coarse Particles) RBFP (Rice Bran Fine Particles);

GGH (Green Gram Husk) GGCP (Green Gram Coarse Particles)

GGFP (Green Gram Fine Particles) BGH (Black Gram Husk)

BGCP (Black Gram Coarse Particles) BGFP (Black gram Fine Particles)

RGH (Red Gram Husk) RGCP (Red Gram Coarse Particles)

RGFP (Red Gram Fine Particles) PPH (Pigeon Pea Husk)

PPCP (Pigeon Pea Coarse Particles) PPFP (Pigeon Pea Fine Particles

From these experiments, we can conclude that the protease

production was affected by the size of the substrate particle employed as well

as the type of substrate used. Finally, wheat bran, rice bran and green gram

husk were selected as the best substrates for further experiments.

Similar result was reported by Renganathan et al (2011) where

among six substrates chosen, wheat bran supported maximum protease

production followed by pigeon pea husk, black gram husk, rice bran, green

85

gram hull, and orange peel but the time required for maximum protease

production was comparatively higher as compared to the present study. Wheat

bran was the best substrate and nutrient source for protease production as

reported by Ramesh and Lonsane (1990), Agrawal et al (2004), Tunga et al

(2001) and Aikat and Bhattacharyya (2000). Prakasham et al (2006) have also

evaluated different agro-industrial wastes for protease production from

alkalophilic Bacillus sp. and reported that green gram husk supported

maximum protease production whereas minimum protease production was

noticed with the red gram husk which is similar to the present study.

Furthermore, they have also studied the effect of particle size on the protease

production and shown that green gram husk material in the range of 1.0-1.4

mm was optimum. In another report, Johnvesly et al (2002) have reported

pigeon pea husk as a substrate for protease production by solid state

fermentation.

3.3.2 Effect of Combination of Substrate on the Protease Production

Protease production was carried out using a substrate combination

of wheat bran with rice bran and green gram husk in different ratios. The

protease activity was 205.7 U/gds at 72 h when wheat bran and rice bran were

used in combination in the ratio of 3:1, which gave higher protease activity

compared to other combinations (Figure 3.2) but interestingly lower than the

single substrate (Control) indicating that wheat bran by itself is the best

substrate for protease production.

86

Figure 3.2 Effect of different ratios of substrates on protease production

3.3.3 Effect of Incubation Time on Protease Production

The inoculated flasks were incubated at 30 °C in a BOD incubator

in order to find out the optimum time for maximum protease production. The

maximum protease production of 238 U/gds was observed at the 48th h and

thereafter it reduced with time. There was very less production at 24 h.

Figure 3.3 Effect of incubation time on protease production

87

This is quite characteristic of SSF because the static nature,

diffusion limitations for oxygen and other nutrients slow down the growth and

hence production.

3.3.4 Optimization of Moisture Level for Protease Production

Initial moisture content of the substrate is an imporatnt factor in the

SSF system that influences the enzyme production and yield (Ramesh and

Lonsane 1990; Baysal et al 2003; Ramachandran et al 2004; Mukherjee et al

2008), because growth of microbes and product (enzyme) formation take

place at or near the surface of moist solid substrate (Pandey et al 2000). Since

the requirement of moisture content (water activity) may vary from microbe

to microbe, optimization of the initial moisture level of the substrate is the

most crucial step for achieving maximum yield of the desirable product.

Figure 3.4 Effect of moisture level on protease production

In the present study, different ratios of water and wheat bran were

taken and the effect of moisture content in media on the protease production

was evaluated. It was observed that there was a little increase in production at

moisture level of 60% (1:1.5) thereafter, further increase in moisture level in

88

fermentation medium resulted in reduction of protease production.

Chellappan et al (2006) have also reported maximum protease production at

moisture content of 60% from E. album BTMF S10 under SSF after 120 h of

incubation.

3.3.5 Effect of Inoculum Size on the Protease Production

Inoculum size is another important factor to be optimized for

protease production. The nature of inoculum as well as its size may affect the

microbial process (Elibol et al 1995). It was varied from 2.5 to 10% (v/w) and

found that enzyme production increased with inoculum size and was

maximum at 5% (228 U/gds). Further, inoculum size of 7.5 and 10% (v/w)

resulted in reduction of 25 and 32% reduction, respectively.

Figure 3.5 Effect of inoculum size on protease production

Renganathan et al (2011) have studied the effect of inoculum

concentration on protease production by Bacillus sp. RRM1 and reported that

15% inoculum supported maximum protease production (1,304 U/g) whereas

further increase or decrease in concentration resulted in the decline of

protease production.

89

3.3.6 Effect of Initial Medium pH on Protease Production

Alkaline protease 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 turn support the cell growth and product production

(Ellaiah et al 2002). Initial medium pH was varied from 6.0 to 10.0 and it was

observed that organism was able to grow and produce protease almost equally

at pH 6-9, then reducing at pH 10.0.

Figure 3.6 Effect of initial medium pH on protease production

Maximum protease production at the end of 72 h incubation was

241 U/gds at pH 8.0 (adjusted with NaOH) followed by 236 U/gds in

production medium with uncontrolled pH and protease production pattern was

almost similar at all pH provided. Elibol and Moreira (2005) have also

reported that production of protease from shipworm bacteria could be possible

in broad optimal pH range from 7 to 9 whereas maximum protease activity

was observed at pH 7.34, in which no pH adjustment was made. Similar to

their finding, the optimal pH range is from 6 to 8 in the present study.

90

3.3.7 Effect of Incubation Temperature on the Protease Production

Temperature is one of the critical parameters that have to be

optimized. The maximum protease production was observed in the range of

240 to 250 U/gds at all incubation temperatures except at 37 °C, where the

maximum protease production was only 42 U/gds (Figure 3.7). This may be

due to reduced growth rate of the bacteria at 37 °C. The protease productivity

(5.05 U/gds/h) was better at 30 °C since the maximum protease production

was achieved at the 48th h itself whereas in the other cases it took 72 h and

hence the optimum temperature for protease production was selected as 30°C.

The production flasks which were incubated at room temperature

(uncontrolled temperature) also shows the maximum protease production

(248.8 U/gds) similar to one obtained at 25 °C

Figure 3.7 Effect of incubation temperature on protease production

Renganathan et al (2011), have also studied the effect of incubation

temperature on protease production from Bacillus sp. and reported that

optimum temperature was 37 °C. If the difference between the optimum and

91

room temperature is minimal, the energy consumption for the process will

also be less.

3.3.8 Effect of Carbon Source on Protease Production

The effect of addition of various carbon sources (0.5 g) to wheat

bran (9.5 g) on protease production was evaluated. The results are given in

Figure 3.8. Starch, sucrose and maltodextrin had a positive influence on

protease production whereas protease production was repressed by the

addition of fructose and glucose. The maximum protease production was 257

U/gds in the presence of starch and sucrose. The effect of carbon sources on

protease production in this study was quite similar to the result reported by

Prakasham et al (2006).

Figure 3.8 Effect of carbon source on protease production

Further, the concentration of starch was optimized in the range of

2-10% (w/w). The result is shown in Figure 3.9, which depicts the enzyme

actitvity profiles for different starch levels. Starch at 5% concentration was

found to be optimum for protease production.

92

Figure 3.9 Effect of starch concentration on protease production

3.3.9 Effect of nitrogen source on the protease production

The influence of addition of different nitrogen sources (0.5 g/10g of

wheat bran) on protease production was evaluated. Commercial casein

supported maximum protease production (269.6 U/gds) followed by soya

flour (240 U/gds) and fish meal (238.2 U/gds) whereas addition of other

nitrogen sources to the medium significantly reduced protease production

(Figure 10).

Figure 3.10 Effect of nitrogen source on protease production

93

The lowest protease production (194.6 U/gds) was observed with

the addition of casein. Chellappan et al (2006) have studied the effect of

addition of organic nitrogen source on protease production by E. album and

reported that all the organic nitrogen source tested has positive effect on

protease except urea.

Figure 3.11 Effect of CC concentration on protease production

Further the optimization of CC concentration shows that there was

similar production at CC concentration of 2 and 5%, whereas protease

production reduced with further increase in concentration of CC

(Figure 3.11).

3.3.10 Scale up of Protease Production

Protease production in the finally optimized media was compared

in flasks (10/100 g substrate) and tray (200 g substrate) to examine feasibility

of scaling up the process. Protease production pattern was similar at all the

levels tried and the maximum protease production was observed in Fern

flasks (313.7 U/gds) with 100 g of WB followed by tray (301.5 U/gds) with

200 g of WB.

94

Figure 3.12 Scale up of protease production

F1: 250 mL Flask; F2: 1 L Flask; F3: Fern Flask (2.8 L)

The reduced protease production in tray may be due to improper

heat dissipation in tray system compared to flask level. Ito et al (2011) that

temperature changes in the Tray system culture was uneven and unstable

likely to be resulted from nonuniform conditions in the substrate. The

microbial heat generation is one of the limitations in SSF and the heat

generated during the growth of the organism in SSF is directly proportional to

the metabolic activities of microorganism as reported by Kwon et al (2011).

3.3.11 Effect of Different Buffers/Solution for Enzyme Extraction

The type of buffer and other solvents may interfere with the

stability of the enzyme during its recovery from the fermented substrate

therefore, it’s necessary to find the most suitable extractant/solvent for

enzyme extraction. Extraction of protease enzyme from fermented broth was

carried out with distilled water, NaOH solution (pH 9.0), carbonate buffer

(9.0), Tris buffer (9.0) and Tris buffer containing 5 mM and 10 mM calcium

chloride. The optimum recovery of the protease enzyme was with Tris buffer

containing 5 mM calcium chloride (232 U/gds) followed by Tris buffer with

10 mM calcium chloride (228.7 U/gds) and Tris buffer (210.4 U/gds). Shata

(2005) has reported increased enzyme extraction with extractant containing

0.05% calcium chloride and 40% glycerol. In a similar study by Mukherjee et

95

al (2008), distilled water containing 0.1% (v/v) Triton X-100, pH 8.0 served

as the best among all extractants used.

Figure 3.13 Effect of extraction solvent

A comparison of protease production using the strain of Bacillus

pumilus MTCC 7514 by submerged fermentation as well as solid state

fermentation is shown in Table 3.1 indicating the positive aspects of SSF system.

Table 3.1 Comparison of protease production in SmF and SSF by

Bacillus pumilus MTCC 7514 at flask level

Characteristics SmF SSF

Protease activity 34 U/mL 270 U/gds

Moisture content 100% 54.4%

Inoculum concentration 2% 5%

Source of Nutrients Soy Flour Wheat Bran

Inorganic salts NaCl, MgSO4,

KH2PO4 and CaCl2

--

Additional carbon source -- --

Additional nitrogen source -- Commercial casein

pH 7.0 7.0

Temperature 30 °C 30 °C

Agitation 120 rpm Static

Fermentation time 36 h 48 h

Cost of production USD 0.95/10 lakh

unit of protease

USD 1.95/10 lakh unit

of protease Cost calculated based on raw materials used in production media

96

3.4 CONCLUSION

In this study, optimization of production parameters for protease

from Bacillus pumilus MTCC 7514 was carried out. Different agro-industrial

residues (WB, RB, PPH, GGH etc.) were screened as a solid substrate for

protease production and the effect of the substrate size on the protease

production was also evaluated. The different physiochemical parameters

influencing protease production was optimized by one at a time approach.

Screening of agro-industrial residues as a solid support for protease

production suggested that wheat bran and wheat bran coarse particles can be

effectively used as a source of nutrients for the protease production followed

by green gram, rice and pigeon pea husk. Wheat bran fine particle leads to

reduced protease production whereas green gram, rice and pigeon pea fine

powder showed comparatively better protease production. The other optimum

conditions for protease production were incubation time, 48 h; moisture level,

1:1.5 (WB:DW); inoculum size, 5% (v/w); initial medium pH, 7.0 (with DW);

temperature, 30 °C; commercial casein, 0.5% and enzyme extractant, Tris

Buffer (5 mM CaCl2).

Scale up studies showed that maximum protease production

increases with increase in the amount of substrate level in the flasks. In tray

system maximum protease production reached 301.5 U/gds which was 11.6%

high when compared to flask level (250 mL) but was slightly less when

compared to the Fern flask (313.7 U/gds). Since the final medium contains

only wheat bran which is an agro-industrial residue easily available

throughout year and as a cheap substrate. Though cost comparison of raw

material needed for 10 lakh unit of protease enzyme production indicated that

SSF is costly over SmF, the ease of down-stream processing and requirement

of less sophisticated instruments for its upstream makes it suitable and

advantageous over SmF for industrial production of protease.