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

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

Date Signature performance Report 7. Design a fermentation process for production of 5000 tonnes L-lysine per annum using microbial strain. 11.coli.S. Fermentative production of α-amylase by Aspergillus nudulans .Coli Page no. 4. 8. 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 α-amylase by Aspergillus nudulans Determination of specific thermal death rate constant (kd) for E. Determine the growth patterns and specific growth rate of E. Determine the effect of peptone concentration on E. 3. 9. 6.coli 2. 5. 10. Name of Experiment No 1.

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

μ =……………. Stationary phase time: d. Specific Growth rate. Lag phase time: b. time (f) Discussion: [Signature of Student] [Signature of Lab. Instructor] .Table: Time in hr OD620nm ln (OD620nm x 100) (e) Results a. Exponential phase time: c.hr-1 Figure: Plot between ln (OD620nm x 100) vs. Death phase time: e.

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

S0. Tabulate your ln (OD620nm x 100) data along with different peptone concentration. 10Mm MgCl2 Procedure: (a) Inoculum preparation of E. Table 1: The effect of initial concentration of peptone of E. g/l OD620nm ln (OD620nm x 100) Results: Figure: Plot of ln (OD620nm x 100) against initial peptone concentration (S0) Discussion: [Signature of Student] [Signature of Lab. (b) 5% inoculum was transferred into five flasks having different concentration of peptone with similar growth media. 2. (c) The sample was collected from each flask at similar time interval. Compute ln (OD620nm x 100) for the values in table 1. Instructor] . Where (OD) is optical density after certain time interval and (OD0) is optical density at time ‘t’= 0. 10mM NaCl.coli on above mentioned growth media.5 % yeast extract.Culture growth medium components as given below: 2% Peptone.5 Mm. (d) Observation & Calculations: a.coli growth Initial Peptone Conc. 0.

(d) The sample was collected from each flask at similar time interval. to which it lends its name. KH2PO4. To find out the optimum temperature of Pseudomonas putida this experiment is required to be planned. including the ability to degrade organic contaminants.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. Cacl2. (NH4)2SO4. It demonstrates a very diverse metabolism. Instrument & Material:       Laboratory glassware A slant of freshly subcultured Pseudomonas putida pH meter UV-Vis Spectrophotometer Colorimeter BOD Incubator cum orbital shaker Reagent: Glucose. MgCl2.2H2O.. P. It is the first patented organism in the world. FeSO4. (b) 5% inoculum was transferred into different flasks which contains suitable growth media. Na2MoO4.2H2O.7H2O. Incubation temperature can effect the growth of any microorganism due to the characteristics of microbes. putida group. Table 1: The effect of temperature on the growth of Pseudomonas putida . K2HPO4. (c) These flask were kept at different temperatures. (e) Optical density (OD) at 620nm was taken by using colorimeter to analyse the effect of temperature on microbial growth. putida has been placed in the P.7H2O Procedure: (a) Inoculum preparation of Pseudomonas putida on above mentioned growth media. Based on 16S rRNA analysis.

0C OD620nm ln (OD620nm x 100) Result: Figure: Plot of ln (OD620nm x 100) against Temperature Discussion: Conclusion: [Signature of Student] [Signature of Lab. Instructor] .Temperature.

12 (d) The sample was collected from each flask at similar time interval. (c) The different pH of solution were maintained in different flask.. FeSO4. MgCl2. 4. Cacl2. It is the first patented organism in the world. putida group.2H2O. K2HPO4. pH of solution can effect on microbial growth due to presence of H+ ion in the solution. including the ability to degrade organic contaminants. Na2MoO4. To find out the optimum pH of Pseudomonas putida this experiment is required to be planned. like pH = 2.7H2O. Table 1: The effect of temperature on the growth of Pseudomonas putida . 8.2H2O. 6. (b) 5% inoculum was transferred into different flasks which contains suitable growth media.7H2O Procedure: (a) Inoculum preparation of Pseudomonas putida on above mentioned growth media. putida has been placed in the P. (e) Optical density (OD) at 620nm was taken by using colorimeter to analyse the effect of pH on microbial growth. 10. KH2PO4. to which it lends its name. (NH4)2SO4. 7. Based on 16S rRNA analysis. It demonstrates a very diverse metabolism. Instrument & Material:       Laboratory glassware A slant of freshly subcultured Pseudomonas putida pH meter UV-Vis Spectrophotometer Colorimeter BOD Incubator cum orbital shaker Reagent: Glucose.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. P.

Instructor] .pH of solution OD620nm ln (OD620nm x 100) Result: Figure: Plot of ln (OD620nm x 100) against pH of solution Discussion Conclusion [Signature of Student] [Signature of Lab.

2H2O.1 ppm in surrounding waters can taint the taste of fish 15]. Therefore. Phenol is very toxic to fish and has been lethal at concentrations of between 5 and 25 ppm while concentrations as low as 0. FeSO4. the phenol was periodically added in increments of 10 mg/l till the cumulative concentration reached 100 mg/l for each of them. Thereafter. Instrument & Material:       Laboratory glassware A slant of freshly subcultured Pseudomonas putida pH meter UV-Vis Spectrophotometer Colorimeter BOD Incubator cum orbital shaker Reagent: Phenol. The most common sources of phenol are in the effluents of oil refineries. This can be in the form of atmospheric pollution or in liquid waste. resin production. paper processing plants.5 g/l in very polluted waters. KH2PO4.7H2O Procedure: Phenol-degrading bacteria are required to be adapted to the phenol environment.7H2O. This is much more important when dealing with toxic compounds such as phenol study. Glucose. Theory: Phenol is a toxic compound even in low concentrations. No sample was taken from these flasks till this time just to avoid contamination and save time. MgCl2. It is frequently found in the wastes from many modern industrial processes. the treatment of phenol effluents is important. To initiate the acclimatization procedure 2% glucose as carbon source in basal salt medium was used for the growth of P.EXPERIMENT NO: 5 OBJECTIVE: Biodegradation of phenol from wastewater. It was planned to transfer the inoculum from these flasks to new flasks for further enrichment of the culture. Phenol is typically found in concentrations up to 1. The degradation was found to be complete for phenol. and coal liquefaction. During acclimatization process certain enzymes in the bacteria are induced so that they are available for taking part in the metabolism reaction. putida (MTCC 1194) at 100 mg/l concentrations. Cacl2.5 g/l but this can rise to 4. Na2MoO4. It is recommended that exposure should not be more than 20 mg of phenol in an average working day. K2HPO4. (NH4)2SO4. The stock solutions of phenol were added to the flasks so as to give 10 mg/l concentrations of the each phenolic compounds in the synthetic medium. it was envisaged to degrade phenol using P. putida (MTCC 1194). .2H2O.

hr Phenol concentration. Instructor] . putida in batch reactor with time Time. mg/l (OD270nm) Result: Biodegradation rate (mg/l/h) = Discussion: Conclusion: [Signature of Student] [Signature of Lab.Table: Table 1: Phenol degradation by P.

EXPERIMENT NO: 6 OBJECTIVE: Upstream & Downstream of bioprocess for the production of Citric acid by Aspergillus niger Citric acid a carboxylic acid.5 Fermentation media: Sucrose 1.02% pH 3. Citric acid produced by fermentation is affected by many factors including nutritional composition of media.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. Filter the solution & take 100 ml of filtrate solution (or define volume) boil on .2% NH4NO3 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. Inoculum preparation: Transfer aseptically one loopfull of A. Note the final volume of neutralized broth. First time in 1923. 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. citric acid was produced by fermentative process using Aspergillus niger.7H2O 0.2% KH2PO4 0.4-3.7H2O 0. / Wt.2% NH4NO3 0. Material & Methods: Organism: Aspergillus niger Stock cultures are reactivated and cultivated by streaking a loopfull of culture on Petri dishes and slants containing medium.02% pH 3. soluble in water with a pleasant taste is the most important acid used in food industries. Inoculum Media: Sucrose 1.1% MgSO4.2% KH2PO4 0. environmental condition pH & dissolved oxygen concentration.1% MgSO4. a fungal strain. To neutralize it we are using phenolphthalein as indicator.4-3. niger to the inoculum media & incubate at 28±2 oC on shake rotating at 120 rpm for 5 days.

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

Its proximate composition was determined. a fungal strain. i. citric acid was produced by fermentative process using Aspergillus niger. Until about 1920. Glucose solution of 5. soluble in water with a pleasant taste is the most important acid used in food industries. For the utilization of waste product into utilized material. Large amount of whey are produced world wide as a by-product of cheese and other dairy products manufacturing. Each flask was incubated with the given spore suspension and incubated at 28±2 0C for up to 8 days. niger to the inoculum media & incubate at 28±2 oC on shake rotating at 120 rpm for 5 days.1% MgSO4.5 Fermentation media: Whey from a dairy plant of the nearby place of IMSEC. 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.e. Inoculum Media: Sucrose 1. First time in 1923.2% NH4NO3 0. environmental condition pH & dissolved oxygen concentration. 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. Whey) in this experiment. the citric acid is produced from dairy waste product (i. Ghaziabad was used as the basal fermentation media. Inoculum preparation: Transfer aseptically one loopfull of A. 10. all commercial citric acid was produced from lemon and lime juices.02% pH 3. Citric acid produced by fermentation is affected by many factors including nutritional composition of media.2% KH2PO4 0.4-3.e.7H2O 0. Waste turns into wealth.1 N NaOH and phenolphthalein as indicator and calculated as % according to the following formula: . At present time citric acid is produced commercially using mutant strains of Aspergillus niger. MATERIAL & METHODS: Organism: Aspergillus niger Stock cultures are reactivated and cultivated by streaking a loopfull of culture on Petri dishes and slants containing medium.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.

Filter the precipitate using previous weighted filter paper.% CA = Normality x Volume of NaOH x Equivalent wt. Filter the solution & take 100 ml of filtrate solution (or define volume) boil on hot plate or water bath for 204 min. The residue washed with boiled water. Note the final volume of neutralized broth. Keep it in the oven at 70 oC for overnight. To neutralize it we are using phenolphthalein as indicator. 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: [Signature of Student] [Signature of Instructor] . Take the final weight and calculate the weight of citric acid produced in the fermentation broth using the weight of Ca-citrate. / 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.

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

[Signature of Student] [Signature of Lab. Instructor] . Instructor] [Signature of Lab.

(7) . Therefore.coli. 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.rd for batch reactor it can be defined by the following equation. Theory: The rate of endogenous decay (rd) is also formulated according to first order kinetics as given below. During the stationary phase.EXPERIMENT NO: 9 OBJECTIVE: Determination of specific thermal death rate constant (kd) for E. the rate of cell growth (rg) follows first order kinetics. the endogenous decay coefficient can be neglected. rd = can be written as dX = − kd X dt (1) Where kd is first order endogenous decay coefficient (h-1). These yields ln X = µg t Xo (6) Where Xo is initial concentration of biomass. equation (2) can be written as  dX  rnet =   = µ g X − kd X = µnet X  dt net Where µnet is net specific growth rate and can be written as µnet = µg . net rate of cell growth (rnet) rnet = rg .kd (4) (5) For exponential growth phase. Therefore. the reduced equation (4) is integrated with boundary condition (X =Xo at t = 0). For this phase. (2) For biological reactions. rg = dX = µg X dt (3) By using equations (1) and (3).

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

Set temperature and shaking speed to 30 0C and 160 rpm respectively. (ii). X.(NH4)2SO4 Concentration (g/l) 3.0 2. (iv). Use distilled water as blank. Results. Sample analysis. Tabulate your ln [OD/OD0] data along with time in another table. [Note: Optical density gives indirect measurement for cell concentration. (iii). Component 1.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).(ii). (ii). hr-1 for that particular micro-organism.Coli cells with Different initial Substrate Concentrations. Analyse them for cell mass by taking optical density at 620 nm on a spectrophotometer. (i). the inoculum will be ready to inoculate in ten conical flasks containing suitable growth media. Growth of E.Dilute the sample 10 times (1ml of sample in 9 ml of distilled water). kd. (c).5 . Allow it for growth in orbital shaker at 30 0C and at 160-170 rpm for 12 hrs after 12 hrs of growth. (d). 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. Endogenous decay coefficient kd = ……………………hr-1 Figure 1: loge (optical density x 100) is plotted against time (t) (f).] (e). (i). Table 1: Culture Medium for Inoculum & Growth. Glucose 2. Place these ten conical flasks in BOD incubator cum Orbital shaker. Take the slope value in the linear exponential decay region which is equal to specific decay rate.Where (OD) is optical density at time‘t’ and (OD0) is optical density at time‘t’= 0. After sampling heat the samples immediately to stop the growth . Tabulate your ten growth optical data in table 2. (iii). 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. Observation & Calculations. if anyone wishes to measure cell mass directly can measure the dry cell weight gravimetrically. Compute ln [OD620nm*100] for the values in table 2.

7 0.CaCl2 8.9 3.MgSO4.Yeast Extract 9. KH2PO4 5.625 0.125 7. Instructor] . Na2HPO4 4.pH Essential graphs & Tabulation: 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: [Signature of Student] [Signature of Lab.625 0.NaCl 7.3.7H2O 6.019 0.

98 tonnes Required volume of broth per batch: 6. Equation for ellipse.EXPERIMENT NO: 10 OBJECTIVE: Design a fermentation process for production of 5000 tonnes L-lysine per annum using microbial strain.5 hrs Total time: 90 hrs Running hrs per annum: 305 x 24 = 7320 hrs Number of batches/year: 7320/90 = 82 (approx.8 = 1. Annual production: 5000 tonnes/ year Strain used: …………………………….5 hrs Media sterilization: 7 hrs Time required for vessel preparation: 8 hrs Extra time (cleaning. Strain capacity: 48 g/l broth (Yield) Time required for one batch: Fermentation time: 60 hrs Empty sterilization time: 1.265 x 106 = 265 m3 Fermentor: It is a cylindrical vessel with elliptical top and bottom.59 x 106/6 = 0. filtration etc): 13. 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 .27 x 106 lit. Let working capacity of fermentor = 80% Then volume of fermentor = 1.59 x 106 Assuming six fermentor is to be installed with each capacity = 1.27 x 106/0.1 x 104 x 103/48 = 1.) Per batch production required: 5000/82 = 60.

89 = 0.Volume of ellipsoidal Ve = 4 π ζob (b2-y2) dy = 4 π [(b2y-y3/3)]ob = 8 π b3/3 = π D3/24………….69 = 11..89 – 2 x 0.81 m Impeller: Diameter of impeller.38 m L’ = 3D/2 = 3 X 5. For single head side (b= D/4) Now total volume of ellipsoidal head (for both heads) = π D3/24 x 2 = = π D3/12 (ii) Volume of cylindrical portion VL = = π D2L’/4 L’ = L-D/2 = 2D-D/2 = 3D/2 Total volume of fermentor: VF = Ve + VL 3 2 = π D /12 + π D /4 x 3D/2 3 3 3 = π D /12 + 3π D /8 = 11π D /24 Since volume is 265 m3 265 = 11π D3/24 D = 5. total length of liquid/ media broth including an elliptical head HL = Hb + D/4 = 8. distance between two opposite flate blade impeller. H = 2D = 2 x 5.89 m Similarly. Di = DF/3 = 5.47 = 0.69/2 = 8.89 Taking standard 6 flat blade turbine impeller The ratio of Li/Di = ¼ = 0.95 m D = DF .38 m Hi/Di = 1 = Hi = Di = 1.4725 = 0.25 x 1.39 m = Height of broth So that. D’F = Di – 2Li = 1.20 Wi = 0.69 m.89 = 0.69/3 = 1.25 Li = 0.47 m Wi /Di = 1/5 = 0.5 m Volume of broth media in fermentor: 265 x 80/100 m3 = 212 m3 Volume of media = π DF3/24 + πHb DF2/4 = 212 Hb = 7. D = 5.20 x 1.29 .

38 – 8.14 x (1. Area of the hole = π D2/4 = 0.81/1.1 Wb = 0.028 = 100 holes Height of sparger plate from bottom = Hi /2 = 1.8 m3 Taking hole diameter (dh) Since 2.89/2 = 0.945 m.028 m2 Number of holes at sparger plate = Area of sparger plate/ area of hole = 2.89 m = 189 cm) for this diameter the hole diameter should be 189 mm. Foam Breaker: Height from liquid level = Height of fermentor – Height of liquid broth/2 HFb = 11.8/0.5 mm dh (1.81/2 = 1. HL/Hi = 8.22/4 = 2.69 Lb = Length of baffle Lb/Di = 3 Lb = 3Di = 3x 1.60 Baffles: Number of baffles = 4 (standard) Wb = Width of baffle Wb/DF = 1/10 = 0.285 m Dimension of a fermentor equipped with two set of standard flat blade turbine & four baffle plates Discussion: [Signature of Student] [Signature of Lab.89 = 4.1 x 5.5 cm = 2. Instructor] .67 m Diameter of sparger plate = Diameter of impeller Ds = Di Area of sparger plate = π D2/4 = 3.89)2/4 = 11.Number of impeller required.89 = 5.

The reaction mixture contained 200 μl soluble starch in phosphate buffer (0. 200 μl of diluted enzyme and 300 μl phosphate buffer. The medium was autoclaved (121 0C for 15 minutes). 1 filter paper and the filtrate was used as a crude α-amylase. then aseptically added to sterile 500 ml Erlenmeyer flasks (100 ml added per flask). The reaction was incubated for 15 minutes at 30 C. citric acid was produced by fermentative process using Aspergillus nudulans. All data points correspond to triplicates of independent experiments. The slurry was squeezed by muslin cloths. environmental condition pH & dissolved oxygen concentration. pH = 6). α-amylase produced by fermentation is affected by many factors including nutritional composition of media. Stock cultures are reactivated and cultivated by streaking a loopfull of culture on Petri dishes and slants containing medium.EXPERIMENT: 11 OBJECTIVE: Fermentative production of α-amylase by Aspergillus nudulans INTRODUCTION: Citric acid a carboxylic acid. Inoculum preparation: Amylae production: Actively growing and heavily sporulating (ten days) old malt agar slant culture was added to 10 ml sterile distilled water. . MATERIAL & METHODS: Organism: Aspergillus nudulans. Enzyme extraction The contents were mixed by shaking for two hours at 28 0C on a rotary shaker at 200 rpm. allowed to cool. soluble in water with a pleasant taste is the most important acid used in food industries. Before cooling 100 μl Rochelle salt (40 % sodium potassium tartarate) was added and colour was measured at 575 nm. They were cultured in a suitable medium and the temperature and pH in all the batch experiments were maintained. One unit of α-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. 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. Enzyme assay The α-amylase enzyme was assayed accordingly to the method described by Miller.1M.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. The extract was filtered with a Whatman No. All chemicals used were of reagent grade. 300 μl dinitrosalicylic acid (DNS) solution were added and boiled for 15 minutes. First time in 1923. A volume of one ml of spore suspension contained more than 106 spores.

Result: DISCUSSION: [Signature of Student] [Signature of Instructor] .

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