Chapter

OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION

OF ALKALINE PROTEASES

~ t h the objective of obtaining high yield of alkaline proteases, factors

liM influencing the production by the selected strains were studied. The

strains selected for production by submerged and solid state fermentation were

Bacillus sp. K 25 and BaciMus purnilus K 242 respectively. The factors

influencing the production were studied one by one, examining one factor at a

time, keeping the other factors constant. Once the optimization has been

done with respect to a factor it was incorporated in the experiment for the

optimization of the next factor. Unless otherwise specified, this was the

strategy followed for designing all the experiments described under this

chapter. Experiments were done in triplicate.

Section A

OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF ALKALINE PROTEASE

BY BACILLUS SP. K 25 BY THE SUBMERGED FERMENTATION METHOD

MATERIALS AND METHODS

The method of preparation of media, inoculation and incubation

followed for performing the experiments under this chapter were same as

described under section A of Chapter 3, except for the alterations or

modifications mentioned under each experiment.

Growth phase

Gm-acellular alkaline protease accumulation at various phases of

growth of Bacillus sp. K 25 was studied. The medium (pH 8.0) which

contained (per litre) 10 g of peptone, 10 g of beef extract, 5 g of sodium

chloride, 1 g of potassium dihydrogen phosphate, 2 g of dipotassium

hydrogen phosphate, 0.2 g of magnesium sulphate and 0.5 g calcium chloride

was inoculated with the bacterium and the culture samples were taken at

regular intervals during the incubation. The relative cell concentrations in the

samples (ODm was taken in a Systronics spectrocolorirneter 103, path

length: 1 cm) and the alkaline protease activity in the culture supernatants

were determined.

Temperature of Incubation

The effect of temperature on the alkaline protease production was

studied using the same medium as in the previous experiment. The medium

was inoculated, incubated at different temperatures and the alkaline protease

activity was determined.

The medium as in the previous experiments, prepared with different

pH was used for studying the effect of pH on the alkaline protease production.

Carbon and nitrogen sources

The effect of different carbon sources on alkaline protease production

by the strain was studied, using the media containing (gil) ammonium

sulphate, 5; sodium chloride, 5; calcium chloride, 0.5; magnesium sulphate,

0.2; dipotassium hydrogen phosphate, 2; potassium dihydrogen phosphate, 1,

and carbon source, 10.

The effect of different nitrogen sources on the enzyme production was

studied using the media containing (gA) starch, 10; sodium chloride, 5;

calcium chloride, 0.5; magnesium sulphate, 0.2; dipotassium hydrogen

phosphate, 2; potassium dihydrogen phosphate, 1, and each of the different

nitrogen sources, 10.

Starch and soya bean meal which were found to be the best carbon

and nitrogen sources respectively were used together at different

concentrations and the effects on the production were studied. The media

used contained (911) sodium chloride, 5; calcium chloride, 0.5; magnesium

sulphate, 0.2; dipotassium hydrogen phosphate, 2; potassium dihydrogen

phosphate, 1 and different concentrations of starch and soya bean meal.

The effect of using different concentrations of sodium chloride in the

medium, on alkaline protease production was studied using media containing

(911) soya bean meal, 20; starch, 20; dipotassium hydrogen phosphate, 2;

potassium dihydrogen phosphate, 1, and different concentrations of sodium

chloride.

The effect of using other salts in addition to sodium chloride was

studied. The media used for this experiment contained (gA) soya bean meal,

20; starch, 20; dipotassium hydrogen phosphate, 2; potassium dihydrogen

phosphate, 1; sodium chloride, 10, and each of the various salts, 0.5.

The effect of using calcium chloride at different levels in addition to

sodium chloride was studied by using media containing (gll) soya bean meal,

20; starch, 20; dipotassium hydrogen phosphate, 2; potassium dihydrogen

phosphate, 1; sodium chloride, 10, and different concentrations of calcium

chloride.

Age of lnoculum

The effect of age of inoculum on alkaline protease production by the

strain was studied using inocula aged 12, 24, 36 and 48 h. The production

medium used (starch-soya bean meal medium) contained (dl) soya bean

meal, 20; starch, 20; dipotassium hydrogen phosphate, 2; potassium

dihydrogen phosphate, 1; sodium chloride, 10, and calcium chloride, 0.2.

Size of inoculum

Starch-soya bean meal medium mentioned under the previous

experiment was inoculated with different volumes of 24 h grown nutrient

broth culture so that the final level of inoculum varied from 0.5-12%.

Agitation

The alkaline protease production by the unagitated culture and the

culture agitated at different rates was determined.

Incubation period

The starch-soya bean meal medium was inoculated with 24 h grown

nutrient broth culture at a level of two percent and the yield of alkaline

protease was determined after incubation for different periods.

RESULTS

Effect of growth phase

Alkaline protease production by the strain Bacillus sp. K 25, as a

function of growth at room temperature is shown in Figure 1.

Figure

Time (h)

1. Time course of growth and alkaline protease production by Bacillus sp. K 25

The production of enzyme by the strain could be noticed from the early

exponential phase of growth. The bacterium was producing only small

quantities of the enzyme in the early stages. A steady increase in production

a u l d be seen as the growth progressed from early exponential to early

stationaly phase. The maximum enzyme accumulation was seen in the early

stationay phase. Thereafter a decline in activity was observed.

Effect of temperature of incubation

The effect of temperature of incubation on the alkaline protease

production by Bacillussp. K 25 is shown in Figure 2.

I 25 30 35 40 45 50 55 60

Temperature of incubation PC)

Figure 2. Effect of temperature of incubation on alkaline protease production by Bacillus sp. K 25

The maximum alkaline protease production was seen when the culture

was incubated at 45°C.

Effect of pH

The effect of initial p H of the medium on alkaline protease production

is shown in Fgure 3.

0.4 4 I 5 6 7 8 9 10 11 12

Initial pH of medium

Figure 3. Effect of initial p H of medium on alkaline protease production by Bacillussp. K 25

The optimum initial p H of the medium for the maximum alkaline

protease production was 9.0.

Effect of carbon and nltrogen sources

Results of studies on the effect of different carbon sources (1% w/v) on

alkaline protease production by the strain is shown in Table 7.

Table 7

Effect of different carbon sources on alkaline protease production by Bacillussp. K 25

Carbon source Alkaline protease production (u mi-' It SEM)

Glucose 0.135 + 0.007

Galactose 0.120 + 0.004

Fructose 0.066 + 0.003

Lactose 0.045 + 0.001

Maltose 0.116 i 0.002

Sucrose 0.123 i 0.004

Mannose 0.060 + 0.002

Mannitol 0.057 + 0.001

Inulin 0.176 + 0.008

Dexbin 0.120 + 0.003

Starch 0.192 + 0.012

Glycerol 0.082 + 0.002 &

The best carbon source for the production was found to be starch

followed by inulin.

The results of studies on the effect of different nitrogen sources on the

alkaline protease production is shown in Table 8.

Table 8

Effect of various nitrogen sources on alkaline protease production by Bacillus sp. K 25

Nitrogen source Alkaline protease production ( u ml-' * SEM)

Ammonium sulphate 0.203 1. 0.006

Ammonium chloride 0.168 kO.011

Potassium nitrate 0.062 +0.003

Beef extract 0.820 5 0.370

Casein 2.608 k 0.084

Peptone 2.191 1.0.117

Tryptone 1.660 -t 0.042

Soya bean meal 3.028 50.126

Yeast extract 1.405 +_ 0.035

The production was better with organic nitrogen sources than with the

inorganic sources. The highest production was obtained when soya bean meal

was used as the nitrogen source.

The effect of using starch and soya bean meal at different

concentrations on the enzyme production is shown in Table 9.

Table 9

Effect of using starch and soya bean meal at different concentrations on alkaline protease production by Bacillus sp. K 25

Concentration of Concentration of Alkaline protease starch (%, wlv) soya bean meal (%, wlv) production (u ml-' f SEM)

1 1 3.054 * 0.131

1 2 4.440 + 0.231

1 3 4.213 + 0.147

2 1 3.427 + 0.082

2 2 4.650 * 0.220

2 3 4.102 k 0.172

3 1 3.690 it 0.215

3 2 4.449 + 0.186

3 3 3.714 + 0.256

The maximum production was obtained when both starch and soya

bean meal were used at 2% (w/v) level.

Effect of salts

The effect of using sodium chloride at different concentrations in the

medium is shown in Table 10.

Table 10

Effect of using different concentrations of sodium chloride on alkaline protease production by Bacillussp. K 25

Sodium chloride Alkaline protease concentration (%, w/v) production (u rnl-' + SEM)

Nil (Control) 3.631 k0.138

0.25 3.861 k0.154

0.5 4.156 k0.216

0.75 4.364 50.117

1.00 4.429 +_ 0.082

1.25 4.322 + 0.264

1.5 4.198 +0.195

The optimum level of sodium chloride for the production was found to

be 1%. With further increase in concentration a slight decline in production

was observed.

The effed of using other salts along with the sodium chloride is shown

in Table 11.

Table 11

Effect of different salts on alkaline protease production by Bacillus sp. K 25

Salts present Alkaline protease

in the medium production (U ml-' k SEM)

NaCI (1 %, wlv) only 4.495 k0.161

NaCI (I%, w/v)+ Calcium chloride (0.05%, wlv) 4.843 k0.219

NaCl (I%, w/v)+ Magnesium sulphate (0.05%, wlv) 4.587 k0.230

NaCI (I%, w/v)+ Ferric chloride (0.05%, wlv) 4.505 k0.181

NaC1 (I%, w/v)+ Zinc sulphate (0.05%, wlv) 3.444 k0.094

NaCl (I%, w/v)+ Cobalt chloride (0.05%, w/v) 1.799 k0.113

NaCI (I%, w/v)+ Manganese chloride (0.05%, wlv) 3.176 k0.141

NaCI (1%. w/v)+ Potassium chloride (0.05%, wlv) 4.592 k0.244

A slight increase in production could be observed in the presence of

calcium chloride. Presence of magnesium sulphate, femc chloride and

potassium chloride were having little or no effed on the enzyme production.

A decline in production was seen in the presence of zinc sulphate, cobalt

chloride and manganese chloride.

Calcium chloride which was found to be favouring the alkaline

protease production was tested at different concentrations. Results are shown

in Table 12.

Table 12

Effed of different concentrations of calcium chloride on alkaline protease production by Bacillussp. K 25

Calcium chloride Alkaline protease concentration in the medium production (u ml-' i- SEM)

(%, w/v)

0.02 5.006 k0.118

0.05 5.044 i- 0.283

There was no considerable difference in the yield of alkaline protease

with the use of calcium chloride in the range 0.02-0.2% (wlv). At higher

concentration (0.3%, w/v), a slight decline in production was seen.

Effect of age of inoculum

The effect of age of inoculum on the production is shown in Table 13.

Table 13

Effed of age of inoculum on alkaline protease production by Baci/us sp. K 25

1 Age of inoculum (h) Alkaline protease production (u mi-' i SEM)

The age of inoculum was found to be having little or no effect on the

production.

Enect of slze of inoculum

The effect of inoculum size on alkaline protease production by the

strain is shown in Table 14.

Table 14

Effect of inoculum size on alkaline protease production by Bacillus sp. K 25

lnoculum level (%) Alkaline protease production (u ml-' + SEMI

0.5 4.430 k 0.223

1.0 4.712 +0.164

2.0 4.875 k0.186

4.0 4.665 i0.219

8.0 4.864 i0.257

12.0 4.532 i0.208

The optimum level of inoculum for the enzyme production was found

to be 1-8%.

Elfect of agitation

The effect of agitation of culture on alkaline protease production is

shown in Table 15.

Table 15

Effect of agitating the culture on alkaline protease production

Agitation rate Alkaline protease (r.p.m) production (u ml-' * SEM) Unagitated 0.603 ~0.026

100 3.802 k0.203

'200 5.325 k0.276

300 5.562 k 0.250

In the unagitated culture alkaline protease was produced only in v e y

low level. The culture showed an increase in production with the increase in

the agitation rate. Vey high levels of alkaline protease could be achieved by

agitating the culture at 200-300 r.p.m.

Effect of period of incubation

The effect of varying the incubation period on alkaline protease

accumulation is shown in Table 16.

Table 16

Effect of incubation period on alkaline protease accumulation in the culture of Bacillus sp. K 25

incubation period (h) Alkaline protease production (u ml-' _+ SEM)

48 2.905 k0.191

72 5.429 k 0.292

96 6.631 k0.347

120 6.086 +0.181

The maximum accumulation of alkaline protease was seen when the

culture was incubated for 96 h. A slight decline in the activity was seen on

further incubation.

DISCUSSION

The influence of various factors on alkaline protease production by

Bacillus sp. K 25 was studied.

The alkaline protease production profiles of the strain as a function of

growth was examined in a complex medium, containing peptone and beef

extract. The production could be seen from the early exponential phase

onwards. It was very low during the early stages of exponential phase. The

alkaline protease production beginning from the early stages of exponential

phase has been reported only rarely (Fogarty and Griffin, 1973; Deane eta/.,

1986; Kaur eta/., 1998). A possible reason for the early secretion of protease

can be the absence of easily metabolizable carbon sources such as sugar in the

medium. It was suggested by Mc Donald and Chambers (1966) that, it was

the primary function of extracellular protease to ensure a supply of carbon for

growth rather than to supply amino acids for the synthetic process in the

absence of easily metabolizable carbon sources. In this study, the complete

absence of easily metabolizable carbon sources might have created a situation

where the peptide bonds in peptone or proteins had to be cleaved for

obtaining carbon for growth and metabolism. The observation of lag in

protease production in Pseudomonas fluorescens in presence of easily

metabolizable carbon source (Mc Keller, 1982) is supportive of this argument.

The culture of Bacillus sp. K 25 was showing a steady increase in

alkaline protease production with the progression of growth from early

exponential to early stationary phase. The production was maximum in the

early stationary phase. Reports are many, on the maximum extracellular

protease production occurring during the later stages of growth. Different

Bacillus species have been reported to be producing the maximum enzyme

during the late exponential (Atalo and Gashe, 1993), post exponential (Ikeda

et al., 1974; Kitada and Horikoshi, 1976; Debabov, 1982; Ward, 1983;

Manachini et a/., 1988) and the stationary (Durham, 1987; Durham ef a/.,

1987; Purva et dl., 1998), phases of growth. The exact reason for the

increased production of protease during the later stages of growth is not

known. A coincidence of reaching of extracellular protease production at the

maximum level with sporulation, the event occurring mainly during the later

stages of growth, has been reported by some workers (Debabov, 1982; Sinha

and Satyanarayana, 1991). The possibility for the existence of a relationship

between the triggering of protease production and sporulation, as observed by

Debabov (1982) in Bacillusspp., cannot be ruled out in this case also. Such a

relationship if any is there can be the reason for the increased production

during the later stages of growth. Detailed studies are required to anive at a

conclusion.

The optimum temperature for alkaline protease production by the

strain was found to be 45°C. Temperatures at or around 45°C have been

reported for the production by bacteria such as Bacillus sp. P-001A (Atalo and

Gashe, 1993) and Bacillus lichenifomis S40 (Sen and Satyanarayana,

1993).

The effed of initial pH of medium on alkaline protease production was

studied. The maximum production was seen at pH 8-10. The most optimum

initial pH of the medium was 9.0. Alkaline protease production using media

with alkaline pH has been reported by Honan Scientific Research Institute for

Leather Industry (1975), Kitada and Horikoshi (1976), Manachini et al.

(1988), Qiu et a/. (1990a, 1990b), Takii et a/. (1990), Sinha and

Satyanarayana (1991), Cheong et af. (1993), Sen and Satyanarayana (1993)

and Putva eta/. (1998).

Results of study on the effect of carbon source on alkaline protease

production shows starch to be the best carbon source followed by inulin.

Starch or starch hydrolysates have been reported as good carbon sources for

alkaline protease production by different Bacillus species (Emtseva, 1975;

Sinha and Satyanarayana, 1991; Sen and Satyanarayana, 1993; Ferrero

eta/., 1996; Purva eta/., 1998). Compared to starch and inulin, glucose and

other easily metabolizable carbon sources were not so good for alkaline

protease production by Bacillus sp. K 25. Sen and Satyanarayana (1993)

who studied alkaline protease production by Bacillus lichenifomis S40 have

also reported similar observation. The repressing effect of glucose on alkaline

protease production has been reported in Wbrio dginol@cus (Long et a/.,

1981) also.

Results of study on the effect of various nitrogen sources on alkaline

protease production by Bacillus sp. K 25 shows that the organic nitrogen

sources are better than the inorganic ones for the production. This

observation conforms with the earlier reports on the repressing effed of

inorganic nitrogen sources on bacterial alkaline protease production (Long

eta/., 1981; Fujiwara and Yamamoto, 1987; Giesecke et al., 1991; Sen and

Satyanarayana, 1993). The reason for the better production with organic

nitrogen sources can be supposed to be their ability to induce protease

production. The inducing effect of organic nitrogen sources on bacterial

alkaline protease production has been reported by Lasure (19801, Ferrero

eta/. (1996) and Kaur eta/. (1998). Of the various nitrogen sources tested

soya bean meal was found to be the best for production. It has been reported

as a suitable nitrogen source for the alkaline protease production by many

bacteria (Honan Scientific Research Institute of Leather Industry, 1975;

Chandrasekaran and Dhar, 1983; Nihete et a/., 1986; Na and Yu, 1988;

Takami et a/., 1989; Lee and Chang, 1990; Purva eta/., 1998).

Starch and soya bean meal found to be the best carbon and nitrogen

sources respectively, were tested together at different concentrations. The

optimal level of both these ingredients for the maximum production was found

to be 2% (wlv).

The results of study on the effect of sodium chloride show that the

presence of sodium chloride can enhance the alkaline protease production by

the strain. The optimum lwel of sodium chloride for the production was

1% w/v. The enhancing effect of sodium chloride on bacterial alkaline

protease production has been reported only rarely. Chandrasekaran and

Dhar (1983) who studied the alkaline protease production by Sb-eptomyces

moderatus NRRL 3150 have observed a beneficial effect of sodium chloride

on the production. The exact reason for the increased production in the

presence of sodium chloride is not known. But it is well-known that sodium

chloride at its optimum level can provide a conducive osmotic environment

for the growth of bacterial cells. The enhanced production of alkaline

protease in the presence of sodium chloride can be supposed to be an

outcome of such an effect.

The effect of using other salts in addition to sodium chloride was

studied. Of the different salts tested only calcium chloride was found to be

enhancing the production. Though calcium chloride is a very common

ingredient of production media for bacterial alkaline proteases, its role in

enhancing the production has not yet been studied. Salts such as magnesium

sulphate, ferric chloride and potassium chloride were found to be having no

effect on the production. The other salts tested, zinc sulphate, cobalt chloride

and manganese chloride were having inhibitory effects.

The effect of using calcium chloride at different concentrations was also

studied. 0.02-0.2% (wlv) level of it was found to be favouring the production.

With the use of higher concentration (0.3% w/v) a slight decline in production

could be noticed. The calcium chloride concentrations used by earlier workers

in the production media for different bacteria range from 0.006% as for a

strain of Bacillus subtihs (Massuco et al., 1980) to 0.3% as for a strain of

Baci//us pumi/us (Honan Scientific Research Institute of Leather Indushy,

1975). In fad the level of calcium chloride required in the medium depends

not only upon the bacterium used, but also upon the presence of other salts

and their concentrations in the medium. This may account for the wide

differences in calcium chloride concentration requirements by the different

bacterial SmF systems.

Studies showed that the alkaline protease production by Bacillus sp.

K 25 was independent of the age of inoculum. Similar observations have

been reported by Miusawa et d. (1969) and Sen and Satyanarayana (1993)

who studied the alkaline protease production by Streptomyces rectus var.

proteolytcus and Bacillus lichenifomis S40 respectively.

The optimum level of inoculum for alkaline protease production by the

strain was found to be 1-8%. This observation is in conformity with the

reports by Sinha and Satyanarayana (1991), Sen and Satyanarayana (1993)

and Gajju et a/. (1996) who studied the alkaline protease production by

Bacillus lichenifonnis N3, Bacillus lichenifomis S40 and Bacillus coagulans

PB 77 respectively.

Agitation of culture was found to be essential for the high production of

alkaline protease by Bacillus sp. K 25. The unagitated culture of this

bacterium was characterized by diminished growth due to pellicle fonation

over the surface of the culture. Only very low levels of alkaline protease was

produced in the unagitated culture. The culture showed an increase in

production with the increase in the agitation rate. Very high yield of alkaline

protease was obtained by agitating the culture at 200-300 r.p.m. Similar

observations have been made on the submerged fermentation systems for the

production of alkaline proteases by Bacillus lichenifomis S40 (Sen and

Satyanarayana, 1993), Bacillussp. (Takami etal., 1989) and Bacillus sp. IS-3

(Purva eta/., 1998).

The incubation period required for obtaining the maximum yield, with

starch-soya bean meal medium providing the optimum conditions was found

to be 96 h. Requirement for such a long incubation period for the maximum

alkaline protease production is not so common for bacterial SmF systems.

The long incubation period observed in the present study can be supposed to

be due to the presence in high concentrations of carbon and nitrogen sources

which are slowly metabolizable. Gajju et a/. (1996) have reported an

incubation period of 96 h for the maximum accumulation of alkaline protease

by Bacillus coagulans PB 77 in a medium containing casein.

As a result of optimization studies, the yield of alkaline protease by the

strain could be increased approximately six-fold. After optimization the

activity was more than 6.0 u ml-'. A precise comparison of the yield obtained

by Bacillus sp. K 25 with the yield in most of the earlier reports is difficult,

because different methodologies have been adopted by different workers for

the assay of alkaline proteases. The composition and pH of the buffer systems

used for assay, temperature of incubation, substrate etc. are different in

different works. Moreover the unit definitions followed by different workers for

expressing the activity were also different. So the yield of alkaline protease

obtained by Bacillus sp. K 25 could be compared only with a few earlier

reports where the methodology of assay and unit definitions were similar or

comparable. The yield by Bacillus sp. K 25 could be found to be better than

the yield reported by Chandrasekaran and Dhar (1983), Tsujibo eta/. (1990),

Sinha and Satyanarayana (1991), Sen and Satyanarayana (1993) and

Gessesse and Gashe (1997) in different bacteria.

Section B

OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF ALKALINE PROTEASE

BY BACILLUS PUMlLUS K242 BY THE SOLID STATE FERMENTATION METHOD

MATERIALS AND METHODS

Various factors influencing the production of alkaline protease by

Bacilluspumilus K 242, by solid state fermentation were studied. Except for

the alterations or modifications described under each experiment, the

procedures followed for performing SSF were same as in the preliminaty SSF

studies described under section B of chapter 3.

Solid substrate

Suitability of different commercially available substrates such as wheat

bran, rice bran, green gram bran, black gram bran, coconut oil cake and

ground nut oil cake for use in SSF was studied. SSF was performed using

these substrates in place of wheat bran and the yield of alkaline protease was

determined.

Particle size

Wheat bran which was found to be the most suitable commercially

available solid substrate was sieved and graded based on the particle size.

SSF was performed using the graded wheat bran and the alkaline protease

yield was determined.

Moisture level, Temperature of incubation and Incubation period

Solid substrate media with different moisture levels were prepared

using different volumes of salt solution for moistening wheat bran (particle size

500-710 p) and the initial moisture content of the media was determined.

The yield of alkaline protease was determined after incubation of inoculated

media for different periods at different temperatures.

The effect of pH on alkaline protease production was studied

performing SSF using moistening solution adjusted to different pH (7.510.5).

The moistening solution used contained (dl) dipotassium hydrogen

phosphate, 2; potassium dihydrogen phosphate, 1; magnesium sulphate, 0.1;

calcium chloride, 0.1 and zinc sulphate, 0.01.

Supplementation with carbon sources

Effect of supplementation of solid substrate medium with different

carbon sources on the alkaline protease production was studied. Different

carbon sources were incorporated into the moistening solution so that their

final levels in the moistened solid substrate media were 1, 2 and 3% (w/w). In

addition to the various carbon sources at different concentrations, the

moistening solution contained (d) dipotassium hydrogen phosphate, 2;

potassium dihydrogen phosphate, 1; magnesium sulphate, 0.1; calcium

chloride, 0.1 and zinc sulphate, 0.01. The pH of the moistening solution was

adjusted to 9.0.

Supplementation with nitrogen sources

Effect of supplementation of solid substrate medium with different

nitrogen sources on alkaline protease production was studied. Different

nitrogen sources were incorporated into moistening solution so that their final

levels in the moistened solid substrate media were 1 and 2% (wlw).

In addition to the various nitrogen sources at different concentrations, the

moistening solution contained (911) dipotassium hydrogen phosphate, 2;

potassium dihydrogen phosphate, 1; magnesium sulphate, 0.1; calcium

chloride, 0.1; zinc sulphate, 0.01 and glucose at a level so as to get the final

concentration of 2% (wlw) in the moistened substrate. pH of the moistening

solution was adjusted to 9.0.

Supplementation with sodium chloride

Effect of supplementing the moistening solution with different

concentrations of sodium chloride, on alkaline protease production was

studied. The moistening solution used contained (gA) dipotassium hydrogen

phosphate, 2; potassium dihydrogen phosphate, 1; magnesium sulphate, 0.1;

calcium chloride, 0.1; zinc sulphate, 0.01, glucose at a level so as to get the

final concentration of 2% (wlw) in the moistened substrate and the different

concentrations (0.1,0.2,0.5 and 1% w/v) of sodium chloride.

Age of inoculum

Effect of age of inoculum on alkaline protease production was studied

using inocula aged 24,48, 72 and 96 h.

Size of inoculum

Effect of size of inoculum on alkaline protease production was studied

vaying the inoculum Levels from 2 to 12% v/w of the moistened substrate.

Medium volume:Flask volume

In order to study the effect of varying the ratio medium vo1ume:flask

volume on the yield, SSF was performed with the different volumes of

moistened medium taken in 250 ml Erlenmeyer flasks.

RESULTS

Effect of different solid substrates

Results of studies on the alkaline protease production by Bacillus

pumilus K 242 by SSF using different commercially available substrates are

given in Table 17.

Table 17

Alkaline protease production by Bacilluspumilus K 242 by SSF, using different commercially available substrates

I Substrates Alkaline protease production (u/g DBB * SEM) ] Wheat bran 50.60 + 2.85'

Rice bran 28.38 + 2.27

Green gram bran 37.72 + 1.03

Black gram bran 28.31 _+ 2.37

Coconut oil cake 8.46 * 0.39

Groundnut oil cake 15.66 * 1.07 'As obtained in the earlier experiment.

Of the various substrates tested wheat bran was found to be the best.

Effect of size of particles of wheat bran

Effed of using wheat bran of different particle size on alkaline protease

1 production is shown in Table 18.

Table 18 Effed of using wheat bran of different particle size on alkaline protease

production by Bacilluspumilus K 242

Particle size of Alkaline protease wheat bran (p) production (ulg DBB * SEM )

Ungraded 50.60 k2.85'

< 100 8.65 k 0.31

100-250 54.12 k2.65

-As obtained in the earlier experiment.

The maximum production was obtained with the use of wheat bran of

particle size 500-710 p. Production was very low with the use of wheat bran

of particle size < 100 p.

Effect of moisture level, temperature of incubation and incubation period

The results of studies on the influence of moisture level, temperature of

incubation and incubation period on alkaline protease production by the

strain are shown in Table 19.

Table 19 Effect of moisture level, temperature of incubation and incubation period on

alkaline protease production by Bacilluspumilus K 242

Percentage moisture level*

60.8

(1:1.3)

64

(1:1.5)

70

(1:2)

Temperature of incubation (OC)

* Wheat bran:moistening solution ratios are given in brackets.

30

37

45

30

37

45

30

37

45

Alkaline protease production (u/g DBB + SEM) when incubated for different periods

28.18 + 0.83

41.15 + 2.45

35.24 + 2.51

37.26 + 2.80

49.68 + 3.12

42.80 + 3.24

40.01 + 3.68

55.40 + 3.72

38.46 + 2.09

96 h 48 h 72 h

43.18 + 2.53

55.54 + 3.44

40.80 + 2.92

48.88 + 3.88

65.02 + 4.87

47.16 + 3.53

45.65 + 0.90

62.14 + 3.10

44.82 + 2.12

51.10 + 3.98

48.32 + 3.90

30.73 k 3.08

58.14 + 4.05

59.84 + 2.83

34.68 + 1.70

46.57 + 1.34

51.22 k 3.66

33.43 + 2.34

The highest production was seen in solid substrate medium with 64%

initial moisture level, after incubation for 72 h at 37°C.

Effect of pH of moistening solution

The effect of pH of the moistening solution on alkaline protease

production is shown in Figure 4.

pH of moistening solution

Figure 4. Effect of pH of moistening solution on alkaline protease production by Bacil/uspumilus K 242

The highest production was obtained when the moistening solution of

pH 9.0 was used.

Effect of extra carbon sources

Effect of supplementation of the wheat bran medium with different

carbon sources, on alkaline protease production is shown in Table 20.

Table 20

Glucose

Lactose

Mannitol

Maltose

Starch

Dextrin

Galactose

Sucrose

Effect of supplementation of wheat bran medium with different carbon sources on alkaline protease production by Bacillus pumilus K 242

Fructose 61.77 + 0.75 65.58 + 3.28 I N. D. - Not determined

Carbon source

Alkaline protease production in the unsupplemented medium, as obtained in the earlier experiment: 64.12 f 4.07 u/g DBB

Alkaline protease production (u/g DBB 4 SEM) at different concentrations (%, wlw of moistened substrate) of extra carbon sources

1 2 3

A considerable increase in production could be noticed by

supplementation of wheat bran medium with many of the carbon sources

tested especially when used at their optimal levels. Carbon sources such as

glucose, dextrin and sucrose at their optimal level of 2% (w/w of the

moistened substrate) were enhancing the production considerably. Maltose

and galactose when used at a level of 1% were also enhancing the

production. The other carbon sources when tested upto a level of 2% were

having little or no effect on the production. Of the different carbon source

supplements tested, glucose at 2% w/w of the moistened substrate was found

to be the best.

Effect of extra nitrogen sources

Effect of supplementation of solid substrate medium with different

nitrogen sources on alkaline protease production is shown in Table 21.

Table 21

Effect of supplementation of solid substrate medium with different nitrogen sources on alkaline protease production by Bacilluspurnilus K 242

Alkaline protease production Nitrogen (ulg DBB + SEM) at different concentrations source (%, W/W of moistened substrate) of extra nitrogen sources

1 2 Soya bean meal 90.76 1t 5.43 78.06 + 5.10

Peptone 93.76+ 6.18 78.24 + 4.41

Casein 88.48+ 5.83 80.31 it 4.13

Yeast extract

Beef extract

Malt extract

Ammonium sulphate 82.08 + 4.66 72.41 i 5.41

Potassium nitrate 75.68 + 1.49 66.14 + 3.51

Diammoniurn hydrogen 91.71 i 5.82 81.86 + 4.04 I phosphate Alkaline protease production in the medium unsupplemented with extra nitrogen source - as obtained in the earlier experiment is 88.16 + 4.99 ulg DBB.

None of the nitrogen sources tested was showing an enhancing effect

on the production. At 1% (w/w of moistened substrate) level, all the nitrogen

sources except potassium nitrate were having little or no effect on the

production. At 2% level all the nitrogen sources were inhibitoty.

Effect of sodium chloride

Effects of supplementation of moistening solution with sodium chloride

at different concentrations are shown in Table 22.

Table 22

Effect of incorporating sodium chloride at different concentrations into the moistening solution, on alkaline protease production

by BaciIIus pumilus K 242

Concentration (%, w/v) of Alkaline protease sodium chloride in the

moistening solution production (u/g DBB * SEM)

Nil 87.03 k3.98

0.10 92.07 k3.80

0.20 94.73 + 5.03

0.50 100.51 k5.21

1.00 87.38 k6.19

Incorporation of sodium chloride into the moistening solution was

found to be enhancing the production slightly. The optimum concentration of

sodium chloride for the production was 0.5% (wlv).

Effect of age of lnoculurn

Results of study on the effect of age of inoculum on alkaline protease

production is given in Table 23.

Table 23

Effect of age of inoculum on alkaline protease production by Bacilus pumilus K 242

- Age of Alkaline protease

inoculum (h) production (u/g DBB rt SEM) 24 98.34 k 7.06

48 100.37 k 6.52

72 105.65 k 6.41

96 96.71 k4.29

The age of inoculum was found to be having little or no effect on the

production.

Effect of size of inoculurn

The effect of size of inoculum on the enzyme production is shown in

Table 24.

Table 24

Effect of inoculum size on alkaline protease production by Bacillus pumilus K 242

Sue of inoculum (%, v/w of Alkaline protease 7

moistened substrate) production (ulg DBB * SEM) 2 104.17 rt6.22

4 102.43 k 7.35

8 107.04 k5.84

12 95.78 k5.50

The inoculum at a level of 2-8% v/w of moistened substrate was found

to be optimum for obtaining the high yield.

Effect of the ratio, medium volume:flask volume

Effect of medium volume:flask volume on alkaline protease production

is shown in Table 25.

Table 25

Effect of varying the ratio medium volume:flask volume on alkaline protease production by Bacilluspumilus K 242

Ratio of medium volume to Alkaline protease flask volume production (u/g DBB + SEM)

1:14.70 96.47 k3.53

1:5.88 101.55 + 5.30

1:2.94 104.38 k4.88

1:1.96 109.93 + 7.02

1:1.47 120.25 k6.80

The maximum yield of alkaline protease was obtained at the lowest

ratio of medium volume to flask volume.

DISCUSSION

With the objective of obtaining maximum yield of alkaline protease by

solid state fermentation using Bacillus pumilus K 242, various factors

influencing the production were studied.

Of the different commercially available solid substrates tested, such as

wheat bran, rice bran, green gram bran, black gram bran, coconut oil cake,

and groundnut oil cake, wheat bran was found to be the most suitable

followed by green gram bran. The production with coconut and groundnut oil

cakes was found to be v e y low. With rice bran and black gram bran the yield

was intermediate. Earlier workers who developed successful bacterial SSF

systems for the production of proteases, also have reported the use of wheat

bran as the substrate for solid state fermentation (Chakraborty and Srinivasan,

1993; George et a/., 1995; Sen, 1995).

Wheat bran with different particle sizes were tested for the suitability for

SSF process. The highest production was obtained when the wheat bran with

particle size 500-710 p was used. The wheat bran with particle sue 250-500 p

was also good. The production was v e y low with the use of bran of particle

size below 100 p. The difference in production with the use of different grades

of wheat bran can be due to the differences in their nutritional value, porosity,

moisture holding capacity etc. Bran of smallest particle size though

nutritionally better often cause stickiness of the medium after autoclaving.

This can in turn lead to the poor growth of the bacterium resulting in the low

yield. Wheat bran of largest particle size though do not cause stickiness after

autoclaving may not have much nutritional value and moisture holding

capacity. The wheat bran of moderate particle sue (500-710 p) can be

supposed to be satisfying the growth requirements by the strain.

Moisture content of the medium, incubation time and incubation

temperature are three important factors that can influence the enzyme

production by SSF. Since the influences of these fadors are interdependent

the effects of these fadors were studied simultaneously. The maximum

alkaline protease production by Baci/us pumilus K 242 was obtained in the

medium with 64% initial moisture level, after incubation for 72 h at 37°C.

Production in the medium with 70% moisture level after incubation for 72 h at

37°C was also good. At all temperatures, i.e., 30, 37 and 45"C, the optimum

moisture level for the production was found to be 64%. Moisture level is a

factor determining the level of available water, solubiliiation of nutrients, gas

exchange and oxygen transfer in the solid substrate medium. Since different

bacteria are requiring these factors at different levels, their moisture level

requirements will also be different. Chakmbotty and Srinivasan (1993) have

reported that Pseudomonas sp. B45 was producing alkaline protease

maximally in medium with initial moisture level 74%, incubated at 37°C for

120 h. George ef a/. (1995) who studied protease production by Bacillus

arnyloliquefaciens ATCC 23844 obtained the maximum yield when wheat

bran moistened at 1:2 ratio was used. The incubation temperature and period

were 37°C and 24 h respectively. In the present study the incubation period

required for obtaining the maximum yield at 37 and 45°C was 72 h. A longer

incubation period, i.e., 96 h was required for obtaining the maximum yield

when incubated at 30°C.

Although pH is a critical factor that can influence the enzyme

production, monitoring and control of pH during the SSF process is not

usually attempted (Lonsane et d, 1985). Good buffering capacity of the

sub&ates used in SSF generally help in eliminating the need for pH control

during the process. This advantage was exploited in this study also.

No attempts were made to control the pH during the fermentation process.

The effect of pH on production was studied by using the moistening solutions

with different pH. The maximum production was obtained with the use of

moistening solution having pH 9.0.

The effect of supplementing solid substrate medium with different

carbon and nitrogen sources were studied. A considerable increase in

production could be noticed by supplementation of wheat bran medium with

many of the carbon sources tested, especially at their optimal levels. While

the optimal level of glucose, dextrin and sucrose was 2% (wlw of the

moistened substrate), it was 1% for maltose and galactose. Of the different

carbon sources tested glucose at 2% (wlw of the moistened substrate) level

was found to be the best as supplement. These observations on the effects of

extra carbon sources were different from the observations reported by earlier

workers in this field. Chakraborty and Srinivasan (1993), George et a/. (1995)

and Sen (1995) who studied on the bacterial SSF systems for protease

production have reported the inability of glucose and some other carbon

sources to improve the yield. The differences in the metabolic characters of

different bacteria may not be the sole reason for such a difference in

observations. It has to be taken into account that the methodology followed in

the present study for determining the effect of carbon source was different

from that followed in the earlier reports. In this study, for determining the

effect of extra carbon sources, they were incorporated into moistening

solution, instead of adding them directly into wheat bran. Due to this reason

pH could be easily adjusted thereafter. On the other hand, if these carbon

sources were added directly into wheat bran, there would not have been such

a facility for the adjustment of pH. In such cases the addition of extra carbon

sources into wheat bran may possibly result in the solid substrate medium with

unfavourable pH. The SSF performed with media having unfavoumble pH

can definitely result in the decreased enzyme production. But in this study,

such a situation could be avoided by adjusting the pH of the moistening

solution after the addition of carbon sources.

Of the different nitrogen sources tested none was found to be

enhancing the production. Though most of them were having little or no

effed on production at 1% (w/w of the moistened substrate) level, they were

inhibitory at 2% level. Potassium nitrate was inhibitory even at the 1% level.

Similar observations have been reported by earlier workers also. Chakmbotty

and Srinivasan (1993) who studied the alkaline protease production by

B. amyloliquefaciens ATCC 23844, have reported a decrease in production

on supplementation of wheat bran with extra nitrogen sources. Sen (1995)

has reported that supplementation of wheat bran medium with extra nitrogen

sources was ineffective in improving the yield of alkaline protease by Bacillus

licheniformis S40. Results of the present study indicate that the nitrogen

sources in the unsupplemented medium itself is sufficient to support the

growth and to induce the enzyme production by the strain. This again points

to the universal suitability of wheat bran as the substrate for the SSF

processes.

The effed of supplementing the moistening solution with different

concentrations of sodium chloride, on alkaline protease production was

studied. Results indicate that sodium chloride can be incorporated into the

moistening solution at a level 0.5% (wlv), to improve the yield of alkaline

protease. Such an observation has not been reported by the earlier workers in

this field.

The effects of both age and sue of inoculum on the protease

production were studied. The age of inoculum was found to be having liffle or

no effect on production. The size of inoculum for obtaining the maximum

yield was 23% (vlw of moistened substrate). At a higher level, i.e., 12% there

was a slight decrease in production. The size of inoculum used for the alkaline

protease production of Bacillus lichenifomis 540 has been reported to be

10% (vlw) (Sen, 1995).

The effect of varying the ratio, medium volume:flask volume was also

studied. It was interesting to observe that the activity was increasing with the

use of larger volumes of moistened medium in the conical flask. The highest

activity was obtained at the lowest ratio tested, i.e., 1:1.47. At this ratio the

moistened solid substrate medium in the flask was having a height of 4 cm

and only a liffle air space was left over it. Chakraborty and Srinivasan (1993)

who studied the effect of medium volume:flask volume on alkaline protease

production by Pseudomonas sp. B45, have observed that there was a

decrease in production with the decrease in the ratio. This difference in the

obse~ations can be ascribed to the differences in the physiological and

metabolic characters of the two bacteria. The comparatively higher production

obtained by BaciJJus pumilus K 242 on using thicker layer of'solid substrate

medium, is of much techno-economic importance. Results indicate that SSF

for the large scale production of alkaline protease by B. pumilus K 242 can be

performed in containers leaving minimum air space over the medium. This

can facilitate the maximum utilization of space in the containers and will be

helpful in reducing the cost of production to some extent.

As a result of optimization of conditions, the alkaline protease

production by Bacilluspumilus K 242 could be increased more than two-fold,

resulting in the production of 120 u of alkaline protease per gram dry bacterial

bran. A comparison of the yield obtained by B. pumilus K 242 with the yields

reported in other bacterial SSF systems is difficult, because the assay

procedures and the unit activity definitions followed in these studies were

different. However taking into account the high yielding nature of B. pumilus

K 242 and the ease and economy for the enzyme production, the strain can

be suggested as suitable for alkaline protease production in a large scale. With

the use of specially designed fennentors under better process control, it may

be possible to improve the yield further.