LABORATORY MANUAL

EBT-553 BIOPROCESS ENGINEERING - I

IMS ENGINEERING COLLEGE, GHAZIABAD

(Affiliated to UP Technical University, Lucknow)

ORIENTATION TO THE LABORATORY RULES OF CONDUCT AND GENERAL SAFETY Many of the microorganisms used in this course (Under Bioprocesses) may be pathogenic for humans and animals. As a result, certain rules are necessary to avoid the possibility of infecting yourself or other people. Anyone who chooses to disregard these rules or exhibits carelessness that endangers others may be subject to immediate dismissal from the laboratory. If doubt arises as to the procedure involved in handling infectious material, consult your instructor. In 1997, the American Society for Microbiology, through its Office of Education and Training, adopted the following on laboratory safety. Each point is considered essential for every introductory microbiology laboratory, regardless of its emphasis. A student successfully completing basic microbiology will demonstrate the ability to explain and practice safe (A) MICROBIOLOGICAL PROCEDURES, including 1. reporting all spills and broken glassware to the instructor and receiving instructions for cleanup 2. methods for aseptic transfer 3.minimizing or containing the production of aerosols and describing the hazards associated with aerosols 4. washing hands prior to and following laboratories and at any time contamination is suspected 5. never eating or drinking in the laboratory 6. using universal precautions (see inside front and end covers of this laboratory manual) 7. disinfecting lab benches prior to and at the conclusion of each lab session 8. identification and proper disposal of different types of waste 9. never applying cosmetics, including contact lenses, or placing objects (fingers, pencils) in the mouth or touching the face 10. reading and signing a laboratory safety agreement indicating that the student has read and understands the safety rules of the laboratory 11. good lab practice, including returning materials to proper locations, proper care and handling of equipment, and keeping the bench top clear of extraneous materials (B) PROTECTIVE PROCEDURES, including 1. tying long hair back, wearing personal protective equipment (eye protection, coats, closed shoes; glasses may be preferred to contact lenses), and using such equipment in appropriate situations 2. always using appropriate pipetting devices and understanding that mouth pipetting is forbidden

(C) Emergency procedures, including

1. locating and properly using emergency equipment (eye-wash stations, first-aid kits, fire extinguishers, chemical safety showers, telephones, and emergency numbers) 2. reporting all injuries immediately to the instructor 3. following proper steps in the event of an emergency In addition, institutions where microbiological laboratories are taught will 1. train faculty and staff in proper waste stream management 2. provide and maintain necessary safety equipment and information resources 3. train faculty, staff, and students in the use of safety equipment and procedures The Workplace Hazardous Materials Information System (WHMIS) requires that all hazardous substances, including microorganisms, be labeled in a specific manner. In addition, there must be a Material Safety Data Sheet (MSDS) available to accompany each hazardous substance. MSDS sheets are now supplied with every chemical sold by supply houses. The person in charge of the microbiological laboratory should ensure that adherence to this law is enforced. All laboratory work can be done more effectively and efficiently if the subject matter is understood before coming to the laboratory. To accomplish this, read the experiment several times before the laboratory begins. Know how each exercise is to be done and what principle it is intended to convey. Also, read the appropriate sections in your textbook that pertain to the experiment being performed, this will save you much time and effort during the actual laboratory period. All laboratory experiments will begin with a brief discussion by your instructor of what is to be done, the location of the materials, and other important information. Feel free to ask questions if you do not understand the instructor or the principle involved. Much of the work in the laboratory is designed to be carried out in groups or with a partner. This is to aid in coverage of subject matter, to save time and expense, and to encourage discussion of data and results. Many of the ASMs recommended precautions are represented by the specific safety guidelines given inside the cover of this laboratory manual. I have read the above rules and understand their meaning.

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INDEX

S. Name of Experiment No 1. Determine the growth patterns and specific growth rate of E.coli 2. 3. 4. 5. 6. Determine the effect of peptone concentration on E.coli. growth Determine the effects of temperature on Pseodomonas putida Determine the effects of pH of solution on Pseodomonas putida Biodegradation of phenol from wastewater Upstream and Downstream of bioprocess for the production of Citric acid by Aspergillus niger Citric acid production from whey with glucose as supplementary carbon source by Aspergillus niger Upstream and Downstream of bioprocess for the production of -amylase by Aspergillus nudulans Determination of specific thermal death rate constant (kd) for E.Coli

Page no.

Date Signature performance Report

7.

8.

9.

10. Design a fermentation process for production of 5000 tonnes L-lysine per annum using microbial strain. 11. Fermentative production of -amylase by Aspergillus nudulans

EXPERIMENT NO: 1 OBJECTIVE: Determine the growth patterns and specific growth rate of E.coli
Theory: Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. Providing no mutational event occurs the resulting daughter cells are genetically identical to the original cell. Hence, "local doubling" of the bacterial population occurs. Both daughter cells from the division do not necessarily survive. However, if the number surviving exceeds unity on average, the bacterial population undergoes exponential growth. The measurement of an exponential bacterial growth curve in batch culture was traditionally a part of the training of all microbiologists; the basic means requires bacterial enumeration (cell counting) by direct and individual (microscopic, flow cytometry, direct and bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most probable number, turbidity, nutrient uptake) methods. Models reconcile theory with the measurements. Instruments and materials: Laboratory glassware A slant of freshly subcultured Yeast/E.coli pH meter UV-Vis Spectrophotometer Orbital Shaker Reagents: Culture growth medium components as given below: 2% Peptone, 0.5 % yeast extract, 10mM NaCl, 2.5 Mm, 10Mm MgCl2 Procedure: (a) Inoculum preparation of E.coli on above mentioned growth media. (b) 5% inoculum was transferred into 10-12 flasks containing required medium. (c) The sample was collected at different time intervals for growth phase study. (d) Observation & Calculations: (i). Tabulate your five growth optical data in table. (ii). Compute ln OD/OD0] for the values in table 2.Where (OD) is optical density at timet and (OD0) is optical density at timet= 0. Tabulate your ln [OD/OD0] data along with time in another table.

Table: Time in hr OD620nm ln (OD620nm x 100)

(e) Results a. Lag phase time: b. Exponential phase time: c. Stationary phase time: d. Death phase time: e. Specific Growth rate, =.hr-1 Figure: Plot between ln (OD620nm x 100) vs. time (f) Discussion:

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EXPERIMENT NO: 2 OBJECTIVE: Determine the effect of peptone concentration on growth of E.coli
Theory: The growth rate of microbial culture can be explained by the following Monods equation = [max.S]/[Ks+S] where, = Specific growth rate, hr-1 ; max = maximum specific growth rate, hr-1; S = limiting substrate concentration, g/l ; and Ks = Saturation constant, g/l. The above Monod equation explains the effect of substrate concentration on specific growth rate. The specific growth rate of a micro organism increases until it reaches a maximum specific growth rate. Further increase in substrate concentration will not result in increased specific growth rate. The saturation 9which is equal to the corresponding substrate concentration at half max value) constant value Ks, indicates the increase in affinity for a micro organism towards the limiting substrate. The Monod equation can be modified to a linear equation as follows: 1/ = 1/ max+ (Ks/ max). 1/S Plotting 1/ against 1/S will lead to straight line with the slope equal to Ks/ max and max and axis intercept equal to 1/ max. To find out Monods parameters, the effect of initial substrate concentration as microbial growth should be investigated and from Lineweaver-Burk plot the Monods parameter can be estimated. Specific Growth rate calculations: The Specific growth rate can be defined as the rate of change in biomass concentration per unit biomass. = 1/X [dX/dt] Rearranging & integrating the above equation will yield, ln (X/X0) = t Where X= biomass concentration at timet hr and X0 = biomass concentration at time t=0. Plotting ln (X/X00 against timet for a particular initial substrate concentration will result in a profile represent in graph. The slope of straight line portion (Exponential growth phase) will yield specific growth rate value, for the initial substrate concentration, S0. Instruments and materials: Laboratory glassware A slant of freshly subcultured Yeast/E.coli pH meter UV-Vis Spectrophotometer Orbital Shaker Reagents:

Culture growth medium components as given below: 2% Peptone, 0.5 % yeast extract, 10mM NaCl, 2.5 Mm, 10Mm MgCl2 Procedure: (a) Inoculum preparation of E.coli on above mentioned growth media. (b) 5% inoculum was transferred into five flasks having different concentration of peptone with similar growth media. (c) The sample was collected from each flask at similar time interval. (d) Observation & Calculations: a. Compute ln (OD620nm x 100) for the values in table 1. Where (OD) is optical density after certain time interval and (OD0) is optical density at time t= 0. Tabulate your ln (OD620nm x 100) data along with different peptone concentration.

Table 1: The effect of initial concentration of peptone of E.coli growth Initial Peptone Conc. S0, g/l OD620nm ln (OD620nm x 100)

Results: Figure: Plot of ln (OD620nm x 100) against initial peptone concentration (S0)

Discussion:

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EXPERIMENT NO: 3 OBJECTIVE: Determine the effects of temperature on growth of Pseodomonas putida
Theory: Pseudomonas putida is a gram-negative rod-shaped saprotrophic soil bacterium. Based on 16S rRNA analysis, P. putida has been placed in the P. putida group, to which it lends its name. It is the first patented organism in the world.. It demonstrates a very diverse metabolism, including the ability to degrade organic contaminants. Incubation temperature can effect the growth of any microorganism due to the characteristics of microbes. To find out the optimum temperature of Pseudomonas putida this experiment is required to be planned. Instrument & Material: Laboratory glassware A slant of freshly subcultured Pseudomonas putida pH meter UV-Vis Spectrophotometer Colorimeter BOD Incubator cum orbital shaker

Reagent: Glucose, K2HPO4, KH2PO4, (NH4)2SO4, MgCl2.7H2O, Cacl2.2H2O, Na2MoO4.2H2O, FeSO4.7H2O Procedure: (a) Inoculum preparation of Pseudomonas putida on above mentioned growth media. (b) 5% inoculum was transferred into different flasks which contains suitable growth media. (c) These flask were kept at different temperatures. (d) The sample was collected from each flask at similar time interval. (e) Optical density (OD) at 620nm was taken by using colorimeter to analyse the effect of temperature on microbial growth.

Table 1: The effect of temperature on the growth of Pseudomonas putida

Temperature, 0C

OD620nm

ln (OD620nm x 100)

Result: Figure: Plot of ln (OD620nm x 100) against Temperature Discussion: Conclusion:

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EXPERIMENT NO: 4 OBJECTIVE: Determine the effects of pH of solution on growth of Pseodomonas putida
Theory: Pseudomonas putida is a gram-negative rod-shaped saprotrophic soil bacterium. Based on 16S rRNA analysis, P. putida has been placed in the P. putida group, to which it lends its name. It is the first patented organism in the world.. It demonstrates a very diverse metabolism, including the ability to degrade organic contaminants. pH of solution can effect on microbial growth due to presence of H+ ion in the solution. To find out the optimum pH of Pseudomonas putida this experiment is required to be planned. Instrument & Material: Laboratory glassware A slant of freshly subcultured Pseudomonas putida pH meter UV-Vis Spectrophotometer Colorimeter BOD Incubator cum orbital shaker

Reagent: Glucose, K2HPO4, KH2PO4, (NH4)2SO4, MgCl2.7H2O, Cacl2.2H2O, Na2MoO4.2H2O, FeSO4.7H2O Procedure: (a) Inoculum preparation of Pseudomonas putida on above mentioned growth media. (b) 5% inoculum was transferred into different flasks which contains suitable growth media. (c) The different pH of solution were maintained in different flask, like pH = 2, 4, 6, 7, 8, 10, 12 (d) The sample was collected from each flask at similar time interval. (e) Optical density (OD) at 620nm was taken by using colorimeter to analyse the effect of pH on microbial growth.

Table 1: The effect of temperature on the growth of Pseudomonas putida

pH of solution

OD620nm

ln (OD620nm x 100)

Result: Figure: Plot of ln (OD620nm x 100) against pH of solution

Discussion

Conclusion

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EXPERIMENT NO: 5 OBJECTIVE: Biodegradation of phenol from wastewater.

Theory: Phenol is a toxic compound even in low concentrations. It is frequently found in the wastes from many modern industrial processes. This can be in the form of atmospheric pollution or in liquid waste. The most common sources of phenol are in the effluents of oil refineries, paper processing plants, resin production, and coal liquefaction. Phenol is typically found in concentrations up to 1.5 g/l but this can rise to 4.5 g/l in very polluted waters. It is recommended that exposure should not be more than 20 mg of phenol in an average working day. Phenol is very toxic to fish and has been lethal at concentrations of between 5 and 25 ppm while concentrations as low as 0.1 ppm in surrounding waters can taint the taste of fish 15]. Therefore, the treatment of phenol effluents is important. Instrument & Material: Laboratory glassware A slant of freshly subcultured Pseudomonas putida pH meter UV-Vis Spectrophotometer Colorimeter BOD Incubator cum orbital shaker

Reagent: Phenol, Glucose, K2HPO4, KH2PO4, (NH4)2SO4, MgCl2.7H2O, Cacl2.2H2O, Na2MoO4.2H2O, FeSO4.7H2O Procedure: Phenol-degrading bacteria are required to be adapted to the phenol environment. During acclimatization process certain enzymes in the bacteria are induced so that they are available for taking part in the metabolism reaction. This is much more important when dealing with toxic compounds such as phenol study, it was envisaged to degrade phenol using P. putida (MTCC 1194) at 100 mg/l concentrations. To initiate the acclimatization procedure 2% glucose as carbon source in basal salt medium was used for the growth of P. putida (MTCC 1194). The stock solutions of phenol were added to the asks so as to give 10 mg/l concentrations of the each phenolic compounds in the synthetic medium. Thereafter, the phenol was periodically added in increments of 10 mg/l till the cumulative concentration reached 100 mg/l for each of them. No sample was taken from these asks till this time just to avoid contamination and save time. It was planned to transfer the inoculum from these asks to new asks for further enrichment of the culture. The degradation was found to be complete for phenol.

Table: Table 1: Phenol degradation by P. putida in batch reactor with time Time, hr Phenol concentration, mg/l (OD270nm)

Result: Biodegradation rate (mg/l/h) = Discussion:

Conclusion:

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EXPERIMENT NO: 6 OBJECTIVE: Upstream & Downstream of bioprocess for the production of Citric acid by Aspergillus niger
Citric acid a carboxylic acid, soluble in water with a pleasant taste is the most important acid used in food industries. First time in 1923, citric acid was produced by fermentative process using Aspergillus niger, a fungal strain. Citric acid produced by fermentation is affected by many factors including nutritional composition of media, environmental condition pH & dissolved oxygen concentration. Material & Methods: Organism: Aspergillus niger Stock cultures are reactivated and cultivated by streaking a loopfull of culture on Petri dishes and slants containing medium. Inoculum preparation: Transfer aseptically one loopfull of A. niger to the inoculum media & incubate at 282 oC on shake rotating at 120 rpm for 5 days. Inoculum Media: Sucrose 1.2% NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.02% pH 3.4-3.5 Fermentation media: Sucrose 1.2% NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.02% pH 3.4-3.5 Add 5-10% inoculum to fermentation medium aseptically using sterile pipette in one litre flask in stationary condition at 28 oC for 7 days & 10 days. Citric acid determination: Citric acid (CA) determined using 0.1 N NaOH and phenolphthalein as indicator and calculated as % according to the following formula: % CA = Normality x Volume of NaOH x Equivalent wt. / Wt. of sample (gm) x 10 By Ca-Citrate precipitate: After separation of mycelium a definite volume of filtrate is treated with saturated Ca (OH)2 solution. To neutralize it we are using phenolphthalein as indicator. Note the final volume of neutralized broth. Filter the solution & take 100 ml of filtrate solution (or define volume) boil on

hot plate or water bath for 204 min. Filter the precipitate using previous weighted filter paper. The residue washed with boiled water. Keep it in the oven at 70 oC for overnight. Take the final weight and calculate the weight of citric acid produced in the fermentation broth using the weight of Ca-citrate. Calculations: CDCW= Concentration of dry cell weight per 100 ml broth =............... Weight of Ca-Citrate = pH of initial broth = pH of final broth =

Discussion:

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EXPERIMENT NO: 7 OBJECTIVE: Citric acid production from whey with glucose as supplementary carbon source by Aspergillus niger
INTRODUCTION: Citric acid a carboxylic organic acid, soluble in water with a pleasant taste is the most important acid used in food industries. Until about 1920, all commercial citric acid was produced from lemon and lime juices. First time in 1923, citric acid was produced by fermentative process using Aspergillus niger, a fungal strain. Citric acid produced by fermentation is affected by many factors including nutritional composition of media, environmental condition pH & dissolved oxygen concentration. At present time citric acid is produced commercially using mutant strains of Aspergillus niger. Large amount of whey are produced world wide as a by-product of cheese and other dairy products manufacturing. For the utilization of waste product into utilized material, the citric acid is produced from dairy waste product (i.e. Whey) in this experiment, i.e. Waste turns into wealth. MATERIAL & METHODS: Organism: Aspergillus niger Stock cultures are reactivated and cultivated by streaking a loopfull of culture on Petri dishes and slants containing medium. Inoculum preparation: Transfer aseptically one loopfull of A. niger to the inoculum media & incubate at 282 oC on shake rotating at 120 rpm for 5 days. Inoculum Media: Sucrose 1.2% NH4NO3 0.2% KH2PO4 0.1% MgSO4.7H2O 0.02% pH 3.4-3.5 Fermentation media: Whey from a dairy plant of the nearby place of IMSEC, Ghaziabad was used as the basal fermentation media. Its proximate composition was determined. Glucose solution of 5, 10, 15 % (w/v) were added to the whey in the fermentation process. Surface liquid culture fermentation process was carried out in a 500 ml Erlenmeyer flask containing 100 ml media. Each flask was incubated with the given spore suspension and incubated at 282 0C for up to 8 days. 5-10% inoculums are added into these fermentation media aseptically using sterile pipette in stationary conditions. Citric acid determination: Citric acid (CA) determined using 0.1 N NaOH and phenolphthalein as indicator and calculated as % according to the following formula:

% CA = Normality x Volume of NaOH x Equivalent wt. / Wt. of sample (gm) x 10 By Ca-Citrate precipitate: After separation of mycelium a definite volume of filtrate is treated with saturated Ca(OH)2 solution. To neutralize it we are using phenolphthalein as indicator. Note the final volume of neutralized broth. Filter the solution & take 100 ml of filtrate solution (or define volume) boil on hot plate or water bath for 204 min. Filter the precipitate using previous weighted filter paper. The residue washed with boiled water. Keep it in the oven at 70 oC for overnight. Take the final weight and calculate the weight of citric acid produced in the fermentation broth using the weight of Ca-citrate. CALCULATIONS: Media Whey Whey + 5% glucose Whey + 10% glucose Whey + 15% glucose Citric acid (g/l) during different incubation time (days) 2 4 6 8

DISCUSSION:

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EXPERIMENT NO: 8 OBJECTIVE: Upstream and Downstream of bioprocess for the production of amylase by Aspergillus sp.
Citric acid a carboxylic acid, soluble in water with a pleasant taste is the most important acid used in food industries. First time in 1923, citric acid was produced by fermentative process using Gliomastix indicus, a fungal strain. -amylase produced by fermentation is affected by many factors including nutritional composition of media, environmental condition pH & dissolved oxygen concentration. Material & Methods: Organism: . Stock cultures are reactivated and cultivated by streaking a loop full of culture on Petri dishes and slants containing medium. Amylae production: Actively growing and heavily sporulating (ten days) old malt agar slant culture was added to 10 ml sterile distilled water. The spores were gently scraped off with the help of a sterile needle and contents were passed through glass wool so as to obtain spore inoculums free from mycelia bits. A volume of one ml of spore suspension contained more than 106 spores. They were cultured in a suitable medium and the temperature and pH in all the batch experiments were maintained. The medium was autoclaved (121 0C for 15 minutes), allowed to cool, then aseptically added to sterile 500 ml Erlenmeyer flasks (100 ml added per flask).These flasks with 100 ml liquid medium were incubated with 2 ml spore suspension with autoclaved distilled water and incubated at 28 0C and 120 rpm for 4-5 days in the preliminary experiments and only four days in all subsequent experiments. All chemicals used were of reagent grade. Enzyme extraction The contents were mixed by shaking for two hours at 28 0C on a rotary shaker at 200 rpm. The slurry was squeezed by muslin cloths. The extract was filtered with a Whatman No. 1 filter paper and the filtrate was used as a crude -amylase. Enzyme assay The -amylase enzyme was assayed accordingly to the method described by Miller. The reaction mixture contained 200 l soluble starch in phosphate buffer (0.1M, pH = 6); 200 l of diluted enzyme and 300 l phosphate buffer. The reaction was incubated for 15 minutes at 30 C, 300 l dinitrosalicylic acid (DNS) solution were added and boiled for 15 minutes. Before cooling 100 l Rochelle salt (40 % sodium potassium tartarate) was added and colour was measured at 575 nm. One unit of -amylase activity was defined as the amount of enzyme that releases 1 mg of reducing sugar as glucose per ml per minute under the assay conditions. All data points correspond to triplicates of independent experiments. Result:

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EXPERIMENT NO: 9 OBJECTIVE: Determination of specific thermal death rate constant (kd) for E.coli.
Theory: The rate of endogenous decay (rd) is also formulated according to first order kinetics as given below.

rd =
can be written as

dX = kd X dt

(1)

Where kd is first order endogenous decay coefficient (h-1). Therefore, net rate of cell growth (rnet) rnet = rg - rd for batch reactor it can be defined by the following equation. (2)

For biological reactions, the rate of cell growth (rg) follows first order kinetics. Therefore,

rg =

dX = g X dt

(3)

By using equations (1) and (3), equation (2) can be written as

dX rnet = = g X kd X = net X dt net

Where net is net specific growth rate and can be written as net = g - kd

(4)

(5)

For exponential growth phase, the endogenous decay coefficient can be neglected. For this phase, the reduced equation (4) is integrated with boundary condition (X =Xo at t = 0). These yields

ln

X = g t Xo

(6)

Where Xo is initial concentration of biomass. During the stationary phase, decay phase equation (1) is integrated which yields

X = X So e kd t
Where XSo is the biomass concentration at the beginning of stationary phase.

(7)

The endogenous decay describes the conversion of cell mass into maintenance energy. This phase starts after the complete consumption of substrate. The decay coefficient affects the growth kinetics because it appears in the mass balance equation of the cell growth. Therefore, in order to calculate the decay coefficient, first the experiment was conducted for initial glucose concentration of 3 g/l. After getting complete conversion of glucose, the batch experiment was continued for 10 days. The cell concentration was estimated at different time intervals shows the typical trend of growth and decay rates of the cells at fixed glucose concentration. Equation (7) is used to calculate kd value graphically. According to this equation loge (optical density x 100) is plotted against time (t). This plot is a straight line which is depicted in Figure 1. The slope of this line gives kd value. The negative slope of the line indicates that the concentration of the cell mass decreases with the passage of time. Instruments and materials: Laboratory glassware A slant of freshly subcultured Yeast/E.coli pH meter UV-Vis Spectrophotometer Orbital Shaker Reagents: Culture medium components as given in table 1. Procedure: (a). Preparation of inoculum (or seed) and growth (or production ) media. (i). Prepare 50 ml of inoculum medium with a nutrient composition given in table 1 in a 250 ml Erlenmeyer flask. (ii). Prepare 100 ml of ten growth media, each having same composition of growth medium (see table 1). Label the flasks by their different time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days). (iii). Set pH to 7 for both inoculum and growth media before sterilization. Autoclave the media. (iv).Sterilization: When medium contains glucose, Phosphates and salts, during sterilization, the cations of salts form a precipitate with phosphates , and glucose is degraded partially in the presence of other medium ingredients particularly Phosphates to certain compounds which are toxic to growth of cells. Therefore, when preparing inoculum and growth medium, the appropriate amount of glucose, Phosphate and remaining nutrients components should be prepared and sterilized separately. After sterilization they can be mixed together aseptically after cooling. (b). Inoculum Preparation. (i). Transfer a loop full of E coli. Aseptically in laminar air flow into inoculum medium from a freshly subcultured slant.

(ii). Allow it for growth in orbital shaker at 30 0C and at 160-170 rpm for 12 hrs after 12 hrs of growth, the inoculum will be ready to inoculate in ten conical flasks containing suitable growth media. (c). Growth of E.Coli cells with Different initial Substrate Concentrations. (i). Inoculate each growth medium with 5 ml of freshly grown inoculum ( 5 % v/v) aseptically in laminar air flow using 10 ml sterilized measuring cylinder. (ii). Place these ten conical flasks in BOD incubator cum Orbital shaker. Set temperature and shaking speed to 30 0C and 160 rpm respectively. (iii).Run the culture for 10 days withdraws approximately 5 ml of samples from ten conical flasks in prelabeled tubes every 24 hour or 1 day aseptically (including zero hr). (d). Sample analysis. After sampling heat the samples immediately to stop the growth .Dilute the sample 10 times (1ml of sample in 9 ml of distilled water). Analyse them for cell mass by taking optical density at 620 nm on a spectrophotometer. Use distilled water as blank. [Note: Optical density gives indirect measurement for cell concentration, X. if anyone wishes to measure cell mass directly can measure the dry cell weight gravimetrically.] (e). Observation & Calculations. (i). Tabulate your ten growth optical data in table 2. (ii). Compute ln [OD620nm*100] for the values in table 2.Where (OD) is optical density at timet and (OD0) is optical density at timet= 0. Tabulate your ln [OD/OD0] data along with time in another table. (iii). Plot ln [OD/OD0] against time t for every initial substrate concentration till you get 5 profile s similar to that of figure for 5 different initial substrate concentrations. (iv). Take the slope value in the linear exponential decay region which is equal to specific decay rate, kd, hr-1 for that particular micro-organism. Results. Endogenous decay coefficient kd = hr-1 Figure 1: loge (optical density x 100) is plotted against time (t)

(f).

Table 1: Culture Medium for Inoculum & Growth. Component 1. Glucose 2.(NH4)2SO4 Concentration (g/l) 3.0 2.5

3. Na2HPO4 4. KH2PO4 5.MgSO4.7H2O 6.NaCl 7.CaCl2 8.Yeast Extract 9.pH Essential graphs & Tabulation:

7.9 3.7 0.625 0.625 0.019 0.125 7.00

Table 2: Batch growth data extended upto decay phase Time (hr) t=0 24 48 72 96 120 144 168 192 216 240 Biomass concentration (OD 620nm) Xo = Xt = Ln (OD620nm*100) Slop of graph = kd

ln Xt*100 = -kd*t + lnXo*100 Discussion:

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EXPERIMENT NO: 10 OBJECTIVE: Design a fermentation process for production of 5000 tonnes L-lysine per annum using microbial strain.
Annual production: 5000 tonnes/ year Strain used: . Strain capacity: 48 g/l broth (Yield) Time required for one batch: Fermentation time: 60 hrs Empty sterilization time: 1.5 hrs Media sterilization: 7 hrs Time required for vessel preparation: 8 hrs Extra time (cleaning, filtration etc): 13.5 hrs Total time: 90 hrs Running hrs per annum: 305 x 24 = 7320 hrs Number of batches/year: 7320/90 = 82 (approx.) Per batch production required: 5000/82 = 60.98 tonnes Required volume of broth per batch: 6.1 x 104 x 103/48 = 1.27 x 106 lit. Let working capacity of fermentor = 80% Then volume of fermentor = 1.27 x 106/0.8 = 1.59 x 106 Assuming six fermentor is to be installed with each capacity = 1.59 x 106/6 = 0.265 x 106 = 265 m3 Fermentor: It is a cylindrical vessel with elliptical top and bottom. Equation for ellipse, x2/a2 + y2/b2 =1 a = major axis b = minor axis Taking 2:1 Ellipsoidal dishead (for large fermentor) a = 2b x2/4b2 + y2/b2 =1

Volume of ellipsoidal Ve = 4 ob (b2-y2) dy = 4 [(b2y-y3/3)]ob = 8 b3/3 = D3/24.. For single head side (b= D/4) Now total volume of ellipsoidal head (for both heads) = D3/24 x 2 = = D3/12 (ii) Volume of cylindrical portion VL = = D2L/4 L = L-D/2 = 2D-D/2 = 3D/2 Total volume of fermentor: VF = Ve + VL 3 2 = D /12 + D /4 x 3D/2 3 3 3 = D /12 + 3 D /8 = 11 D /24 Since volume is 265 m3 265 = 11 D3/24 D = 5.69 m. H = 2D = 2 x 5.69 = 11.38 m L = 3D/2 = 3 X 5.69/2 = 8.5 m Volume of broth media in fermentor: 265 x 80/100 m3 = 212 m3 Volume of media = DF3/24 + Hb DF2/4 = 212 Hb = 7.39 m = Height of broth So that, total length of liquid/ media broth including an elliptical head HL = Hb + D/4 = 8.81 m Impeller: Diameter of impeller, Di = DF/3 = 5.69/3 = 1.89 Taking standard 6 flat blade turbine impeller The ratio of Li/Di = = 0.25 Li = 0.25 x 1.89 = 0.4725 = 0.47 m Wi /Di = 1/5 = 0.20 Wi = 0.20 x 1.89 = 0.38 m Hi/Di = 1 = Hi = Di = 1.89 m Similarly, distance between two opposite flate blade impeller, DF = Di 2Li = 1.89 2 x 0.47 = 0.95 m D = DF ; D = 5.29

Number of impeller required, HL/Hi = 8.81/1.89 = 4.60 Baffles: Number of baffles = 4 (standard) Wb = Width of baffle Wb/DF = 1/10 = 0.1 Wb = 0.1 x 5.69 Lb = Length of baffle Lb/Di = 3 Lb = 3Di = 3x 1.89 = 5.67 m Diameter of sparger plate = Diameter of impeller Ds = Di Area of sparger plate = D2/4 = 3.14 x (1.89)2/4 = 11.22/4 = 2.8 m3 Taking hole diameter (dh) Since 2.5 cm = 2.5 mm dh (1.89 m = 189 cm) for this diameter the hole diameter should be 189 mm. Area of the hole = D2/4 = 0.028 m2 Number of holes at sparger plate = Area of sparger plate/ area of hole = 2.8/0.028 = 100 holes Height of sparger plate from bottom = Hi /2 = 1.89/2 = 0.945 m. Foam Breaker: Height from liquid level = Height of fermentor Height of liquid broth/2 HFb = 11.38 8.81/2 = 1.285 m Dimension of a fermentor equipped with two set of standard flat blade turbine & four baffle plates Discussion:

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EXPERIMENT: 11 OBJECTIVE: Fermentative production of -amylase by Aspergillus nudulans

INTRODUCTION: Citric acid a carboxylic acid, soluble in water with a pleasant taste is the most important acid used in food industries. First time in 1923, citric acid was produced by fermentative process using Aspergillus nudulans. -amylase produced by fermentation is affected by many factors including nutritional composition of media, environmental condition pH & dissolved oxygen concentration. MATERIAL & METHODS: Organism: Aspergillus nudulans. Stock cultures are reactivated and cultivated by streaking a loopfull of culture on Petri dishes and slants containing medium. Inoculum preparation: Amylae production: Actively growing and heavily sporulating (ten days) old malt agar slant culture was added to 10 ml sterile distilled water. The spores were gently scraped off with the help of a sterile needle and contents were passed through glass wool so as to obtain spore inoculums free from mycelia bits. A volume of one ml of spore suspension contained more than 106 spores. They were cultured in a suitable medium and the temperature and pH in all the batch experiments were maintained. The medium was autoclaved (121 0C for 15 minutes), allowed to cool, then aseptically added to sterile 500 ml Erlenmeyer flasks (100 ml added per flask).These flasks with 100 ml liquid medium were incubated with 2 ml spore suspension with autoclaved distilled water and incubated at 28 0C and 120 rpm for 4-5 days in the preliminary experiments and only four days in all subsequent experiments. All chemicals used were of reagent grade. Enzyme extraction The contents were mixed by shaking for two hours at 28 0C on a rotary shaker at 200 rpm. The slurry was squeezed by muslin cloths. The extract was filtered with a Whatman No. 1 filter paper and the filtrate was used as a crude -amylase. Enzyme assay The -amylase enzyme was assayed accordingly to the method described by Miller. The reaction mixture contained 200 l soluble starch in phosphate buffer (0.1M, pH = 6); 200 l of diluted enzyme and 300 l phosphate buffer. The reaction was incubated for 15 minutes at 30 C, 300 l dinitrosalicylic acid (DNS) solution were added and boiled for 15 minutes. Before cooling 100 l Rochelle salt (40 % sodium potassium tartarate) was added and colour was measured at 575 nm. One unit of -amylase activity was defined as the amount of enzyme that releases 1 mg of reducing sugar as glucose per ml per minute under the assay conditions. All data points correspond to triplicates of independent experiments.

Result: DISCUSSION:

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