BIOCHEMICAL PROCESS INDUSTRY
BIOCHEMICAL PROCESS INDUSTRY MANUFACTURING OF PRODUCTS FORMULTIPLE MARKETSFOOD & BEVERAGEHEALTH CARETHERAPEUTICSDIAGNOSTICSDEVICESPECIALTY CHEMICALWASTE TREATMENT MANUFACTURING BY MULTIPLESYNTHETIC & EXTRACTIVETECHNOLOGIESBIOSYNTHETIC - MICROBIAL, ANIMALEXTRACTIVECHEMICAL SYNTHESIS PRODUCTS BELONG TO MULTIPLECLASSESPROTEINSNUCLEIC ACIDSCARBOHYDRATESCATABOLITES AND ANABOLITESCELLS AND VIRUSES
Selling Price vs. VolumePrice (cents/lb)H. Scherfler, 10th International Biotech Symposium, Sydney AustraliaAnnual Production (ton/y)
Revenue Rate AnalysisProductConcentrationCitric AcidEthanolGlutamic AcidGluconic AcidLactic Xanthan GumVitamin B12ProductivityPriceRevenue Rate(g/liter)(g/liter-day)( /kg)( ageStd. Dev.0.160.11
Supply ChainUTILITIESRA WM A T’LSSY N TH ESISD SPFIN A LPRO D U CTM A RK ETWASTESD ETERM IN ES PLA N T SIZEA N D PRO D U CT SPECS
Relationship of Profit toPriceSelling price isFixed by utilityAnd competitionMarket size determinedby problem solvedPROFIT VFM (SPSA CM )Market share is afunction of ovation speedManufacturingcostSpecificActivitySensitivity of Profit to Sp, Sp & CmSaSp(Units/lb) ( ( /lb) MM55355555552103309010205070V 30 b lb sugar & Fm 0.2
Outline for microbial growthGROWTH AND METABOLISMA QUANTITATIVE OVERVIEWCHARACTERISTICS OF CELL GROWTHBacteria, Yeast and Mycelial FormsMEASUREMENT OF GROWTHDirect Measurement of Cell NumberDirect Measurement of Cell MassIndirect Assessment of GrowthTHE RATE LAW FOR GROWTH AND PRODUCT FORMATIONExponential GrowthCell DeathCell LysisDependence on Cell TypeDEPENDENCE OF GROWTH RATE ON ENVIRONMENTTemperaturepHIonic Strength and Os molalityNutrient Supply and ConcentrationREQUIREMENTS FOR GROWTH - MEDIA DESIGNProcess ObjectivesNutritional RequirementsEnvironmental RequirementsRegulatory ConstraintsTechno-Economic Constraints
THE GOAL IS TO PRODUCE AN ADEQUATE AMOUNT OF AQUALITY PRODUCT CONSISTANTLY AND AT A LOW COSTProcess Constraints: Product is made by organism Product is intra- or extra-cellular The culture environment must meetspecific conditions The equipment exists or needs to bedesigned FDA, EPA, OSHA constraints The cost of product in broth cannotexceed /kg or liter. kg/mo of product is needed formarketP Q p d t q p Xd tProcessProductivityOxygen or HeatTransferLimitationCell Mass Concentration
FERMENTATION AND CELL CULTURPRODUCT AND PROCESSDEVELOPMENTPROCESS DEVELOPMENT GOALSMAXIMIZE THE AMOUNT OF "ACTIVE" CELL MA- IN AN AFFORDABLE MANNARMAXIMIZE THE SPECIFIC PRODUCTIVITY, qp,ASSOCIATED WITH THE CELL SGenetic ControlEnvironmental ControlMATCH PROCESS GOALS WITH EQUIPMENTCABABILITY - OXYGEN AND HEAT TRANSFERMINIMIZE THE PROCESS TIMEREPRODUCIBLE PERFORMANCE - ROBUSTNESSSTRATEGY:REMOVE OPERATIONAL BARRIERSCONTROL AT CRITICAL POINTSSYSTEMS APPRAOCH TO MEDIUM, CULTUREGROWTH, PROCESS OPERATION
CHARACTERISTICS OF CELL GROWTHYEAST GROWTH - CELL DIVISION BY BUDDINGDaughter CellMother CellPSEUDOMYCELIATEM of S. cerevisiae grown inYEPG medium in stationary phase(T. Srinorakutara J Ferm Bioeng 86 253 1998)
Fermentation ProcessDevelopmentMolecular BiologyExpression systemPlasmid design and copy numberControl of metabolismA B -- CA C --- D ESet of Enzymes andExperimental ParametersHost cell selectionExpression systemMedia designFermentation conditionsAeration strategyCell harvesting strategyReactionsMetabolic pathwaysProcess kineticsO2CellEnvironmentCO2GlucoseH2OProductNH4 Net BiochemistryElemental balancesSolubility & EquilibriaPerformance AssessmentGrowth RateProduct ConcentrationBy-Product ConcentrationRaw Materials UtilizationCornSryupReactor s.FlowD.O.Mass transferWater balanceEquipment correlationsAirTempProductComp.FlowWt
Cell compositionCELL PROPERTIES AND COMPOSITION*Size (µ)ShapeVolume (µ3)Weight (g/cell)DensityDoubling 4Molds5- 50mycelia,pelletsActino's2- 12-202-35- 305-105-745-557-121-210- 50105-740-507-121-210- 30105-755-6010-152-35-105-105-760*Composition is based on Dry Cell Weight which is approximately 20% of Wet CellWeight.
MEASUREMENT OF CELL GROWTHMETHODS FOR MEASURING AND ESTIMATING CELLGROWTH - CRITERIA FOR TECHNIQUE SELECTIONDIRECT MEASUREMENT OF CELL NUMBERApplies to single cell, non-aggregating cells in suspensionRequires separation of individual cellsCan distinguish between viable and non-viable cellsOpportuni ties for cell sortingDIECT MASUREMENT OF CELL MASSApplicable to all cells and cell aggregatesRequires separation of cells form non-cellular solidsDoes not distinguish between viable and non-viable cellsINDIRECT ASSESSMENT OF CELL GROWTHApproach is based on stoichiometry of growth andmetabolismAccuracy depends on the technique and assumptionsApplicable to all cell typesTechniques can be used in systems with non-cellular solids
Direct Estimation of Cell NumberDIRECT MICROSCOPIC COUNTAREAPETROFF - HAUSSER SLIDED 0.02 mmD DEPTHHEMOCYTOMETER SLIDED 0.1 - 0.2 mmVIABLE PLATE COUNTSAMPLEDILUTEPLATEAgar MediumINCUBATESLIDE CULTURESAMPLEDILUTEINCUBATE2-3 Td
Cell Mass Estimation by ViscosityEinstein equation for viscosity of suspensionsµc viscosity of cell suspensionµc (1 0.5C)µr supernatant viscosity 2C volumetric particle conc.µr(1 C)Note: weakdependenceon conc.µc432 (2.43x10 )C (2.79x10 )C 1.01µrPerley, et. Al (Biotech. Bioeng. 21,519, 1979)
DIRECT MEASUREMENT OF CELL MASSDRY CELL WEIGHT (DCW)Microbial cells are typically about 20-25% solids when dried to con stanmass. Thus,Wet Cell Weight is approximately 4.5(DCW)PROTOCOL FOR DRY CELL WEIGHT MEASUREMENTSAMPLESOLID/LIQUIDSEPARATIONWASHDRYWhat is the optimal size?Soluble and insolubleimpuritiesPossible leaching vs.contamination with saltsOverdrying?
CULTURE BROTH TURBIDITYdIdLtln ECItItIo ECLIo Intensity of initial lightIt Intensity of transmited lightE Molar extinction coefficientC ConcentrationL Length of light pathBeer - Lambert LawOpticalDensity2 OD Units/g DCW250 KU/g DCWConcentration
Effect of Temperature onGrowthµ Ae ERTE 60-70 kcal/mol
µ1/ 2 bT bToRatkowsky, et al., Jbacteriol 149 1-5 (1982)
Aspergillus amstelodamiSalmonella newportStaphyloccus aureus vmφln aw 55.5aw water activityv number of solute ionsm molar concentrationø molar osmotic coefficient
Allocation Model forSubstrate Utilizationq pXdS µX mX dt YmaxYp/ sµ f(cell, Cs, Cp, pH, T, aw, t)Use hyperlinks to other slides
Strategies for MetaboliteOverproduction Random mutagenesis and selection– Example of penicillin Environmental control & optimization Metabolic engineering– Enhance or alter metabolite or electron flow– E.g. AAs, isoprenoids (kiesling), polyketide(chethan) Alteration of control– Flux control (inhibition or acceleration)– Transcriptional control
Influence of Glucose on Fermentation
H. Scherfler, 10th International Biotech Symposium, Sydney Australia
Mycelial Pellet GrowthPenicillium crysogenumt 0ht 11 ht 23 h
Mycelial GrowthHyphal tipelongationBranchingMycelia growth onSolid straw substrate
Penicillin ProductionGlucoseCell Mass 0.45 g cell/g gluMaintenance 0.02 g glu/g cell-hPenicillin 1.1 g pen/g glu
Kinetics of PenicillinFermentation
Pathway for Penicillin SynthesisCys Val PAA PenicillinG2.5Glucose 3NH3 H2 SO4 PAA PenG CO2 α AAAComponent***GlucoseH2SO4AmmoniaWeight Yield*(g Pen/g Sub.)1.13.610.5Molar YieldUnit Yield**(mole Pen/mole)(Bill. Units/kg Su0.71.02.01.86.017.5*MW Na-salt of benzyl penicillin is 356.4 & 6-APA is 216.3µg benzyl** One international unit of penicillinis 0.6 sodium penicillin*** Assumingα -AAA is recycled
MEDIA DESIGNMEETING THE REQUIREMENTS FOR GROWTH APRODUCT FORMATION1. FERMENTATION PROCESS OBJECTIVESCell mass vs. Product synthesisSubstrate allocation modelPhysiological ModelAvoid C, N, S or PO4 catabolite repressionSpecific precursors, inducors, or repressors2. NUTRITIONAL REQUIREMENTSElemental requirementsSpecific nutrients, e.g. vitamins. minerals, aminoacids, etc.Energy requirements - Carbon source and OxygenGrowthProduct SynthesisMaintenance3. ENVIRONMENTAL REQUIUREMENTSpH profileTemperature profileDissolved oxygen profileCatabolite repressionPhysiological constraints, e.g. ionic strength,product inhibition
MEDIA DESIGNMEETING THE REQUIREMENTS FOR GROWTH APRODUCT FORMATION(continued)4. REGULATORY CONSTRAINTSQualification of vendorsMultiple sourcesTraceabilityPotential impurities or contaminantsConsistency5. TECHNO-ECONOMIC CONSTRAINTSCostMaterials availabilityProduct recoveryEnvironmental impact
Estimation ofMaintenance EnergySubstrate allocation modelq pXdS µX mX dt YmaxYp / sAssume that qp is small, thusdS µX µX mXdtYYmaxm1Y1Ymaxm11 Y Ymax µ1/µTypical values of m 0.02 to 0.1 g glucose/g cell-h
FER M EN TA TIO NM ED IU M C O M PO N EN TS PEN ICILLINM olasses0.2% Soybean O il1% Cottonseed Flour STREPTO M Y CIN2.5% Cerelose4% Soybean O il LA CTIC A CID BA CTERIAPhosphate buffer0.5% Tryptone0.5% Y eastExtract BA CITRA CIN3% Corn Steep3% G lucose Baker’s Y eastM olassestyyirupImH ighntC onteseahpitM ul nentspoC ombleairaVityQ ual
FER M EN TA TIO NM ED IAN U TR IEN TC A R BO N SO U R C EG LU CO SER A W M A TER IA LCERELO SEPR ETR EA TM EN TH Y D O LY ZED FRO M STA RCHM O LA SSESIN V ERSIO N(SU CRO SE TO FRU CTO SE A N D G LU CO SE)FA TS/O ILSSTA RCHSO LU BILZIA TIO NCELLU LO SEG RIN D IN G A N D H Y D RO LY SISSO Y BEA N O ILCO TTO N SEED O ILN ITR O G EN SO U R C EA M M O N IAPRO TEIN H Y D RO LY SA TESA CID O R EN ZY M E CA TA LY ZEDH Y D RO LY SISCO STCO RN SU G A RSTA RCHM O LA SSESCELLU LO SIC BIO M A SSPU RITY
Process WaterR. A. Governal & C. P. Gerba, J Ind Micro & Biotech1999 23 166-172
Pancoast and Junk. Handbook of Sugars, 1980SucroseMolassesProduction ofM olasses from Sugar C aneBagasse
Cane Sugar Molasses
PROFLO Finished Product Standard (www.tradersprotein.com)Net Weight as Packed:25 Kilos .25 Kilos or500 Kilos 5 Kilos or1000 Kilos 10 Kilos%Moisture as Packed% Protein as Packed (N x 6.25)% Oil (P.E. Extract)Color (Minimum reflectance through agreen filter on a Photovolt reflectometer)Fineness (To pass No. 200 U.S. St’d Screen)Total Plate Count3.0% Maximum58.0% Minimum (Dry Weight Basis)5.0% Maximum4590.0%Less than 5,000 colonies per gramPROFLO Typical AnalysisTotalProtein, Dry Weight Basis (N x 6.25)FatMoisture asAshFiberGossypol (Free)Gossypol (Total)pH (Aqueous Solution)Total Plate CountColor (Reflectance)Fineness (Thru 200 5615 colonies per g ram4991.72%PROFLO is a premium quality, finely ground , yellow flour made from the embryo ofcottonseed. The principal component of PROFLO is nonhyd rolyzedglobular protein. This is a natural protein of excellent quality which comes from Traders'special oil extraction process.PROFLO is guaranteed to contain a minimum of 58% protein on a dry weight basis andhave a total plate count of less than 5,000 colonies per g ram. PROFLOis made only from excellent quality, high protein cottonseed. This selectivity of rawmaterial helps provide a greater degree of uniformity in the finished product
PROFLO OILPROFLO OIL is a non-edible, crude co ttonseed oil. The free fatty acid content is lessthan 2.0% and normally runs 1.6%. PROFLO OIL contains approximately 70%unsaturated fatty acids. Its iodine value is 105. PROFLO OIL is recommended for use asa carbon source and antifoamer in production of antibiotics and steroids by sub mergedfermentation. PROFLO OIL is considerably more resistant to oxidation than refined oil.The remarkable stablity of PROFLO OIL is due to the presence of natural antioxidants.PROFLO OIL has been stored at room temperature for as long a s seven months withoutappreciable deterioration or development of rancidity.Fatt y A c idA mo cPalmitoleicOleicLinoleicLinolenicUnknownNone Found0.69%22.11%2.43%None FoundNone Found0.60%17.67%46.98%3.15%6.37%PROFLO OIL is miscible at ordinary temperatures in all proportions with most organicsolvents. Its solubility in water is very slight. PROFLO OIL dissolves 4-10% of itsequivalent volume of air at ordinary temperatures. The solubility of most gases inPROFLO OIL increases with increasing temperatures.T emp erat ureVi sc osi 3743321Dens it y0.910.9060.8860.8380.799The density of PROFLO OIL at 15 C. is usually between 0.918 and 0.922. Th e density ofthe oil change s about 0.000638 for each degree C. change in temperature.The specific heat for PROFLO OIL is approximately 0.475 Cal . /G at 19.3 C . and 0.495Cal . /G. at 50.7 C.
Typical values of m 0.02 to 0.1 g glucose/g cell-h. FERMENTATION MEDIUM COMPONENTS PENICILLIN Molasses 0.2% Soybean Oil 1% Cottonseed Flour STREPTOMYCIN 2.5% Cerelose 4% Soybean Oil LACTIC ACID BACTERIA Phosphate buffer 0.5% Tryptone 0
(iii) Biochemical tests The staining is followed by use of various biochemical reagents and tests to get closer to the identification of bacteria. There are many biochemical tests available for bacterial identification. Few of them are required to be carried out depending upon the bacteria. The commonly used biochemical tests are as mentioned below
Biochemical tests To identify bacteria, we must rely heavily on biochemical testing. The types of biochemical reactions each organism undergoes act as a " thumbprint " ماببلإا ةمصب for its identification. Purposes of biochemical tests 1. Test for metabolism of carbohydrates and related products. Sugar fermentation test 2.
In this lab, students identify an unknown bacteria using a biochemical method and a molecular method. For the biochemical method, students use a combination of differential growth tests and enzyme tests developed for clinical use. For the molecular method, students PCR amplify and sequence the 16S
SCHEME OF EXAMINATION B.TECH (BIOCHEMICAL ENGINEERING) 1 Bachelor of Technology (Biochemical Engineering) Program: BCE Program Code: 004 Duration – 4 Years (Full time) Program Scheme and Syllabus (1st to 8th semester) University School of Chemical Technology GGS INDRAPRASTHA UNIVERSITY SECTOR 16C, DWARKA, NEW DELHI-110078
Biochemistry Biochemical Techniques FTIR Description of Module Subject Name Biochemistry Paper Name 12 Biochemical . Infrared spectroscopy is supposed to be one of the most robust analytical techniques. The greatest benefits of infrared spectroscopy is that it can almost analyse any sample in any state may be powder, film, liquid, gas .
Biochemical characterization of Bacillus isolates Biochemical characterization of Bacillus isolates was carried out by using standard HiBacillusTM Identifications kit and KB009 HiCarbohydrateTM Kit. The main principle of this test is change in pH and substrate utilization after incubating,
Biochemical tests was performed to identify the unknown bacterial isolates [11]. Biochemical tests applied in this study was catalase test, citrate utilization test, methyl red test, Voges-Proskauer test, Indole test, glucose fermentation test, and lactose fermentation test by using the standard protocols [13].
g) Normal serum biochemical parameters in camels Serum biochemical parameters of camels are shown in Tables 11‒18. Table 6 Ranges of blood values for dromedary and Bactrian camels28 C. dromedarius C. bactrianus Total leucocytes 2.9 - 9.7 8.6 - 16.5 Neutrophils 33.0 - 70.0 55.0 - 79.0 Eosinophils 0 - 4.0 0 - 9.0
