Chapter OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF ALKALINE PROTEASES

Transcription

1 Chapter OPTIMIZATION OF CONDITIONS FOR THE PRODUCTION OF ALKALINE PROTEASES

2 ~th 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.

3 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.

4 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

5 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 %. 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.

6 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 auld 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.

7 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 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.

8 Effect of ph The effect of initial ph of the medium on alkaline protease production is shown in Fgure I Initial ph of medium Figure 3. Effect of initial ph of medium on alkaline protease production by Bacillussp. K 25 The optimum initial ph of the medium for the maximum alkaline protease production was 9.0.

9 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 Galactose Fructose Lactose Maltose i Sucrose i Mannose Mannitol Inulin Dexbin Starch Glycerol The best carbon source for the production was found to be starch followed by inulin.

10 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 Ammonium chloride ko.011 Potassium nitrate Beef extract Casein k Peptone Tryptone t Soya bean meal Yeast extract _ 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.

11 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) * * k it The maximum production was obtained when both starch and soya bean meal were used at 2% (w/v) level.

12 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 concentration (%, w/v) Alkaline protease production (u rnl-' + SEM) Nil (Control) k k k _ 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.

13 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 in the medium Alkaline protease production (U ml-' k SEM) NaCI (1 %, wlv) only k0.161 NaCI (I%, w/v)+ Calcium chloride (0.05%, wlv) k0.219 NaCl (I%, w/v)+ Magnesium sulphate (0.05%, wlv) k0.230 NaCI (I%, w/v)+ Ferric chloride (0.05%, wlv) k0.181 NaC1 (I%, w/v)+ Zinc sulphate (0.05%, wlv) k0.094 NaCl (I%, w/v)+ Cobalt chloride (0.05%, w/v) k0.113 NaCI (I%, w/v)+ Manganese chloride (0.05%, wlv) k0.141 NaCI (1%. w/v)+ Potassium chloride (0.05%, wlv) 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.

14 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 concentration in the medium (%, w/v) Alkaline protease production (u ml-' i- SEM) k i There was no considerable difference in the yield of alkaline protease with the use of calcium chloride in the range % (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)

15 production. The age of inoculum was found to be having little or no effect on the 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 k k i i i0.208 to be 1-8%. The optimum level of inoculum for the enzyme production was found

16 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 (r.p.m) Alkaline protease production (u ml-' * SEM) Unagitated ~ k0.203 ' k k In the unagitated culture alkaline protease was produced only in vey 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 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) k k k

17 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.

18 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).

19 The effed of initial ph of medium on alkaline protease production was studied. The maximum production was seen at ph 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

20 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

21 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 % (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

22 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 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).

23 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.

24 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.

25 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 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 ( ). 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, 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, The ph of the moistening solution was adjusted to 9.0.

26 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.

27 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. Effect of different solid substrates RESULTS 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 ' Rice bran Green gram bran Black gram bran _ Coconut oil cake 8.46 * 0.39 Groundnut oil cake * 1.07 'As obtained in the earlier experiment. Of the various substrates tested wheat bran was found to be the best.

28 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 k2.85' < k k2.65 -As obtained in the earlier experiment. The maximum production was obtained with the use of wheat bran of particle size 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.

29 Percentage moisture level* Table 19 Effect of moisture level, temperature of incubation and incubation period on alkaline protease production by Bacilluspumilus K 242 Temperature of incubation (OC) Alkaline protease production (u/g DBB + SEM) when incubated for different periods 48 h 72 h 96 h 60.8 (1:1.3) (1:1.5) (1:2) * Wheat bran:moistening solution ratios are given in brackets k k

30 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.

31 Carbon source Table 20 Effect of supplementation of wheat bran medium with different carbon sources on alkaline protease production by Bacillus pumilus K 242 Alkaline protease production (u/g DBB 4 SEM) at different concentrations (%, wlw of moistened substrate) of extra carbon sources Glucose Lactose Mannitol Maltose Starch Dextrin Galactose Sucrose Fructose I N. D. - Not determined Alkaline protease production in the unsupplemented medium, as obtained in the earlier experiment: f 4.07 u/g DBB

32 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 Nitrogen Alkaline protease production (ulg DBB + SEM) at different concentrations source (%, W/W of moistened substrate) of extra nitrogen sources 1 2 Soya bean meal t Peptone Casein it 4.13 Yeast extract Beef extract Malt extract Ammonium sulphate i 5.41 Potassium nitrate Diammoniurn hydrogen i I phosphate Alkaline protease production in the medium unsupplemented with extra nitrogen source - as obtained in the earlier experiment is ulg DBB.

33 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 sodium chloride in the moistening solution Alkaline protease production (u/g DBB * SEM) Nil k k k 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).

34 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 inoculum (h) Alkaline protease production (u/g DBB rt SEM) k k k k 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 moistened substrate) production (ulg DBB * SEM) rt k k k5.50 7

35 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 flask volume Alkaline protease production (u/g DBB + SEM) 1: k3.53 1: : k4.88 1: : 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,

36 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 vey 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 p was used. The wheat bran with particle sue p was also good. The production was vey 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 ( 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

37 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 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

38 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

39 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.

40 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.

41 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.