CM4120 Bioprocess Experiments 1 Fermentation; D. Shonnard MEMORANDUM Department of Chemical Engineering Michigan Technological University TO: BioUO Lab Team Cycle 1, Weeks 1-3: L-lysine Production in a Bioreactor Experiment starts Tue. Jan. 11, 2005 in room 205 (CSEB) FROM: David R. Shonnard Associate Professor and Faculty Supervisor Email: [email protected] Phone: 487-3468, Shu Yat Graduate Student Laboratory Supervisor Email: [email protected] DATE: 11 January, 2005 SUBJECT: L-Lysine Fermentation Objectives Introduction Fermentation involves the growth of microorganisms in various bioreactor configurations for the purpose of obtaining some product. The product may be a biomolecule (protein, enzyme, amino acid, etc.) or the cells themselves. The growth characteristics of the microorganisms as they metabolize the carbon source is a very important aspect of the process. In this experiment, you will grow an auxotrophic (the cell lacks the ability to biosynthesize one or more key amino acids required for protein / enzyme production and growth) mutant strain of the bacterium, Corynebacterium glutamicum, on a six carbon sugar (glucose), using an automated bioreactor operated in batch mode over a 2-3-day period. You will monitor for the change in concentration of the cells, glucose, and L-lysine in the culture over time. The task of the Tuesday group will be to set up and start the experiment. Both groups will take samples periodically over the run. The Thursday group will analyze the samples for glucose and lysine and shut down the experiment and clean up. One report will be submitted by the Team. Cycle Objectives Week 1: 20 g/L glucose, and base case concentrations of amino acids, Week 2: Choose among, i. temperature effects on enzyme kinetics ii. gas to liquid mass transfer kinetics. Week 3: Choose the other, i. temperature effects on enzyme kinetics ii. gas to liquid mass transfer kinetics.CM4120 Bioprocess Experiments 2 Fermentation; D. Shonnard For Week 1 you are to; 1. Conduct a batch growth experiment of this bacterium utilizing the carbon substrate glucose in a defined growth medium over a 2-3-day period. Culture the bacteria under the following set of conditions; 30 ˚C, pH = 7.0, dissolved oxygen at a setpoint of 50% of saturation value (in contact with air), The media preparation instructions are provided as part of this assignment. a) Periodically measure the cell concentration of the samples (about 5 ml) take from the bioreactor at 500 nm wavelength (A500 for cells) using a visible spectrophotometer (Milton Roy 21D). The conversion between absorbance and cell numbers is y = 1.034x109 x, where y is the cell numbers per millileter of solution and x is A500. If you wish to express cell concentration in units of mg dry cell weight, you can convert from cell numbers (y) to mg by knowing that there are 0.5 mg dry cell wt. for each 109 cells. A filtered sample from the bioreactor is taken to detect L-Lysine using an enzyme assay and absorbance at 340 nm (A340). Glucose is monitored using blood test strips. From the data over this time period, identify i) the main growth stages and then ii) calculate the maximum specific growth rate (µmax) for this bacterium growing on glucose and iii) it's doubling time at maximum growth rate (hrs). Your group will develop the calibration curves for glucose and L-lysine detection. iv) From data for glucose and L-lysine concentrations, calculate the yield coefficients for cell growth on glucose (YX/ S) and for L-lysine production on glucose (YP/ S). For this yield calculation, only consider the period of time when cells are actively growing for (YX/ S) and when lysine is being produced for (YP/ S). 2. Using the first draft procedures provided to you, conduct the laboratory experiment (in the presence of the faculty advisor and TA) and note improvements to be made. Integrate these improvements into the existing procedures (this statement of objectives and procedures will be attached to email) paying particular attention to the safety and operational aspects. For example, are there ways to speed the preparation for the experiment safely? You should submit these improved procedures as appendices to your final report and mark them in bold print. Also, you should thoroughly discuss the procedure improvements in the body of the final report and address why they are needed. 3. Discuss your cell growth and L-lysine production results in the context of the known metabolism of Corynebacterium glutamicum. For example, does final cell concentration in culture increase or decrease with increase or decrease in initial amino acid concentration? ToCM4120 Bioprocess Experiments 3 Fermentation; D. Shonnard answer this you will need to have access to the results from previous cycles. I will provide you with those results, but please remind me. Is L-lysine production growth associated or non-growth associated and which of these would you expect from your knowledge of Corynebacterium glutamicum metabolism and pathway for L-Lysine production? Are measured yields in the range expected from the literature? Compare to data from your handouts. You were given a tour of the laboratory and the fermentation equipment. However, you may wish to contact me well in advance of preparing your laboratory proposal in order to answer any questions with the equipment, the location of any chemicals used in this experiment, and the use of the computer data acquisition equipment. In addition, read over the handout material prior to writing your proposal in order to gain a better understanding of microbial growth kinetics. The proposal, a team effort, is due on the Monday prior to the experiment, January 24, 2005. For Weeks 2 and 3, you are to prepare shorter proposals for the objectives and for the experimental methods / data analysis in these experiments. Your handout materials contain background information on both temperature effects on enzyme kinetics and gas to liquid mass transfer kinetics. After finishing the fermentation experiment in Week 1, you should have a good idea about how the equipment in the lab can be used to investigate the topics for Weeks 2 and 3. As you conduct the Week 1 experiment, think ahead to the experiments in Weeks 2 and 3 and how the procedures can be modified to accomplish the goals of these additional "mini" experiments. The proposals for these "mini" experiments are