PRACTICAL MANUAL BABS3031/BABS3631 Biotechnology and Bioengineering Biotechnology and Bioengineering (Advanced) Term 2, 2023 Student Name: Student Number: Demonstrator:Practical schedule: [Practical 1 Oxygen Transfer and Uptake] 1 [Practical 2 Cellulases] 8 [Practical 3 Crossflow filtration and diafiltration] 16PRACTICAL 1 1 PRACTICAL 1: [Oxygen Mass Transfer and Oxygen Uptake] Learning outcomes: • Understand how to measure dissolved oxygen and calculate kLa from data • Understand the influence of changing aeration rate and turbulence on oxygen mass transfer • Learn what microbioreactors are and their utility in bioprocess research • Understand the concept of catabolite repression and the impact on oxygen uptake and respiratory/fermentative metabolism Contents: [Introduction]…………………………………………….. Error! Bookmark not defined. [Background Theory]……………………………………………………………………………Error! Bookmark not defined. [Oxygen transfer rate] ……………………………………………………………………………. Error! Bookmark not defined. [Measurement of dissolved oxygen] …………………………………………………….. Error! Bookmark not defined. [Oxygen uptake rate] ……………………………………………………………………………………………………………………………………….2 [Catabolite repression] ………………………………………………………………………………………………………………… 2 [Part 1 Laboratory Measurements – Oxygen Transfer]………………………………………………………………… 2 [Part 2 Laboratory Measurements – Oxygen Uptake]…………………………………………………………………… 4 [Other Useful Information]………………………………………………………………………………………………………………… 5 [Example of sample kLa calculation]……………………………………………………………………………………………………………..5 [Graph: Estimating Saturated Oxygen Concentration (CO2,l*) as a function of temperature] …………… 5 [The polarographic electrode] …………………………………………………………………………………………………….… 6 [Analysis of the results and Report requirements] ………………………………………………….… 7PRACTICAL 1 2 Introduction The calculation of the mass transfer of oxygen in bioreactors is a very important parameter which enables us to estimate the ability of a particular reactor to supply oxygen to the fermentor and support the growth of aerobic cells in the bioreactor. This is especially important in those systems involving high cell densities and rapid growth rates. It also enables us to compare the relative effectiveness and efficiency of different reactors and reactor configurations. The aim of Part 1 of this practical is to study the effect of variables such as the stirrer speed, the number of impellors, baffles, air flow rates, etc on oxygen transfer rates in laboratory-scale fermenters. Background theory Oxygen transfer rate The oxygen transfer rate (OTR) may be defined as follows: (1) ( ) O2, l O2, l L O2, l C*Cak dt dC OTR − == Integrating gives the following relationship: (2) t ak *C C 1 – ln L l O2, l O2, =       − where t = time period of measurement (h) CO2,l* = saturated [O2] in the liquid (mM or mg l-1) at that temperature CO2,l = actual [O2] in the liquid (mM or mg l-1) kLa = O2 mass transfer coefficient (h-1) Rearranging the above also gives: (3) )e(1*CC a.tk lO2,lO2, L − − = Alternatively, data can be re-arranged graphically to solve for kLa: (4) lO2, lO2, * L lO2, C dt dC ak 1 C +         − = Measurement of dissolved oxygen Oxygen electrodes do not measure absolute amounts of dissolved oxygen in gl-1 or mol l-1 rather they measure the partial pressure of the dissolved gas (see the Appendix). The electrodes need to be calibrated in zero and oxygen saturated solutions, to produce a scale from 0-100 % dissolved oxygen tension (DOT). (5) * O2,l O2,l C DOT = 100 * C Therefore we can go from (2) to get: PRACTICAL MANUAL −= Solubility of Oxygen Solutes can affect the absolute dissolved oxygen concentration at saturation (DOT = 100%). See charts below from the lecture notes, showing differences in air at different pressures/temperatures in fresh and salt water.PRACTICAL MANUAL 3 Oxygen Uptake Rate Aerobic cells in culture consume oxygen to function as a terminal electron acceptor in the respiratory chain. Oxygen is generally supplied to fermenters in the form of air or air/oxygen mixes. Oxygen consumption can be described by • the specific rate (QO2 in mmol/cell/h) • the oxygen uptake rate, OUR (mmol/l/h) = QO2.X o where X = cell concentration (cell/l or gX/l) Catabolite repression This term is used to describe the repression of respiration by excess levels of catabolite, usually a sugar. E.coli is subject to catabolite repression in the presence of excess glucose. For high cell density fermentations, this necessitates the implementation of fed-batch processes to avoid or limit the repression of respiration. The main fermentative end product of E.coli is acetic acid; hence, a falling pH suggests some repression of aerobic metabolism due to catabolite repression. Laboratory measurements for Part 1 The procedure will involve your group alternately purging the vessel of oxygen using nitrogen and aerating it under the specific condition being examined. The re-aeration will be monitored via an oxygen electrode connected to an oxygen meter. You will record the increase in DOT using a stopwatch. Working with your fermenter, you should aim to perform 3-6 runs under different conditions (stirrer speed and/or aeration rate). Each run will involve purging the vessel prior to the run with nitrogen, establishing your experimental conditions, then aerating and recording the DOT trace. This data will be used to estimate the kLa for that particular run. Prior to starting, check calibration For this to work, it is vital that you do a few things prior to beginning. 1. Become familiar with the actual controls at your disposal – stirrer speed and aeration rate. 2. Record the temperature 3. “Zero” the system by sparging with nitrogen until no further decrease is observed in the signal on the meter and on the chart recorder. If necessary, adjust the zero on the meter. 4. Now aerate the fermenter by connecting the air line. You should see the DO value on the meter begin to increase. Depending on the conditions you set, this could take 2-10 minutes. Once there is no further increase, ensure the meter reading is 100% (adjust if necessary by adjusting the “span”). How we run this In smaller groups, you should aim to move around the various fermenters so you get at least 3- 4 measurements from at least 3 different fermenters. One will be a bubble column fermenter (you can only adjust flowrate of air) and the others will be stirred tanks (you can adjust stirrer speed and/or air flow rate). Data needs to be entered into a spreadsheet, available in the lab on a desktop PC.PRACTICAL MANUAL 4 In summary, for each run 1. Zero the fermenter by purging it with nitrogen 2. Set-your experimental conditions. Record these clearly in your lab book 3. Switch air on and start your stop watch 4. Record data until at least 80% of air saturation is reached. You will want to record DO data every 5 to 10s 5. One of your group add this to the spreadsheet being collected on the lab PC Part 2 – Microbioreactor cultures and oxygen uptake In this experiment we will operate two “microbioreactors” for each class using the Presens system to look at both oxygen uptake and respirative metabolism. Please have a look at this link so you understand the background: https://www.presens.de/products/detail/sdr-sensordishr reader-basic-set.html . Also have a look at the pre-lab video. The Presens plates contain fluorescence emitting “dots” on the bottom of each of the 24 wells. Plates are made that are either responsive to dissolved oxygen or to pH. For the DO plate, the dissolved oxygen quenches the fluorescence signal, so as oxygen is consumed by the cells, the fluorescence emitted (read by the plate reader on which the plates are situated) increases. Software allows this data to be converted into a measurement of dissolved oxygen. Similarly, for the pH plate, fluorescence emitted depends on the solution pH value. We will be culturing E.coli in LB media (Tryptone, yeast extract and salt) supplemented with Glucose (0, 1,2 and 5 g/l) Below are some examples of readouts using the system: DO versus time. It shows the oxygen signal over time for 12 of the 24 wells.PRACTICAL MANUAL 5 For each lab class we will establish cultures prior to the class commencing. Data will be collected during the class and you can observe the data collection. Parallel cultures will be set up to enable an estimation of cell density in a geometrically identical 24-well plate without the sensor dots. This cell count data and the data from the Presens experiment will be provided for analysis. Other useful information Example of Sample treatment for kLa calculation Sample kLa calculation Time (s) DO% (1-DO/100) =-LN(1/DO/100) 0.00 4.37 0.956 0.045 10.00 25.56 0.744 0.295 20.00 51.33 0.487 0.720 30.00 71.92 0.281 1.270 40.00 83.63 0.164 1.810 50.00 91.89 0.081 2.512 60.00 96.34 0.037 3.308 70.00 97.88 0.021 3.854 80.00 99.00 0.010 4.605 First 2 data points removed. Straight line fitted. Slope = kLa = 0.0654 s-1 = 235 h-1 0.00 20.00 40.00 60.00 80.00 100.00 120.00 0.00 20.00 40.00 60.00 80.00 100.00 Raw Data: DO vs time 0.000 2.000 4.000 6.000 0.00 50.00 100.00 Time (s) -ln(1-DO/100)PRACTICAL MANUAL 6 Graph: Estimating Saturated Oxygen Concentration (CO2,l*) as a function of temperature The polarographic electrode The probe used in bioreactors consists of a platinum electrode (cathode) and a silver electrode polarised with a voltage of 0.7 VDC. The electrodes are immersed in a KCl solution. Dissolved oxygen from the solution is reduced at the surface of the cathode. This sets up a flow of electrons (current) which is proportional to the partial pressure of the gas. Cathodic reaction: O2 + 2 H2O + 2e-  H2O2 + 2 OH- H2O2 + 2e-  2 OH- Anodic reaction Ag + Cl-  AgCl + e Overall 4 Ag + O2 + 2 H2O + 4 Cl-  4 AgCl + 4 OH y = 0.0654x – 0.6888 R² = 0.9967 0.000 0.500 1.000 1.500 2.000 2.500 3.000 3.500 4.000 4.500 5.000 0.00 20.00 40.00 60.00 80.00 100.00 Time (s) “filtered” data -ln(1-DO/100)PRACTICAL MANUAL 7 Analysis of the results and Report requirements 1. A brief introduction describing the aims of the experiment 2. Methods – only note any deviation from what is described here 3. Results 3.1 Oxygen transfer • Briefly summarise the meaning of the terms OUR, kL.a, OTR and QO2 • Using a reference, determine “typical” kLa values for bubble column and stirred tank fermenters • Using the data set collected on your lab day from the different fermenters, calculate kLa values; you will need to manipulate your data, plot it and identify a linear region in order to do this calculation. Present one full sample calculation to show how you did it. Hint: From this equation, t ak 100 1 -ln L =       − DOT you can see that there should be a linear relationship between       − 100 1 DOT and kLa when plotted on a semi-log graph. The slope is the kLa value. You may need to exclude early data points for reasons we will discuss in class. Using graphs if possible, see if you can interpret the effect of stirrer speed, aeration rate and reactor configuration on kLa 3.2 Oxygen uptake and acid metabolite generation using the Presens microbioreactors The experiment involves cultivating E.coli in a growth media supplemented with different levels of glucose. You will see a trend of declining dissolved oxygen in all examples and a decline then possible increase in pH. 1. Note and discuss the differences in the profiles of dissolved oxygen and pH and relate these to different levels of glucose in the media and also batch metabolism of E.coli. If there are differences, explain the role catabolite repression may play in causing these differences. 2. Determine the specific growth rate of E.coli in each condition using the data from the sampled plate 3. Explain why fed-batch culture is used to achieve high cell densities, by overcoming the catabolite repression effect. How do you think feed rate could be controlled to achieve fully aerobic respiration?PRACTICAL MANUAL 8 PRACTICAL 2: [Industrial cellulases for biofuels] Learning outcomes: • Understand how to apply an industry standard Filter Paper Unit assay is used to characterise an industrial cellulase • Undertake a more precise cellobiase assay to look at the cleavage of the beta-1,4 glycosidic linkage between glucose monomers • The role of cellulases in second generation biofuel production Contents: [Introduction]……………………………………………………………………………………. 9 [Background]………………………………………………………………………………………………………………………………………10 [Celluclast] ………………………………………………………………………………………………………………………………………………………11 [Cellobiose and Cellobiase]……………………………………………………………………………………………………………………………11 [Lab Aims] ……………………………………………………………………………………………………………………………………………………….12 [Laboratory Measurements – FPU Assay]……………………………………………………………………………………….12 [Laboratory Measurements – Cellobiose Assay; Glucose Method]……………………………………………..14 PRACTICAL MANUAL 9 Introduction Intensive research has been carried out over decades to generate industrial cellulases for the realisation of second generation biofuels; that is, converting cellulose from low-cost resources into fermentable glucose. In this practical we characterise one such industrial enzyme. The practical is divided into two main parts. Both are centred around batch processing of lignocellulosics to create fermentable glucose for the production of bioethanol and other products by fermentation • Part 1 – Hydrolysing cellulose (Whatman filter paper: FPU Assay) • Using the NREL filter paper unit assay to assess a commercial cellulase preparation for application in the biofuels industry • Part 2- Determining cellobiase activity • Determining the activity of the commercial enzyme preparation with respect to cellobiase activity Background The conventional method to produce industrial grade bioethanol, known as first generation bioethanol, is to utilise sugar and starch crops as the source of the fermentable carbohydrate. Food sources used to provide fermentable carbohydrates include: • Sugar and molasses require little or no pretreatment • Starches (α-1,4 and a-1,6 polymers of glucose) are hydrolysed using enzymes such as amylases However, these resources are costly and use for fuel production may put upward pressure on resource prices, as there are other markets for these products.PRACTICAL MANUAL 10 In second generation biofuels, lignocellulosic resources such as forestry or timber milling waste, agricultural byproduct (eg. wheat or rice straw) or dedicated energy crops such as switchgrass are processed to release cellulose and then enzymically convert this cellulose to glucose using cellulases. A summary of the process is the conversion of lignocellulose into: • Cellulose (β-1,4 polymer of glucose) • Hemicellulose • Lignin Cellulose fraction hydrolysed to produce fermentable glucose Hemicellulose hydrolysed to produce a number of sugars, some of which are not normally fermentable; eg xylose Lignin purified to generate a range of products or combusted Celluclast This enzyme is used in this practical as an example of an industrial cellulase. The figures below show the stability and activity profiles at different pH and temperature values. The enzyme is used in a range of applications, including lignocellulosic processing for biofuels, textile processing and in the food/beverage industry. Here are some useful links from Novozymes, the company that makes Celluclast (and many other industrial enzymes): https://biosolutions.novozymes.com/en/bioenergy Cellic range for bioethanol: https://biosolutions.novozymes.com/en/bioenergy/ethanol/biomass-conversionPRACTICAL MANUAL 11 Cellobiose and Cellobiase Cellobiose is a dimer of glucose linked by a β-1,4 ester bond. This is the bond that links glucose monomers in cellulose. The hydrolysis reaction is described here: Cellobiases hydrolyse the β-1,4 ester bond found in cellulose. Using cellobiose as a substrate, liberated glucose can be used to estimate cellobiase activity. Alternatively, we can use an analogue of cellobiose and use the enzyme to undertake this reaction in the laboratory. CellobiosePRACTICAL MANUAL 12 The p-nitrophenol released is yellow in colour at pH values > 7.5 and can be measured at 410 nm using a spectrophotometer. Aims of this lab The overall aim is to characterise an industrial cellulase (Celluclast) enzyme using two different assays 1. FPU assay 2. Cellobiase assay The end product of the hydrolysis reaction (FPU assay) and the cellobiase reaction is glucose. This will be measured using an enzyme based assay on a separate lab day. Methods 1. FPU Assay This assay is derived from the NREL method (NREL/TP-510-42628, January 2008: https://www.nrel.gov/docs/gen/fy08/42628.pdf) whereby pieces of Whatman paper #1 are subjected to enzyme treatment for 60 minutes and then assayed for released glucose. The adapted procedure that you will follow is described below. 1. Weigh 4 tubes using a 4 place balance. Label the tubes 1-4 & with your initials. 2. To each, add 1 strip of ~50mg Whatman paper and reweigh. 3. Record the weight. Calculate the mass of paper in each tube. 4. Add 1.0ml of Citrate buffer (pH 4.8) to each tube. 5. Place tubes in a rack in the heat blocks in the 50 o C incubator to equilibrate to temperature for 5 minutes 6. After 5 minutes, add the following to each tube: • Tube 1: 500 µl buffer only • Tube 2: 20 µl enzyme + 480 µl buffer • Tube 3: 40 µl enzyme + 460 µl buffer • Tube 4: 80 µl enzyme+ 420 µl buffer 7. Incubate the tubes in heat blocks for 60 minutes. Ensure paper is submerged. Periodically remove tubes and vortex for 5s to mix (2-3 times during the 60 minutes) 8. Remove tubes and place on ice for 5-10 minutes to stop the reaction 9. Remove 1.0 ml of each sample into labelled microcentrifuge tubes 10. Centrifuge at 13000g for 3 minutes. 11. Carefully remove the supernatants and pipette into fresh labelled tubes. You also need to prepare dilutions of each sample using citrate buffer is diluent. You will have the following tubes for each sample: • Undiluted • 1:5 diluted – add 100 µl of sample and 400 µl buffer to a new tube and label • 1:10 diluted – add 50 µl of sample and 450 µl buffer to a new tube and label • 1:20 diluted – add 25 µl of sample and 475 µl buffer to a new tube and labelPRACTICAL MANUAL 13 In all, you should now have 16 tubes, clearly labelled and placed in a rack 12. Place rack for frozen storage. The glucose assay will be performed in the following week. 2. Cellobiase activity by determining release of p-nitrophenol from p-nitrophenyl glucopyranoside Cellobiase creates 2 moles of glucose per mole of cellobiose hydrolysed. In this assay you will add dilutions of Celluclast and a standard cellobiase to a buffered solution, containing the cellobiose analogue, p-nitrophenylglucopyranoside. The assay determines the release of p-nitrophenol, which can be measured at 410 nm. Once the reaction starts, you will add aliquots from the reaction tube to an equal volume of a stop solution (carbonate buffer) at different time points so a graph of p-nitrophenol vs time can be generated. CELLOBIOSE CONTROLPRACTICAL MANUAL 14 Cellobiase Assay (for each group) You will prepare 14 microcentrifuge tubes for this experiment • Controls: 2 Tubes; B and E • Standard: 6 tubes (B-Glucosidase from almonds, Sigma G0395, 2U/mg where 1 U releases 1 µmol of glucose per min at pH 5, 37 degrees) • Celluclast: 6 tubes Add 14 x 150 µl aliquots of Stop solution (pH 9.6 Carbonate buffer) into labelled microcentrifuge tubes. Label these as follows: • Control samples, Beginning and End; B, E • Cellobiose Standard: S0, S1, S2, S4, S8, S16 • Celluclast: Cell0, Cell1, Cell2, Cell4, Cell8, Cell16 Solutions Citrate buffer: 0.05M, pH 4.8 Stop buffer: 0.1M Sodium carbonate, pH 9.6 Substrate: 2 mM p-nitrophenylglucopyranoside (600 mg/l) in citrate buffer (pNPG solution) Standard: A dilution of a standard cellobiase in citrate buffer Celluclast: A dilution of Celluclast in citrate buffer 1. Pipette 1350µl of substrate solution (2 mM pNPG in citrate buffer) into each reaction tube (Control, Standard and Celluclast). Incubate at 50 o C for 10 minutes in the supplied heating block. 2. Add 150 µl of citrate buffer to the Control reaction tube. Mix and remove a 150 µl sample and add to the tube called Beginning (B) 3. Add 150 µl of the Standard Enzyme to the reaction tube labelled Standard. Start timer and immediately remove a 150 µl sample, adding to the stop solution in the tube labelled S0. After 1 minute, remove a 150 µl sample from the reaction and add to tube S1. After 2,4,8,16 minutes do the same adding to S2, S4, S8, S16 respectively. 4. Now repeat using the diluted Cellculast enzyme. Start by adding 150 µl of the Celluclast Enzyme to the reaction tube labelled Celluclast. Start timer and immediately remove a 150 µl sample, adding to the stop solution tube labelled Cell0. After 1 minute, remove a 150 µl sample and add to tube labelled Cell1. After 2,4,8,16 minutes do the same adding to Cell2, Cell4, Cell8, Cell16 respectively 5. Finally, remove a 150 µl sample from the control tube and add to the tube called End Yellow, absorbs at 410 nm When pH>7.5PRACTICAL MANUAL 15 At the end of this, you should have 14 tubes (B, E, S0, S1, S2, S4, S8, S16, Cell0, Cell1, Cell2, Cell4, Cell 8, Cell 16) with 300 ul of solution in each tube. If the reaction has worked, you should see a yellow colour increasing in intensity over time.PRACTICAL MANUAL 16 Data Recording and treatment FPU Assay 1. You will undertake the glucose assay in the Week 7 lab and perform the glucose assay while we are doing the Filtration Practical (Lab 3) in Week 8. You will receive glucose analysis data in the form of a spreadsheet. Here is what you need to do. 2. The plates will have glucose standard loaded in rows A and B. You need to add 50µl of samples to your plate as per the diagram below 3. Create a standard curve: Absorbance values on the x-axis and amount of glucose (nmol/well) on the y-axis. Note 50µl of standard at 2mM = 100 nmol/well, 40µl of standard at 2mM = 80 nmol/well, ……… 4. Determine which of your sample readings “fit” on the standard curve. Covert these into values of nmol/well then nmol/ml. Now multiply by the sample dilution (1,5,10 or 20). 5. Determine a mean result for each sample. For the FPU assay, determine the #FPUs in the Celluclast. Suggested Plate loading for glucose analysis for FPU experiment Example of a Glucose Assay standard curve y = 0.0145x + 0.031