SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Experiment 1

Aim: To prepare broth media for microbial growth. Theory: Bacteria in contrast to fungi are often cultured in a liquid broth (i.e. media lacking Agar). The most common constituents of basic media used in routine bacteriological laboratory are beef extract (a beef derivative which is a source of organic carbon, nitrogen, vitamins and inorganic salts) and peptone (a semi digested protein). These may be modified in a variety of ways by supplementing with some specific chemicals or materials to provide a medium suitable for the cultivation or demonstration of a reaction for specific types or groups of bacteria. Nutrient broth and glucose broth have been considered as basic liquid media for cultivation of bacteria. Materials Required: Constituents for Nutrient broth Peptone Beef extract Distilled water 5.0g 3.0g 1000.0ml

Constituents for Glucose broth Peptone Glucose Sodium Chloride Distilled water 10.0g 5.0g 5.0g 1000.0ml

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

1 N HCl 1N NaOH pH meter Distilled water Hot plate or Heater Autoclave Beaker corning, 50ml, 250ml Measuring cylinder Cotton Culture tubes Glass rod Weighing balance Spatulas

Procedure: Nutrient broth 1. Put the weighed amount of peptone (5g) and beef extract (3g) in 500ml of distilled water in a beaker. 2. Heat with agitation to dissolve the constituents. 3. Add more distilled water to make the total volume as 1 liter. 4. Adjust pH of the medium to 7.0, using a pH meter, by adding either acid or alkali, as the case may be. 5. Pour 10 ml of media in to 50ml corning flask. 6. Apply cotton plugs on the mouth of coning flasks 7. Cover the cotton plugs with aluminum foils tightly. 8. Autoclave at 121oC, 15 psig pressure for 20 minutes. 9. Put off the power supply for the autoclave and remove the plug from the socket. 10. Allow the autoclave to cool to room temperature (make sure that the pressure in side the autoclave is shown zero). 11. Release the pressure valve completely and wait for a minute before opening the autoclave lid.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

12. Take out the nutrient broth corning flasks and label them using a marker pen and put your name along with date and incubate at 37oC temperature in the incubator. 13. After 48 hrs check for any contamination or turbidity in the broth and if there is any turbidity noted in the broth then repeat the above steps. 14. If no turbidity is observed then store it in the refrigerator for further use.

Glucose broth Glucose broth can be prepared in the same way as nutrient broth.

Observations: The media flasks are to be kept under observation / incubation for 48 hrs at room temperature or in an incubator at 37oC and check weather there was any contamination in the broth media. If there is any contamination then discard the media and repeat the experiment till you succeed to have a sterile broth.

Result: Briefly write about the conclusion and result of the experiment performed.

Note: Clean all your glassware and the work bench after finishing your experiment.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Experiment 2 Aim: To culture the microbial organisms in a shake flask using orbital shaker incubator. Theory: Microbial culturing is performed by using broth as a liquid media in the laboratory on a small scale or in an industrial fermenter on a large scale. The media termed broths, milks, or nutrient solutions are made by dissolving various solutes in distilled water and later sterilizing (autoclaving) it. Appropriate physical and chemical environments are required for growing microorganisms in a broth. Temperature is the most vital requirement for growing cultures which is supplied by incubator in the laboratory. Many cultures also require oxygen, a certain amount of which is absorbed in to the surface of broth in the flask placed in an undisturbed rack on the incubator shelf; this condition is termed as static incubation. Now a shaker incubator having shaking platform fitted with conical flask holders, where the culture flasks can be put up, are used to provide oxygen to broth cultures and at the same time maintains the desired or set temperature. For large volumes of broth, air is filter sterilized and pumped in to large broth filled vessels, fermentors.

Growth of microorganism occurs throughout the container and may present a dispersed cloudy or particulate appearance. Some microorganisms uniformly increase the turbidity (milkiness or cloudiness) of broth, due to increase in their numbers. Pellicle, a mat of cells, is formed by some microorganisms which grow only at the surface of a static broth culture. Other microbial cultures may settle to the bottom of the flask/ tube to form a sediment or button of cells that stick together. Filamentous fungi form intertwined mycelia (clumps) suspended within a broth.

The prime use of broth cultures is to study the characteristics of microorganisms and for the production of various industrial products by the microorganisms, e.g. production of antibiotics, mass production of bakers yeast etc.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Materials Required: Sterile broth media ( Nutrient broth / Glucose broth ) Transfer loop Non adsorbent cotton 70% alcohol Microbial culture ( Bacillus subtilis ) Spirit lamp Match box Laminar hood Shaker incubator Tissue paper Marker

Procedure: 1. Take the transfer loop, non-adsorbent cotton, 70 % alcohol, Bunsen burner & tissue paper and place them in the laminar air flow chamber. 2. Put on the UV light in the laminar hood and wait for 15 minutes. Make sure that the laminar hood is closed properly before the UV light is on. 3. Put off the UV light after 15 minutes and put on the fluorescent (white) light and the laminar air flow. 4. Open the lower lid (part) of the laminar hood and place the sterile media and the culture microbe on the work bench of laminar hood. 5. Fold your both hand clothes beyond the elbows and wipe the palms and hands up to the elbows with 70% alcohol. 6. Spray the 70% alcohol on the surface of work bench and further wipe the surface using non adsorbent cotton or tissue paper and keep the cotton /tissue paper at a corner. If required take the assistance of the instructor. 7. Light the spirit lamp / Bunsen burner and adjust the flame to an appropriate level. 8. Flame the transfer loop to red hot condition and then wait for a minute to cool the loop.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

9. Open the cotton plug of the microbial strain in the vicinity of flame and take a loopful of the culture and close the culture tube with the same cotton plug. Caution, do not keep the loop on the work bench and hold it in your hand for the next step. If required, take the help of the instructor. 10. Take the sterile media flask and open the cotton plug in the vicinity of flame and transfer the loopful of microorganisms in to the media by dipping and shaking of the loop in the media. Then immediately close the mouth of the media flask with the cotton plug in the vicinity of the flame only. 11. Now flame the transfer loop to red hot and then keep it on the work bench. 12. Before closing, put off the burner flame, clean the work bench and wipe the spills using the tissue paper and drop it in to the dust bin. 13. Take out the inoculated media and the culture strain from the laminar and put off the laminar air flow and white light and then close the laminar lid. 14. Place the microbial strain in the refrigerator 15. Label the inoculated media flask with your name and put the date and time using marker pen and place the flask on the plat form of shaker incubator. 16. Set the temperature at 30oC and shaker speed at 90 rpm. If required take the help of instructor. 17. Wait for 24 hours to observe the growth of microbial culture.

Observations: Observe the turbidity of the broth as the indication of growth of microorganisms. Store it in the fridge for future use. Result: Briefly write about the conclusion and result of the experiment performed. Note: Clean all your glassware and the work bench after finishing your experiment.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Experiment 3 Aim: To estimate the Microbial biomass produced through shake flask culturing. Theory: Estimation of bacterial growth requires inoculation of a sterile broth medium and incubation of the culture under optimum temperature and gaseous conditions. The bacteria will reproduce rapidly under these conditions and the microbial growth may be measured by variety of techniques like wet weight determination, dry weight determination, determination of total nitrogen by Kjeldahl method, direct counting of cells under a light microscope, or indirectly by viable count (plate count) which detects living organisms by their ability to form colonies on agar surfaces, and turbidity measurements, which relate cell number to the turbidity (cloudiness) of a broth culture.

Here we will estimate the biomass concentration by wet weight method and it can be extended to dry weight as well. Initially it is better to start with the determination of wet weight of biomass using centrifuge to make a pellet of the particle like bacterial cells suspended in the broth. To weight the biomass simple digital balances can be used with higher sensitivity as the biomass collected would be of very small quantity of mg order.

Materials Required:

Microbial broth culture Pipettes ( 5/10 ml ) Centrifuge tubes Centrifuge Weighing balance Blotting paper Tissue paper Marker pen

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Procedure: 1. Take the shake flask containing the broth from the refrigerator and keep it in on the work bench to bring it to normal room temperature. 2. Take the centrifuge tubes and label them as 1, 2, 3 & 4 on the tube as well as on the cap and then weigh them using the digital balance. Note the readings. 3. Evenly distribute the broth from the shake flask using the pipette in to centrifuge tubes (10 ml in each tube). 4. Put the centrifuge tubes in to the centrifuge rotor and make sure that the tubes are arranged in the opposite holdings / holes. 5. Close the centrifuge lid and make sure that it is locked. 6. Put the power plug in the socket and switch it on. 7. Put on the mains switch of centrifuge. Here you can observe the indicator glow as an indication to power supply to the machine and the time display will be blinking. 8. Switch on the rotor by rotate the speed controlling knob clockwise and observe the speed display for the rotor speed and then finely keep rotating the knob till you get the speed of 5000 rpm. 9. Now set the time for the centrifugation period by pressing the time button step by step till you get 15 min display. 10. Now, wait till the centrifuge is stopped after 15 minutes and observe the displays of both time and speed as zero as an indication for the completion of centrifugation process. 11. Put off the centrifuge mains and power supply, then open the centrifuge lid. If required take the help of the instructor. 12. Take out the tubes and decant the supernatant (liquid portion) in to the sink and make sure that the biomass pellet is retained in the tube undisturbed. 13. Use the blotting paper to remove the droplets from the tube with out touching the biomass pellet. 14. Close the centrifuge tubes with caps and weight them using the digital balance and note the corresponding readings. 15. Clean the centrifuge tubes using the detergent water and put them in the hot air oven at 50 oC for drying. If required take the help of instructor. 16. Wash the pipette also using the detergent water and put up in the hot air oven.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Observations S. No Sample Sample Volume (ml) 1 Microbial broth culture 2 3 10 Initial weight (gm) Final weight (gm) Wet weight Microbial of biomass concentration (gm) (mg/ml)

Initial weight: Weight of empty centrifuge tube with cap Final weight: Weight of centrifuge tube with microbial biomass pellet after centrifugation Wet weight of biomass = (Final weight) (Initial weight) Microbial concentration = (wet weight of biomass) / Sample volume

Result: Report the final result as the average concentration of the microbial culture in the shake flask for 24hrs culturing using the shaker incubator.

Note: Clean all your glassware and the work bench after finishing your experiment.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Experiment 4

Aim: To plot Microbial growth curve for shake flask culturing using turbidity method.

Theory: Estimation of bacterial growth requires inoculation of a sterile broth medium and incubation of the culture under optimum temperature and gaseous conditions. The bacteria will reproduce rapidly under these conditions and the microbial growth may be measured by variety of techniques and one of the indirect methods of measure is turbidity measurement, which relates cell number to the turbidity (cloudiness) of a broth culture. Turbidity or optical density (OD), being an indirect measure of bacterial number, the increase in turbidity indicates bacterial growth. OD is directly proportional to cell concentration. To measure the OD spectrophotometer can be used. Bacterial growth curve plotting requires the samples of a 24hr shake flask culture to be measured for population sizes at regular time intervals during the incubation period. The bacterial growth curve shows four distinct phases of growth, lag phase, logarithmic phase, stationary phase and death phase, the actual length of each phase varies with the organism and with environmental conditions.

Materials required: Sterile Nutrient broth media (100ml) Inoculum Laminar hood Spirit lamp Match box Micro Pipettes (1000l) Sterile micro tips (1000l) Centrifuge( SPINWIN ) Distilled water Blotting paper Tissue paper Marker pen

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Procedure: 1. Prepare 100ml of nutrient broth in a 250ml of conical flask and sterilize it using autoclave. 2. Inoculate the sterile media with the given inoculum of 2ml aseptically in the laminar hood and shake well for uniform distribution of inoculum. 3. Aseptically remove 1.5ml of culture broth from the shake flask using sterile micropipette and add it to a centrifuge tube. (Make duplicate of samples.) 4. Incubate the conical flask in the shaker incubator at 30oC and 110 rpm. (Here initially note the time to be zero.) 5. Centrifuge the broth solution at 10000 rpm to get the pellet of biomass for 10min. 6. Decant the supernatant and add 1.5 ml of distilled water to the pellet. 7. Place the centrifuge tubes in to the holes of vertex mixer platform and spin it vigorously for two minutes to suspend the microbial pellet in the water. 8. Switch off the vertex mixer and take out the tubes and here you can observe the turbid solution of cells. 9. Take the OD of this turbid solution at 600nm using spectrophotometer and note it in the record. 10. After one hour stop the shaker of the orbital shaker and take out the shake flask carefully and put it in the laminar hood. 11. Repeat steps 3-10 till 12 hrs of incubation time. 12. Plot the growth curve with time on X-axis and OD on Y-axis.

Observations 10000 rpm for 10 @ S. No Time (hr) 1 2 0 0.5 Sample Addition of distilled OD @ Remarks water to pellet 1.5 ml min 620nm

volume 1.5ml Centrifugation

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

1.0 1.5

10hr

Plot the graph between OD600 & Time (hr) and identify the lag phase, log phase etc. Result: write about the growth curve and mention about lag period and log period etc. and also put your remarks if any. Note: Clean all your glassware and the work bench after finishing your experiment.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Experiment 5 Aim: To Estimate the Monod Parameters for microbial growth kinetics Theory: Kinetic equations, which describe the growth of microorganisms on a particular substrate, are crucial in understanding many phenomena in biotechnological processes. So, quantitative experimental data is used for the design and optimization of biological transformation processes. A variety of mathematical models have been proposed to describe the kinetics of substrate nutrients exposed to pure cultures of microorganisms or microbial populations of natural environment. Among those models, Monod model has been widely used to describe growth-linked substrate utilization as shown in the following equation:

max s
Ks s

Where,

= max Ks S

Specific growth rate of microbial cells = = = Maximum specific growth rate of microbial cells Substrate constant Concentration of substrate

Characterization of the microbe-substrate interactions involves estimation of several parameters in the kinetic models from experimental data. In order to describe the true behavior of the system, it is important to obtain accurate estimates of the kinetic parameters ( max , Ks ) in this model.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Materials required: Sterile Glucose broth media (100ml) Inoculum ( E.coli ) Laminar hood Spirit lamp Match box Micro Pipettes (1000l) Sterile micro tips (1000l) Centrifuge( SPINWIN ) Distilled water Blotting paper Tissue paper Marker pen Spectrophotometer

Procedure: 1. Prepare 100ml of glucose broth in a 250ml of conical flask and sterilize it using autoclave. 2. Inoculate the sterile media with the given inoculum of 2ml aseptically in the laminar hood and shake well for uniform distribution of inoculum. 3. Aseptically remove 1.5ml of culture broth from the shake flask using sterile micropipette and add it to a pre-weighed centrifuge tube. (Make duplicate of samples.) 4. Incubate the conical flask in the shaker incubator at 30 oC and 100 rpm. (Here initially note the time to be zero.) 5. Centrifuge the broth solution in the centrifuge tubes at 10000 rpm for 10min to get the pellet of biomass. 6. Collect 1ml of supernatant in a vial and discard the remaining supernatant. 7. Weigh the pellet along with centrifuge tube using a balance. (note the reading for calculating the wet weight) 8. Use the collected supernatant in the test tube as a sample to estimate the glucose concentration using anthrone method.

16

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

9. After one hour stop the shaker of the orbital shaker incubator and take out the shake flask carefully and put it in the laminar hood. 10. Repeat steps 3-7 till 12 hrs of incubation time. 11. Plot the growth curve with substrate consumed (mg/ml) on X-axis and specific growth rate (min as well as wet weight of Biomass on Y-axis.

Observations: Make note of all your readings and the observations in the following two tables. Table - 1 S. No Time Sample volume (ml) 1 2 3 Centrifugation @ 10000 rpm for 10 min 1.5ml Wet weight Cell of (mg) Cell growth Sp. Cell

pellet concentration rate, (X/t) ^, X (mg/ml)

growth rate , (X/t)/X

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

^ Wet weight of cells per ml of sample volume X = Xi Xi-1 ; t = ti - ti-1

Table -2 Sl. No Time (hr) Sample volume (ml) 1 2 3 Centrifugation @ 10000 rpm for 10 min 1.5 1.0 Supernatant volume (ml) Follow the anthrone method to determine the glucose concentration OD620 Reading Substrate concentration (mg/ml)*

* The values must be obtained from the standard plot of glucose concentration corresponding to the obtained OD values.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Result: Write about the monads growth curve and mention the values of monad parameters i.e. max and Ks from the graph. Note: Clean all your glassware and the work bench after finishing your experiment.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Experiment - 6 Aim: To get familiarized with the lab scale fermenter (bench top fermenter) Theory: Fermenters are the reactors modified and designed for the operation and maintenance of fermentation process using microbial organisms. The vessel used for the fermentation process is generally of cylindrical shape with a round bottom and a round top made up of stainless steel at pilot and industrial scale. In case of laboratory bench top fermenters the body of the vessel is ideally made up of transparent glass of appropriate thickness with a stainless steel top accommodating the provisions for the monitoring of process parameters such as temperature, pressure, dissolved oxygen concentration, pH, antifoam agents etc. and maintenance of sterility on the fermenter. The process parameters are usually monitored through probes inserted from the top stain less steel plate (in case of bench top fermenter) and these probes are in turn connected to an electronic control panel where the potentiometric signals of the probes converted in to digital signals for the display during the process for real time monitoring. In addition, the fermenter is also connected with an air sparger through which the sterile air is spurged in to the fermenter. This sterile air is generated using a compressor which sucks the air from the atmosphere and pumps it through an air filter, and this sterile air pipe line is connected to a flow regulation unit, where the air flow rate can be regulated through a knob fitted to the rotameter. For the regulation of the temperature, a chilled water supply unit is connected to the fermenter externally through a rectangular coil, and the control is provided by the flow control unit as it also connects the chilled water supply unit and the fermenter. And these all parameters are controlled automatically trough the set values of process parameters in the control panel. The bench top fermenter can be represented diagrammatically as shown in the figure below.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Figure: Bench top fermenter

Figure: Control panel and stocking rack of Bench top fermenter

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Required Materials Bentch top fermenter o Cylindrical glass vessel o Control panel o Compressor o Chilled water supply unit o Flow regulatory unit o Sensors o Silicone rubber Piping o Buffering bottles ( Acid /Base) o Peristatic pumps o Distilled water etc Procedure: 1. Take a lab note book, pencil, eraser and a pen and go to the bench top fermenter. 2. With the help of the instructor identify the major parts of the fermenter and familiarize yourself. 3. Note all the probes present in the fermenter and identify the connections with the fermenter and control panel 4. Draw and Label the fermenter unit along with the control panel, compressor, flow regulator and Chilled water supply units. ( Ask the instructor for any clarification and suggestions) 5. Write briefly about the major parts of the fermenter in your observations.

SPSU UDAIPUR Department of Biotechnology

LAB MANUAL- BIOPROCESS ENGINEERING BT -303

Observations: Draw the neat and labeled diagram of the bench top ferment and give a brief description about the major parts of the different units in the bench top fermenter.

Result: Briefly write about your familiarization with the bench top fermenter.