1CM4120 Chemical Pilot Operations LabBioprocess (Fermentation) ExperimentDr. David R. Shonnard, [email protected] Yat, Teaching [email protected]: http://www.chem.mtu.edu/~drshonna/cm4120/index.htmlFermentation Experiment Plan for Spring SemesterPresentation OutlinevBio in the ChE Curriculum at MTUqLecture and Laboratory ComponentsvIntegrated Bioprocess ExperimentsvPedagogical Objectives: FermentationvResults from Student TeamsvStudent Response2Bioengineering in the ChE at MTU:Lecture ComponentCM4710 Biochemical Processes (3 cr. lecture)• Introduction to Biochemistry• Cell growth, kinetics, and yield• Bioreactor analysis• Bioseparations analysis• Applications of molecular biology→→→→ genetic engineering basics→→→→ ethanol from cellulosic biomass“Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 lnXXotooooSlope = µµµµmax5-L Batch BioreactorMobile AutoclaveAirN2Air FilterAir FilterMicrofilterIon ExchangerColumn Fraction Collectoruv-visible spectrophotometerFERMENTATIONBIOSEPARATIONSugar SterilizerO2Integrated Bioprocess Experiments3Integrated Bioprocess ExperimentsFermentationSampling for cell growth,glucose, and L-LysineChromatographyCollecting fractions duringelution of L-LysineLearning Objectives:Fermentation for Production of L-LysinevIntroduction to Cell Growth and Metabolismq Assume no background in biochemistryvIntroduction to biochemical process equipmentq Key steps in production of a bio-productvIntroduction to feedback inhibitionq Overcome L-lysine production limitation through DNA mutationvTeamwork experiencevBiosafety issuesvInvestigate system response to changes / improvement in production4L-asparticacidaspartylPhosphateaspartylsemialdehydeL-homoserineL-threonineαααα-ketobutyratedihydrodipicolinatediaminopimelateL-lysine L-methionine L-isoleucineConcerted feedback inhibitionEnzyme rxnstepsDesired productL-methionine (40 mg/L), L-threonine (150 mg/L), L-leucine (100 mg/L)Overcoming Concerted Feedback InhibitionCorynebacterium glutamicum ATCC no. 21253 Glucose Concentration (g/L) Amino Acid Concentration 20 30 1. 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) Tasksv 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 intervalsv Wednesday (both groups, 8 am until midnight)q sample at 4 hr intervalsv Thursday group (8 am until 4 pm)q measure L-Lysine using saccharopine dehydrogenaseDesign of Fermentation ExperimentsEach Experiment Lasts 48-52 hr5vCell Growth RatevYieldsµ=1XdXdtAKASKSAS++=maxµµlnXXo=µmaxtYX/S= ∆X/∆S, over exponential and deceleration phasesYP/S= ∆P/∆S, over stationary phaseTheory for Fermentation ExperimentResults from Fermentation Experiments05101520250 5 10 15 20 25 30 35 40 45 50Time (hours)Concentration (g/L)1,E-021,E-011,E+001,E+01Cell Concentration (g/L)Glucose Lysine Cellsexponentialdeceleration stationary decline6Summary of Results: 2001 Team 1 Team 2 Team 3 Team 4 Team 5 Team 6 Initial Glucose Concentration (g/L) 20 20 20 30 30 30 Initial Amino Acid Concentration Basecase 1/2 Basecase 150% Basecase Basecase 1/2 Basecase 150% Basecase Maximum L-lysine Concentration (g/L) 2.09 2.21 7.51 2.55 10.0 0 Maximum Cell Concentration (g/L) 9.5 4.0 8.5 8.0 3.9 2.1 µµµµmax (1/hr), Max. Specific Growth Rate 0.38 0.50 0.33 0.43 0.42 0.30 ττττd (hr) Doubling Time 1.82 1.39 2.09 1.6 1.64 2.31 YX/S (g cells/g glucose) 0.47 0.33 0.35 0.27 0.99 0.02 YP/S (g L-lysine/g glucose) 0.14 0.60 0.32 0.23 0.60 0 End of semester summary emailed to each teamqSupplemental amino acids limits the maximum cell concentration qCell growth and L-lysine production occur in separate phases qIt is possible to increase L-lysine concentration > basecaseResults: 2002Contamination of Culture7Summary of Results: 2002Contamination 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) 20 20 20 30 30 30 Initial Amino Acid Concentration Base 150% Base 1/2 Base Base 1/2 Base 150% Base Maximum Lysine Concentration (g/L) 6.3 2.21 1.07 2.57 1.84 Maximum Cell Concentration (g/L) 5.9 10.2 7.8 11.9 12.6 µµµµmax (hr-1) 0.39 0.49 0.48 0.50 0.43 ττττd (hr) 1.77 1.41 1.54 1.38 1.61 YX/S (g cell/g glucose) 0.3 0.34 0.48 0.35 0.078 YP/S (g lysine/g glucose) 0.34 0.15 0.055 0.25 0 Results: 200420 g/L glucose, basecase amino acidsLysine, Glucose, and Cell Conc. vs. Time05101520250 102030405060Time (hr)Concentration (g/L)LysineGlucoseCells8Results: 200420 g/L glucose, 150% basecase amino acids0510152025300 102030405060Time (hrs)Concentration (g/L)Glucose ConcentrationCell ConcentrationLysine ConcentrationResults: 200420 g/L glucose, 50% basecase amino acids024681012141618200 102030405060Time (hours)Concentration (g/L)CellsGlucoseL-Lysine9Student ResponsevStudents enjoy the transparency and accessibility of the fermentation experiment, and derive satisfaction from producing a commercial product.vStudents 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