Results and Discussion

Transcription

1 4.0. RESULTS AND DISCUSSION: The results obtained during the course of present study are presented and discussed in this chapter. This part of the thesis elucidates isolation of microbes from different habitats, screening for amylase activity, identification of the isolate by Bergey s manual of Bacteriology and ribotyping, media optimization for amylase production in submerged and solid state fermentation. In addition, purification and characterization of the amylase and its application potentials are discussed ISOLATION AND SCREENING FOR AMYLASE PRODUCING ORGANISMS Soil samples from different exotic environments were collected, and used for isolation of bacterial strains producing amylase enzyme. One gram of soil was suspended in sterile distilled water and mixed thoroughly for one hour at room temperature before using for isolation studies. Sterile 1 % Potato dextrose starch agar plates were prepared and incubated at 4 o C for 15 min to solidify the agar solution. The plates were brought to room temperature and spread with 0.1 ml of soil solution under sterile conditions. These plates were incubated at 30 o C in an incubator. After 24 hrs of incubation, the plates were checked for microbial colonies with clear zone of hydrolysis. More than 30 microbial strains from different plates were selected and grown in the starch containing agar slants. Each isolate was further confirmed for its production pattern using 1 % Potato dextrose starch agar plates. Depending on the zone of clearance, 17 strains were further screened and among them one colony which showed a very clean zone was selected for further studies and designated as MK CHARACTERIZATION OF THE ISOLATE MK 07 The strain MK 07 was characterized for various physiological properties. The results indicated that this strain was gram negative in nature and produces endospores. Colonies are initially white, quickly Murali Krishna. CH Page 62

2 becoming black with conidial production and growth produced radial fissures in the agar. The growth pattern (colony morphology) on the agar medium was observed to be round with mycelial flower like, rough surface and opaque density (Table 4.1.1). On starch agar plates, the strain produced clear hydrolysis zone. The isolate growth was studied at different temperatures (4 C to 65 C) and ph (2 to12) environments. It was noticed that growth was seen in the temperature range of 20 to 35 C and in the ph range of 5 to 6.5 only with optimum at 30 o C and ph of 5.0. Further, the growth pattern of the selected strain was studied in the presence of different concentrations of Sodium Chloride. It was found that growth was seen up to 8.5 % Sodium Chloride concentration (Table 4.1.2). The biochemical characteristics of the strain and ability to produce acid using carbohydrates were investigated according to Bergey s manual of Bacteriology and is presented in Tables (4.1.3) and (4.1.4), respectively. Based on the studies the selected strain was identified as Aspergillus niger. Murali Krishna. CH Page 63

3 TABLE MORPHOLOGICAL CHARACTERISTICS OF THE MK 07 TESTS RESULTS Colony Morphology MK 07 Configuration Margin Elevations Surface Density Round Wavy Convex Rough and Black Powdery Pigments - Gram s Reaction Cell Shape Size Arrangement Negative Mycelial Long Chain/Multiple mycelial Spore(s) Endospore + Position Shape Terminal Oval Sporangia Bulging + Capsule Motility Fluorescence (UV) Murali Krishna. CH Page 64

4 TABLE PHYSIOLOGICAL CHARACTERISTICS OF THE ASPERGILLIS STRAIN MK 07 TESTS RESULTS Growth at Temperatures MK 07 4 C - 10 C - 15 C + 25 C + 30 C + 37 C + 42 C + 45 C + 55 C ± 65 C - Growth at ph ph ph ph ph ph 9.0 ph 11.0 Murali Krishna. CH Page 65

5 Growth on Sodium Chloride (%) 2.5 % 5.0 % % + 8.5% Growth under Anaerobic Condition BIOCHEMICAL CHARACTERISTICS OF THE STRAIN MK 07 TESTS RESULTS Growth on Mac Conkey Agar + Indole Test - Methyl Red Test - Voges Proskauer Test - Gas Production From Glucose + Citrate Utilization + Casein Hydrolysis _ Starch Hydrolysis + Gelatin Hydrolysis + Urea Hydrolysis - Murali Krishna. CH Page 66

6 Nitrate Reduction - Nitrite Reduction - H2S Production - Cytochrome Oxidase + Catalase Test + Oxidation/Fermentation/Negative (O/F/-) Arginine dihydrolase Lysine decarboxylase - Ornithine decarboxylase - Table Acid Production from Carbohydrates by the Strain MK 07 TESTS RESULTS Adonitol - Arabinose _ Cellobiose + Dextrose + Dulcitol + Fructose + Galactose + Inositol _ Murali Krishna. CH Page 67

7 Inulin + Lactose - Maltose + Mannitol + Mannose Melibiose Raffinose _ Rhamnose - Salicin + Sorbitol Sucrose Trehalose Xylose 4.3. OPTIMIZATION OF SUBMERGED FERMENTATION CONDITIONS FOR AMYLASE PRODUCTION Any product production by biological system mainly depends on genetic nature of the organism, fermentation medium components and their concentration, physiological growth conditions and interactive influence of all above factors. Hence, medium development (formulation) is an essential prerequisite to get higher productivity (Pandey et al, 2000). In fact, several authors reported increased productivity values of microbial enzymes under optimized environment (Prakasham et al, 2005). In general, ideal production medium must meet as many criteria as possible according to Stanbury et al, Murali Krishna. CH Page 68

8 (1997). Therefore, in the preliminary stage, it was planned to develop/formulate a suitable production medium for amylase production from Aspergillus niger MK 07 by studying the effect of various culture factors on amylase production in a conventional methodology followed by optimization of nutritional parameters by Plackett-Burman (PB) design. Initially, the following major physiological fermentation parameters which influence the production of amylase by Aspergillus niger MK 07 were investigated by conventional methodology while the selection of carbon and nitrogen sources were performed using PB design and the composition of the production media used was Sucrose 30 g/l, Magnesium sulphate 1 g/l, Potassium chloride 1 g/l, Ferrous sulphate 0.05 g/l, Peptone 5 g/l, Potassium phosphate 1 g/l, Corn steep liquor 10 g/l and to the media added 1000 ml of distilled water and ph was adjusted to 5.0 and the media was sterilized in an autoclave for 15 min at 121 o C. The media was inoculated with a loop-full of spore suspension of Aspergillus niger isolated and then incubated in 30 o C at 200 RPM for 96 h. The media was centrifuged at 5000 RPM for 15 min and the crude enzyme isolate was used for amylase analysis. Further studies were carried out with ph 5.0 and 2 % (v/ v) inoculum, which was grown overnight by incubating at 200 rpm at 30 o C. The parameters were optimized in such a fashion that one parameter optimized was used for subsequent experiments. The following Parameters were optimized: Effect of Incubation time Effect of substrates Effect of Temperature Effect of ph Effect of inoculum level Effect of age of inoculum Effect of RPM Screening of Carbon and Nitrogen sources Screening of Initial moisture Content. Murali Krishna. CH Page 69

9 ROLE OF INCUBATION TIME Maximum enzyme production could be obtained only after a certain incubation time which allows the culture to grow at a study state (Pandey et al, 2000).Enzyme production of each strain is based on the specific growth rate of the strain. Growth rate and enzyme synthesis of the culture are the two main characteristics which are mainly influenced by incubation time (Ellaiah et al 2002).The results of the present experiment reveals that the isolated strain MK 07 when incubated for a period of 120 h the strain showed an increase in amylase activity till a period of 72 h and upon further increase in incubation period the growth started declining. The maximum amylase activity by the isolated Aspergillus niger strain was observed at 72 h and the maximum amylase activity obtained was 55 U/ml. Samples were collected at regular intervals of 12 h periods. The growth pattern revealed an increase in enzyme production from 12 h to 72 h and on further incubation the amylase production declined indicating 72 h as optimal incubation period for the isolated strain. Kathiresan et al, (2005) with strain Penicillium reported similar kind of enzyme production pattern with increase in enzyme production till a certain period of 96 h and on further incubation, enzyme production decreased. Maximum amylase production obtained with that strain at 96 h was 136 U/ml. figure and table reveals the amylase enzyme production pattern over a period of 120 h by the isolated Aspergillus niger MK 07 strain. Maryam et al, (2010) also made similar kind of observation stating that 72 h was the optimal incubation time for Bacillus sp.kr-8104 and on further incubation no significant difference in enzyme production was observed. Anto et al, (2006) reported that the ideal incubation time for maximum enzyme production by Bacillus cereus MTCC 1305 was 72 h and the yield of enzyme decreased with further incubation. Murali Krishna. CH Page 70

10 INFLUENCE OF TEMPERATURE Temperature is one of the most important parameter to be optimized for maximum enzyme production. Optimum temperature for maximum enzyme production depends on the characteristics of the strain (Adinarayana et al, 2005). In submerged and SSF fermentation, temperature plays a very important role in enzymatic synthesis (Lonsane et al 1990). The isolated Aspergillus strain was tested in a wide range of temperatures ranging from 20 o C to 40 o C. In the present experiment with increase in temperature enzyme production increased up to a certain level and upon further increase of temperature, production decreased. Mukherjee et al (1993) also reported 30 o C as optimum temperature for Aspergillus flavus. Hayashida et al (1998) also expressed similar kind of results stating 30 o C as optimum for Aspergillus flavus. The role of incubation temperature on growth and amylase production by Aspergillus sp MK 07 was studied by incubating the culture broth at different temperatures ranging from 20 to 40 o C with an increment of 5 o C. The fermentations and assays were carried out in triplicate and average values are reported as described earlier. The results indicated that the organism had potential to grow in all the tested incubation temperatures and produced amylase enzyme. However, effective enzyme production was noticed in the temperature range of 30 to 35 o C. The maximum growth was observed at 30 o C with amylase production of (59 U/ml) in 24 h as shown in figure and table Hence fermentation experiments for optimization were performed at this incubation temperature INFLUENCE OF ph Amylase production by microbial strains strongly depends on the extracellular ph as culture ph strongly influences many enzymatic reactions and also for the transport of various components across the cell membrane Murali Krishna. CH Page 71

11 (Nahas et al; 2002 & Ellaiah et al 2000). Growth and metabolism along with enzyme production is governed by an important factor called ph (Sivaramakrishnan et al, 2006).Different organisms have different ph optima and any modification in their ph optima could result in a decrease in their enzyme activity (Adinarayana et al, 2005). The results in the present experiment revealed that the strain isolated had an optimum ph of 5.0 with a maximum enzyme activity of 63 U/ml with increase in ph value from strong acidic phase to a neutral phase enzyme activity increased up to a ph of 5.0 and upon further increase in ph, enzymatic activity decreased. Olama et al (1989) stated that ph 7.0 was optima for Aspergillus flavus. Hayashida et al,(1986) reported that Aspergillus sp. Such as Aspergillus oryzae and Aspergillus niger were found to give significant yields of alpha amylase between a ph of 5.0 and 6.0. The effect of initial medium ph on biomass growth and amylase yield was studied at 30 o C. Different initial medium ph values ( ) were used for the study. The fermentations and assays were carried out in triplicate as per the general procedures. Growth and amylase production were observed in all studied ph environments. However, the influence of ph on growth and enzyme production could be seen. Maximum biomass and enzyme production (63 U/ml) were observed at ph 5.0 and minimum production (<10 U/ml) was noticed at ph 6.5. These results are shown in figure INFLUENCE OF VARYING CONCENTRATIONS OF INOCULUM Varying concentrations of Inoculum levels were studied in this experiment and the various range of inoculum (%) selected in this experiment ranged from 3% to 15%. Enzyme production varied with percentage of inoculum and the maximum enzyme production was 61 U/ml with 5% Inoculum. Increase of inoculum level from 5% to 10% or 15% showed a marginal decrease in amylase production. Murali Krishna. CH Page 72

12 Initial microbial load to a medium does affect the growth and inturn metabolite production. To study the effect of inoculum level, experiments were conducted using 3-15 % of inoculum (v/v) using fermentation medium adjusted to a ph of 5 and by incubating at 30 o C. The culture was grown for 18 hrs and having optical density of 0.8 was used for experimentation. The results indicated that the growth and amylase production increased with increase in the level of inoculum up to 5 % and further increase resulted in a decrease in both (biomass and enzyme) production values. Maximum enzyme production (61 U/ml) was noticed with inoculum (5%) and further increase in inoculum level did not increase any enzyme production. Approximately 30 % variation in enzyme production was observed with increase of inoculum concentration from 3 to 5 %. Kunamneni et al. (2005) reported highest enzyme production with an inoculum level of 10% (v/v) and the maximum amylase production obtained was 281 U/ml. The results are shown in figure and table EVALUATION OF DIFFERENT CARBON SUBSTRATES IN THE PRODUCTION MEDIUM Several carbon substrates like Glucose, Starch, Maltose, Lactose, and Sucrose were tested to evaluate the enzyme production by SmF. On supplementation of various carbon substrates maximum enzyme production was exhibited by Sucrose (3% w/v). Results showed different impact on enzyme production with different substrates. The maximum enzyme production obtained was 69 U/ml with 3% w/v Sucrose. Glucose and starch when supplemented as additional carbon substrate to the medium has resulted in enhanced enzyme production. Among the tested substrate sucrose and glucose resulted in enhanced enzyme production. Maryam et al, (2010) with Bacillus strain reported lactose as an additional carbon supplementation for increasing amylase production. Maximum amylase Murali Krishna. CH Page 73

13 production obtained with Bacillus strain by Maryam et al was U/ml. Srivastava et al, (1986) reported soluble starch as best substrate for production of alpha amylase by Bacillus stearothermophilus. Goyal et al, (2005) reported starch and glycerol as best carbon substrate for amylase production by Bacillus sp Several researchers have tested various carbon sources for enhancing amylase enzyme production such as orange waste, starch, tapioca etc (Haq et al, 2005). Varying carbon substrates effect on enzyme production by the isolated Aspergillus niger strain are reported in figure and table EVALUATION OF VARYING CONCENTRATIONS OF CSL TO THE PRODUCTION MEDIUM The effect of varying concentrations of CSL as nitrogen sources was studied with supplementation to the production medium and the results were further analysed. A control flask was maintained without additional CSL supplementation. CSL supplementation of 1%, 2% 3%, 4% were tested in the study. Among the varying percentages of CSL, 2% supplementation of CSL has showed an enhanced enzyme production. Maximum enzyme production was obtained with CSL (2% v/v). Maximum enzyme production obtained was 84 U/ml. 1% CSL supplementation also showed a considerable amount of increase in amylase production compared to the control. 3% and 4% supplementation of CSL to the medium has not resulted in enhanced enzyme production. Shah et al, (1990) has also used CSL for alpha amylase production from a mutant of Bacillus subtilis. The results of this experiment are shown in table and figure Murali Krishna. CH Page 74

14 Figures (Submerged Fermentation Parameters Optimization) Figure Figure Murali Krishna. CH Page 75

15 Figure Figure Murali Krishna. CH Page 76

16 Figure Figure Figure Comparitive Contribution Profile of Each Component Murali Krishna. CH Page 77

17 Table Effect of Incubation Period on Amylase Activity Incubation period (Hrs) Amylase activity (U/ml) Table Amylase Activity at Different Temperatures Temperature ( C) Amylase activity (U/ml) Table Amylase Activity At Different ph ph Amylase activity (U/ml) Murali Krishna. CH Page 78

18 Table Varying Percentages of Inoculum levels Inoculum Levels (%) Amylase activity (U/ml) Table Effect of Different Carbon Substrates Carbon substrate Amylase activity (U/ml) Sucrose 69 Glucose 63 Starch 58 Maltose 42 Lactose 13 Murali Krishna. CH Page 79

19 4.4. MODEL DEVELOPMENT FOR SCALE UP STUDIES IN THE FERMENTOR Bio-processing involves a multitude of complex enzyme-catalysed reactions within specific microorganisms and these reactions are critically dependent on the physical and chemical conditions that exist in the immediate environment of the microorganisms. In this context, scale up studies assumes importance in conversion of lab scale experimental results to industrial production levels. In fact, bio processing succeeds only when all the essential factors are brought together. It is necessary to know how an organism responds to a set of measurable environmental conditions to understand and/or control a fermentation process. However, it is difficult to know the complete metabolic processes of any microbial strain hence, scientists developed different mathematical models based on experimental values (Panda et al., 1990). Any mathematical model development for a particular fermentation process requires the basic information regarding consumption pattern of medium components, growth of the microbial strain and product formation especially in terms of metabolite production even after the growth is completed. The growth rate at different substrate concentrations is also to be evaluated. Since, the Aspergillus niger MK 07 has shown the potential in industrial use, further modelling experiments were carried out to understand the relation between the substrate consumption with respect to biomass growth and amylase production values. And the results were analysed for economic and effective productivity using the developed unstructured model. Murali Krishna. CH Page 80

20 4.4.1 FERMENTER STUDY Two culture media A and B were tested in Fermentor for the production of alpha amylase by isolated Aspergillus niger MK 07 strain. The production of enzyme following growth of the organism was obtained after 36 and 48 hours by media A and media B respectively. Kinetic study and volumetric rates of enzyme formation and biomass revealed that the medium B gave better results compared to medium A and hence for further studies medium B was selected. Table Media A Ingredients g/l Sucrose 30 Sodium Nitrate 3 Magnesium sulphate 0.5 Potassium chloride 0.5 Ferrous Sulphate 0.01 Di potassium hydrogen phosphate 1.0 Table Media B Ingredients g/l Sucrose 30 CSL 20 Magnesium sulphate 0.5 Potassium chloride 0.5 Potassium phosphate 1 Ferrous sulphate 0.01 Peptone 5 g/l Murali Krishna. CH Page 81

21 EFFECT OF VOLUME OF THE MEDIA: The effect of Fermentor volume on the production of alpha amylase by isolated MK 07 strain was evaluated. Maximum production of 1675 U/ml was achieved when the reactor volume was kept at 70%. As the volume of the Fermentor media was increased, the enzyme production was decreased. The kinetic values of Y p/x, and Qp was also found to be significant when the volume of the Fermentor was maintained at 70%. When the medium volume percentage in the Fermentor was increased to 80% a decrease in amylase production was obtained. Most of the fermentation studies for enhanced amylase production were carried out by synthetic media (Haq et al. 1997). Hence, for further studies the volume of the media was maintained at 70% as shown in figure and table EFFECT OF RATE OF AGITATION: Effect of varying rate of agitation was investigated for alpha amylase production in Fermentor by the isolated strain. The fermentation was carried out at 150, 200, 250 and 300 rpm. The production of enzyme following growth of the organism was found to be maximum of 1734 U/ml at an RPM of 250. When the agitation rate of the Fermentor was increased above 250 rpm a decrease in enzyme production was observed. Hence, for further studies the RPM was maintained at 250 as shown in figure and table Kinetic study revealed the values of product yield coefficient (Y p/x), volumetric rate of enzyme (Qp), biomass formation (Qx) and specific rate of product formation (qp) was found maximum when the agitation intensity was maintained at 250 rpm. Agitation intensity influences the mixing and oxygen transfer rate in many fungal fermentations thereby influencing mycelial morphology and product formation (Justen et al, 1996). Agitation intensities of up to 300 rpm have normally been reported in the literature for the production of amylase from various micro organisms (Ellaiah et al. 2002). Murali Krishna. CH Page 82

22 EFFECT OF DIFFERENT VOLUME OF AIR SUPPLY: Effect of different range of air supply ( LPM) to the fermentation medium for the production of alpha amylase by isolated MK 07 was studied. The production of alpha amylase enzyme following growth of the organism was optimum and maximum at 2.5 LPM (1576 U/ml) and further increase or decrease in air supply decreased the enzyme production. The production of amylase enzyme steadily increased with increase in air supply, indicating the isolated strain to be an aerobic organism. Jin et al. (1999) has reported that no morphological changes occur in Aspergillus strain in air-life bioreactors and that pellet size decreased considerably as the air velocity increased. Hence, for further studies the air supply was maintained at 2.5 LPM as shown in figure and table EFFECT OF DIFFERENT INOCULUM SIZES: Effect of different sizes of inoculum on the production of alpha amylase by the isolated strain MK 07 was investigated. The production of enzyme following growth of the organism was increased with increase in inoculum volume up to 10% and upon further increase in inoculum volume enzyme production decreased. At low level of inoculum the production of enzyme was insignificant and at higher levels of inoculum percentage did not had any considerable increment in enzyme production. Under optimized inoculum sizes the amylase production increased as well as an increase in biomass was also observed. Kunamneni et al. (2005) reported highest enzyme production with an inoculum level of 10% (v/v) and the maximum amylase production obtained was 281 U/ml. And the maximum amylase production obtained was 1691 U/ml. The results are shown in figure and table EFFECT OF ph: Effect of different ph on the production of alpha amylase by the isolated strain Mk 07 was investigated. Among the physical parameters, the ph of the growth medium place an important role by inducing morphological Murali Krishna. CH Page 83

23 change in the organism and in enzyme secretion. The ph change observed during the growth of the organism also affects products stability in the media (Ellaiah et al. 2002). The production of enzyme increased with increase in ph up to 5.0 and upon further increase in ph the enzyme production decreased drastically. Various other researchers have reported that a ph of 6 was optimal for amylase production by Bacillus strains which are used commercially. A lot of work on the morphology and physiology of alpha amylase production by Aspergillus strain during batch cultivation has been done (Adinarayana et al. 2005). The results of the same experiment are shown in figure and table Under all optimized conditions a high enzyme production of 1723 U/ml was obtained in 48 hrs. Figure Standard Plot of Maltose Figure Standard Plot of BSA Murali Krishna. CH Page 84

24 Figure Figure Murali Krishna. CH Page 85

25 Figure Figure Murali Krishna. CH Page 86

26 Figure Table Effect of Volume of Media Volume (%) Amylase activity (U/ml) Table Amylase Activity At Different RPM's RPM Amylase activity (U/ml) Murali Krishna. CH Page 87

27 Table Amylase Activity At Different LPM's Air Supply (LPM) Amylase activity (U/ml) Table Varying percentages of Inoculum levels Inoculum Percentage Amylase activity (U/ml) Table Effect of different ph ph Amylase Activity (U/ml) Murali Krishna. CH Page 88

28 4.5. FERMENTOR USED Murali Krishna. CH Page 89

29 4.6. SOLID STATE FERMENTATION Most of the microbial products at industrial scale are generally produced using submerged fermentation due to its apparent advantages in consistent enzyme production characteristics with defined medium and process conditions (Maryam et al, 2010). Further, it has advantages in downstream processing in spite of the cost-intensiveness for medium components (Reeta et al. 2009). However, solid-state fermentation has gained renewed interest and fresh attention from researchers because of its edge in biomass energy conservation, solid waste treatment and its application to produce secondary metabolites over submerged fermentation (Sivaramakrishnan et al, 2006). Production of biocatalysts using agrobiotech substrates under solid-state fermentation conditions provide several advantages in productivity, cost-effectiveness in labour, time and medium components further the effluent production is less and thus it is eco friendly (Pandey et al., 2000). However, these production characteristics would have to offer a competitive advantage over existing products (Lonsane et al, 1990). Hence, the present study was aimed to exploit the locally available, inexpensive agro-substrate for amylase production using Aspergillus sp. MK 07 under solid-state fermentation conditions EVALUATION OF DIFFERENT AGRICULTURAL RESIDUES AS SUBSTRATES FOR AMYLASE PRODUCTION The selection of a suitable agricultural residue as a substrate for SSF is one of most critical factor to be considered. Several substrates have to be screened for high enzyme production through SSF (Prakasham et al, 2005). The availability and the cost of the raw material are the two important parameters that have to be considered while selecting a raw material in SSF (Pandey et al 2000). The substrate selected should allow the maximum growth of the organism and also should ease in high product formation. In the present study three agricultural residual substrates like rice bran, wheat Murali Krishna. CH Page 90

30 bran and green gram husk were evaluated for their maximum enzyme production. Wheat bran supported the highest enzyme production compared to the other two substrates and these results were similar to the observations made by Ellaiah et al (2002). The results in the present study have indicated that amylase production pattern varied with the type of raw material used. Maximum enzyme production obtained with wheat bran as substrate was 128 U/g. To carry out further experiments wheat bran was used as the substrate. Adinarayana et al, (2005) reported maximum enzyme enzyme production of 261 U/g with wheat bran as substrate. Lonsane et al. (1990) reported wheat bran as efficient substrate for alpha amylase production by thermophilic strains. These results are shown in figure and table INFLUENCE OF VARYING INOCULUM LEVELS Varying concentrations of Inoculum levels were studied in this experiment and the various range of inoculum (% spores) selected in this experiment ranged from 3% to 15%.Enzyme production varied with percentage of inoculum and the maximum enzyme production was 106 U/g with 5% Inoculum. Increase of inoculum level from 5% to 10% or 15% showed a marginal decrease in amylase production. Kunamneni et al. (2005) reported highest enzyme production with an inoculum level of 10% (v/v) and the maximum amylase production obtained was 281 U/g. The results are shown in figure and table ROLE OF INCUBATION PERIOD Maximum enzyme production could be obtained only after a certain incubation time which allows the culture to grow at a study state.enzyme production of each strain is based on the specific growth rate of the strain. Growth rate and enzyme synthesis of the culture are the two main characteristics which are mainly influenced by incubation time (Ellaiah et al 2002).The results of the present study showed that amylase production Murali Krishna. CH Page 91

31 increased with increase in incubation time as shown in figure and table linearly till 120 hrs and on further incubation there was a decrease in the amylase production. Maximum amylase production was obtained at 120 h (132 U/g). Adinarayana et al (2005) reported 120 h as optimal incubation period for amylase production by Aspergillus strain. Ellaiah et al. (2002) reported that for the starin T. lanuginosus the optimal incubation period for high amylase production was 120 h and the maximum amylase enzyme production obtained was 262 U/g. Maryam et al. (2010) reported highest amylase production with strain Bacillus sp. KR 8104 at 72 h and the maximum enzyme production obtained was 96 U/g. The Results are shown in figure and table INFLUENCE OF INITIAL MOISTURE CONTENT Initial Moisture content plays an important role in enzyme production during SSF. In SSF most of the microbial growth and product formation takes place at or near the surface of the solid substrate, thus it is very crucial to provide optimized water level that controls the water activity of the fermenting substrate for achieving maximum product, Prakasham et al (2005). In the present study maximum enzyme production was obtained with an initial moisture content of 70%. And the maximum enzyme production obtained was 116 U/g.Till 70% there was a linear increase in enzyme production and upon further increase there was a marginal decrease in enzyme production. Ellaiah et al (2002) reported in their studies that 80% of initial moisture content was optimum for their Aspergillus strain. Kunamneni et al (2005) reported that 90% of the initial moisture content as optimum for Amylase production by Thermomyces lanuginosus and the maximum enzyme production obtained was 298 U/g. And the influence of moisture content on the isolated Aspergillus niger strain are shown in figure and table Murali Krishna. CH Page 92

32 INFLUENCE OF ph Growth and metabolism along with enzyme production is governed by an important factor called ph (Sivaramakrishnan et al 2006).Amylase production by microbial strains strongly depends on the extracellular ph,as culture ph strongly influences many enzymatic reactions and also for the transport of various components across the cell membrane (Ellaiah et al 2000). Different organisms have different ph optima and any modification in their ph optima could result in a decrease in their enzyme activity. The results in the present experiment revealed that the strain isolated had an optimum ph of 5.0 with a maximum enzyme activity of 119 U/g. With increase in ph value from strong acidic phase to a neutral phase enzyme activity increased up to a ph of 5.0 and upon further increase in ph, enzymatic activity decreased. Anto et al (1997) observed ph 6.5 as optimum for Bacillus sp. Our results were also in accordance to the observations made by Nahan et al, (2002) stating that ph 5.0 was optima for Aspergillus sp. Olama et al (1989) stated that ph 7.0 was optima for Aspergillus flavus. The influences of ph on isolated Aspergillus stain are shown in figure and table INFLUENCE OF TEMPERATURE Temperature is one of the most important parameter to be optimized for maximum enzyme production. Optimum temperature for maximum enzyme production depends on the characteristics of the strain.in solid state fermentation temperature plays a very important role in enzymatic synthesis (Lonsane et al, 1990).The isolated Aspergillus strain was tested in a wide range of temperatures ranging from 20 o C to 40 o C. Maximum amylase production was obtained at a temperature of 30 o C (114 U/g) as shown in figure In the present experiment with increase in temperature enzyme production increased up to a certain level and upon further increase of temperature, production decreased. Mukherjee et al (1993) also reported Murali Krishna. CH Page 93

33 30 o C as optimum temperature for Aspergillus flavus. Sadhukhan et al (1990) reported that 45 o C was optimum for Myceliophthora thermophila. The present investigation reports were comparable to the results expressed by Ellaiah et al (2000). Olama et al (1989) reported maximum enzyme activity by Aspergillus flavus at 45 o C. Hayashida et al (1998) also expressed similar kind of results stating 30 o C as optimum for Aspergillus flavus. Some thermophilic strains like Thermotoga maritima also showed maximum enzyme production at 30 o C, as stated by Vieilli et al (2001). The influence of temperature on the isolated strain is shown in figure and table EFFECT OF ADDITIONAL CARBON SUPPLEMENTATION TO THE SUBSTRATE Several carbon substrates like glucose, starch, maltose, lactose, and sucrose were tested along with control to evaluate the enzyme production by SSF. On supplementation of various carbon substrates maximum enzyme production was exhibited by starch (1% w/w). Results showed different impact on enzyme production with different substrates. The maximum enzyme production obtained was 139 U/g with 1% w/w starch. Supplementation of sucrose also aided in enhanced enzyme production. These results were in accordance to the observations made by Kunanmeni et al (2005). Ellaiah et al (2002) reported maximum enzyme activity on supplementation with Fructose. Anto et al (2005) reported maximum enzyme production on supplementation with Sucrose. Sadhukhan et al (1990) results were also in accordance to the results of our experiment stating that starch was the best supplement for M.thermophila. And the results of the present experiment are shown in figure and table EFFECT OF ADDITIONAL NITROGEN SOURCE SUPPLEMENTATION TO THE SUBSTRATE The effect of additional nitrogen sources like peptone, urea, sodium nitrite, yeast extract and ammonium nitrate at 1% supplementation to the Murali Krishna. CH Page 94

34 medium was tested and the results were further analyzed. A control flask was maintained without additional nitrogen supplementation. Among all the nitrogen sources tested the maximum enzyme production was obtained with peptone (1% w/w). Maximum enzyme production obtained was 137 U/g. Urea also showed a considerable amount of increase in amylase production compared to the control. And these results showed some similarity with the observations made by Pandey et al (1994). Pandey et al (1990) reported high enzyme production on supplementation of sodium nitrate as nitrogen source for Aspergillus niger. H.Anto et al (2006) reported maximum enzyme production on supplementation of yeast extract to the medium for fungal culture. Ellaiah et al (2002) obtained maximum enzyme production on supplementation of Urea. Cherry et al (2004) obtained maximum enzyme production upon supplementation of ammonium phosphate as a nitrogen source. Shaista et al (2003) reported maximum enzyme activity upon supplementation with 0.2% peptone as a nitrogen source for Bacillus species. And the influence of the additional nitrogen supplementation to the medium results is shown in figure and table CONCLUSION The results obtained in the present study indicated that Aspergillus species could be a potential strain for Amylase production by SSF with wheat bran as the substrate. The enzyme production was influenced by various physiological and chemical nature of the substrate as well as the conditions during SSF. Among the various parameters screened for SSF, ph, temperature, supplementation of carbon and nitrogen sources along with Incubation period played an important role for higher amount of amylase production. After optimization of individual parameters separately when further experiment was carried out with all optimized parameters, amylase production increased to a maximum of 164 U/g. figure shows a comparative profile contribution by each factor for high enzyme production. Murali Krishna. CH Page 95

35 Figure Figure Murali Krishna. CH Page 96

36 Figure Figure Murali Krishna. CH Page 97

37 Figure Figure Murali Krishna. CH Page 98

38 Figure Figure Murali Krishna. CH Page 99

39 Figure Murali Krishna. CH Page 100

40 Table Agricultural Residues as Substrates for SSF Substrate Amylase activity ( U/g) Wheat bran 128 Rice bran 96 Green gram bran 74 Table Optimization of Inoculum Level (%) Inoculum Level (%) Amylase activity ( U/g) Table Optimization of Incubation Period (Hrs) Incubation Period (Hrs) Amylase activity ( U/g) Murali Krishna. CH Page 101

41 Table Optimization of Initial Moisture Content (%) Moisture content (%) Amylase activity ( U/g) Table Optimization of ph ph Amylase activity ( U/g) Table Optimization of Temperature ( C) Temperature ( C) Amylase activity ( U/g) Murali Krishna. CH Page 102

42 Table Supplementation of additional carbon Source Carbon Supplement Amylase activity ( U/g) Glucose 89 Starch 139 Sucrose 117 Lactose 81 Maltose 79 Control EFFECT OF CALCIUM CHLORIDE ON THE THERMOSTABILITY OF ALPHA AMYLASE Different concentrations of calcium chloride were added to the fermentation medium and the enzyme produced by the fungal species was tested for the thermostability of alpha amylase. The thermostability of the crude enzyme was increased with increase in the concentration of calcium ion. The enzyme produced by Aspergillus sp.was found to be most thermostable at o C at 0.3 M concentration of calcium chloride. Further increase in the concentration levels decreased the thermostability of the enzyme. Murali Krishna. CH Page 103

43 Table M Calcium Chloride Temp ( o C) Activity (U/ml) Table M Calcium Chloride Temp ( o C) Activity (U/ml) Table M Calcium Chloride Temp ( o C) Activity (U/ml) Murali Krishna. CH Page 104

44 Table M Calcium Chloride Temp ( o C) Activity (U/ml) Figure Figure Murali Krishna. CH Page 105

45 Figure Figure Murali Krishna. CH Page 106

46 4.8. PARTIAL PURIFICATION OF ALPHA AMYLASE The alpha amylase was partially purified by ammonium sulphate precipitation and Sepahdex G 100 X Chromatography. The specific activity of the enzyme was gradually increased after purifying the alpha amylase and found optimum after ammonium sulphate precipitation. The specific activity of the enzyme was found to increase after ammonium sulpahte precipitation. Table Partial Purification of Alpha Amylase from Fermented broth Protein (mg/ml) Step Amylase activity (U/ml) Fermentation Broth Ammonium Sulphate Precipitation Sephadex G 100 Chromatography EFFECT OF TEMPERATURE ON THE ACTIVITY OF THE ENZYME The residual activity of the partially purified alpha amylase enzyme was measured by incubating the enzyme at different temperatures. The results showed that the activity of the enzyme increased with increase in temperature.the partially purifed enzyme activity was found to be highly active between o C. As the optimum activity was obtained at 75 o C thus this temperature was optimzed for the conversion of starch to oligosacharides Murali Krishna. CH Page 107

47 EFFECT OF ph ON THE ACTIVITY OF THE ENZYME The residual activity of the partially purified alpha amylase enzyme was measured by incubating the enzyme at different ph. The results showed that the activity of the enzyme increased with increase in ph.the partially purifed enzyme activity was found to be highly active between ph As the optimum activity was obtained at ph 5 thus this ph was optimzed for the conversion of starch to oligosacharides in further experiments. Table 4.9 Effect of Temperature on Enzyme Activity Temp ( o C) Starch Hydrolysis ( mg/ml) Table Effect of ph on Enzyme Activity ph Starch Hydrolysis ( mg/ml) Murali Krishna. CH Page 108

48 4.10. ALPHA AMYLASE AS DESIZER EFFECT OF ENZYME CONCENTRATION The effect of enzyme concentration on the desizing of the cotton cloth was studied by partially purified enzyme. The concentration of enzyme was varied from U/ml/min. The desizing (separation of starch from the cloth ) of the cloth was increased with increase in enzyme concentration and was found to be optimum at 300 U/ml/min. Further increase in the concentration had no significant effect on the desizing of the cloth. Table Effect of enzyme Concentrations on the desizing of cotton cloth by partially purified alpha amylase Enzyme Concentrations (U/ml/min) Percent of Desizing Murali Krishna. CH Page 109

49 Figure 4.9 Figure Murali Krishna. CH Page 110

50 Figure Murali Krishna. CH Page 111