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 agroindustrial 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 agroindustrial 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, Agaragar, 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 24 th 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 CaCl 2 and 10 mm CaCl 2. 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) WBFP (Wheat Bran Fine Particles) RBCP (Rice Bran Coarse Particles) GGH (Green Gram Husk) GGFP (Green Gram Fine Particles) BGCP (Black Gram Coarse Particles) RGH (Red Gram Husk) RGFP (Red Gram Fine Particles) PPCP (Pigeon Pea Coarse Particles) WBCP (Wheat Bran Coarse Particles) RB (Rice Bran) RBFP (Rice Bran Fine Particles); GGCP (Green Gram Coarse Particles) BGH (Black Gram Husk) BGFP (Black gram Fine Particles) RGCP (Red Gram Coarse Particles) PPH (Pigeon Pea Husk) 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 48 th 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 48 th 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, MgSO 4, KH 2 PO 4 and CaCl 2 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 CaCl 2 ). 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.