CM4120 Chemical Pilot Operations Lab Bioprocess (Fermentation) Experiment. Presentation Outline

Transcription

1 CM4120 Chemical Pilot Operations Lab Bioprocess (Fermentation) Experiment Dr. David R. Shonnard, Instructor Shu Yat, Teaching Assistant Website: Fermentation Experiment Plan for Spring Semester Presentation Outline v Bio in the ChE Curriculum at MTU q Lecture and Laboratory Components v Integrated Bioprocess Experiments v Pedagogical Objectives: Fermentation v Results from Student Teams v Student Response 1

2 Bioengineering in the ChE at MTU: Lecture Component CM4710 Biochemical Processes (3 cr. lecture) Introduction to Biochemistry Cell growth, kinetics, and yield Bioreactor analysis Bioseparations analysis Applications of molecular biology ln X X o o o o o Slope = µ max t genetic engineering basics ethanol from cellulosic biomass Bioprocess Engineering: Basic Concepts Shuler and Kargi, Prentice Hall, 2002 Integrated Bioprocess Experiments Sugar Sterilizer Air Filter Mobile Autoclave 5-L Batch Bioreactor Air Filter Microfilter Ion Exchanger N 2 Air O 2 uv-visible spectrophotometer Column Fraction Collector FERMENTATION BIOSEPARATION 2

3 Integrated Bioprocess Experiments Fermentation Sampling for cell growth, glucose, and L-Lysine Chromatography Collecting fractions during elution of L-Lysine Learning Objectives: Fermentation for Production of L-Lysine v v v Introduction to Cell Growth and Metabolism q Assume no background in biochemistry Introduction to biochemical process equipment q Key steps in production of a bio-product Introduction to feedback inhibition q Overcome L-lysine production limitation through DNA mutation v Teamwork experience v Biosafety issues v Investigate system response to changes / improvement in production 3

4 Overcoming Concerted Feedback Inhibition Corynebacterium glutamicum ATCC no Concerted feedback inhibition L-aspartic acid aspartyl Phosphate aspartyl semialdehyde L-homoserine L-threonine Enzyme rxn steps dihydrodipicolinate α-ketobutyrate diaminopimelate Desired product L-lysine L-methionine L-isoleucine L-methionine (40 mg/l), L-threonine (150 mg/l), L-leucine (100 mg/l) Design of Fermentation Experiments Each Experiment Lasts hr Amino Acid Concentration Glucose Concentration (g/l) low (50% lower) Team 2 Team 5 2. Base case Team 1 Team 4 3. high (50% higher) Team 3 Team 6 Team (2, 4-student groups) Tasks v Tuesday group ( 8 am until midnight) autoclave fermenter formulate 4 L of defined media, calibrate ph and DO probes, set-up data acquisition, inoculate from flask (35 ml), sample at 2 hr intervals v Wednesday (both groups, 8 am until midnight) q sample at 4 hr intervals v Thursday group (8 am until 4 pm) q measure L-Lysine using saccharopine dehydrogenase 4

5 Theory for Fermentation Experiment v Cell Growth Rate µ = 1 X v Yields dx dt S A µ = µ max K + S K A ln X = µ X max t o S A + Y X/S = X/ S, over exponential and deceleration phases Y P/S = P/ S, over stationary phase Results from Fermentation Experiments Concentration (g/l) exponential deceleration stationary decline 1,E Time (hours) 1,E+01 1,E+00 1,E-01 Cell Concentration (g/l) Glucose Lysine Cells 5

6 Summary of Results: 2001 Team 1 Team 2 Team 3 Team 4 Team 5 Team 6 Initial Glucose Concentration (g/l) Initial Amino Acid Concentration Basecase 1/2 Basecase 150% Basecase Basecase 1/2 Basecase 150% Basecase Maximum L-lysine Concentration (g/l) Maximum Cell Concentration (g/l) µ max (1/hr), Max. Specific Growth Rate τ d (hr) Doubling Time Y X/S (g cells/g glucose) Y P/S (g L-lysine/g glucose) End of semester summary ed to each team q Supplemental amino acids limits the maximum cell concentration q Cell growth and L-lysine production occur in separate phases q It is possible to increase L-lysine concentration > basecase Results: 2002 Contamination of Culture 6

7 Summary of Results: 2002 Contamination of Culture Affected Many Runs Comparison of Data From All 2002 Runs Cycle 1 Cycle 2 Cycle 3 Week 1 Week 2 Week 1 Week 2 Week 1 Week 2 Initial Glucose Concentration (g/l) Initial Amino Acid Concentration Base 150% Base 1/2 Base Base 1/2 Base 150% Base Maximum Lysine Concentration (g/l) Maximum Cell Concentration (g/l) µ max (hr-1) τ d (hr) Y X/S (g cell/g glucose) Y P/S (g lysine/g glucose) Results: g/l glucose, basecase amino acids 25 Lysine, Glucose, and Cell Conc. vs. Time Concentration (g/l) Lysine Glucose Cells Time (hr) 7

8 Results: g/l glucose, 150% basecase amino acids Concentration (g/l) Glucose Concentration Cell Concentration Lysine Concentration Time (hrs) Concentration (g/l) Results: g/l glucose, 50% basecase amino acids Time (hours) Cells Glucose L-Lysine 8

9 Student Response v Students enjoy the transparency and accessibility of the fermentation experiment, and derive satisfaction from producing a commercial product. v Students state that they like the idea of working on an experiment where their results are a part of a larger experiment plan. Acknowledgments National Science Foundation, Instrumentation and Laboratory Improvement (ILI) program Michigan Technological University and the Davis W. Hubbard Memorial Fund James and Lorna Mack Endowment Fund 9