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J , z’xy'4' 3:33:71" *‘Wefix ,. rfizgfimrxzfin .7 46‘" 'N’ "tr”. 4 1w». . . :L . «- 28:30 3&5; \llllljl‘l ll; ll Llljll LIBRARY Michigan State University This is to certify that the thesis entitled CHARACTERIZATION OF ION EXCHANGE RESINS FOR THE IMMOBILIZATION OF ESCHERICHIA COLI presented by Leonard Matthew Czupski has been accepted towards fulfillment ‘ of the requirements for : M. S . degree in Chemical Engineering mow Major professor Date A w3®* <5 ; \‘l %q 0-7639 MS U i: an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before duo din. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Adlai/Equal Opportnnlty Imitation oWMt WWW OF 1cm mom RESDB Fm MWCNG’WGMM ImrdMatthewCZupski A'IHESIS in partial fulfillment of the requirements of msmzs 01" em Department of Chemical Engineering Michigan State University 1989 Am WWW OF ICN Exam RFSDB Fm 1113 DIDBILIZATICN OF ESCHERICHIA (DLI By IeonardMatthavCZupski Adsorption of bacteria is a very easy and mild technique of immobilization. Though studies show that adsorption on to ion exchange resins is an applicable technique none, define the resin characteristics needed for high cell loading. Escherichia coli was used as a test organism since common organism for product expression genetic engineering. Viable cells were suspended in a non growth solution at favorable living conditions, 37°C and pH 7.0 - 7.5, and contacted with the Cl' form of ion exchanger. 'lhe geometric characteristics found to be favorable are high external surface areas with small porosity and small percentage of crosslinking. Charge characteristics favor a TYpe I strong base anion exchanger. Adsorption of bacteria follow a Iangmuir isotherm with the mechanism being a second order irreversible reaction of cells with open "sites" on the resin. The mass transport follows normal transport phenomena for constant surface composition. mums I Wild like to thank Dr. Eric A. GrUlke and Dr. Patrick J. Oriel support and expertise shared with me during this project. Allen Greenberg of Dow dieinical Company for his expertise on ion exchange and the use of the ion exchange resins studied. The use of Dr. Dennis J. Miller's mercury porosimeter and the Composite Center's scanning electron microscope is greatly appreciated. As is Doug Degaetano of the Center for Electron Optics, Michigan State University, for the preparation of the immobilized resins and the photographs taken of the immobilized resinsJI‘he financial assistance fran the State of Michigan's Research Excellence Fund and from the Department of Chemical Engineering was greatly appreciated. A special thanks to my wife Trish, for her patience during this study . ii Title MEOFGNTENI'S gage msror'nams...... .................... . ........................ ...v IJSI'OP‘FIm ..OOOOIOOOOOIOOO..OOOOOOOOOOOOOOOOOOO 000000000000 ooViii m1: wwOOOOOOO OOOOOOOOOOOOOO 0 ...... O ..... 0.0.0.00000001 mnzmm...” ........................... . ....... 4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Adsorption for Immobilization ..... . ........................... 4 Ion Exchange ................................................ ..6 Exchange Mechanism .......... . ....... . ............. ...........8 Mechanism of ImmObilization ............... ........ ....... .....8 Cell Adsorption .............................................. 9 Equilibrium .................. .............. ..... .............11 Kinetics ................... . ................................ .11 Cell Age ...................... . ................... . ...... ....12 Surface Area ................................................ 12 Porosity.. ................ . .......... .......................12 Physiological Effects ....................................... 13 ImmObilized Fermentations ........... ...... ..................14 Recycle.. .......... . ..... .. .............................. ....18 Summary ................. . ......................... . .......... 18 mm:mmms&m ....... .... ......................... 20 1. Organism and Cultures ....... .. ............................... 20 Organism ............................ . ...................... 20 Growth Plate ...............................................21 Overnight Cultures ...... . ..... ................ ..... ........22 Measuring cell Cbncentration..................................22 Optical Density .............. .... ................ .... ....... 22 Ion EXChange Resin............................................24 Resin Preparation.... ........ . ................. .............24 Adsorption....................................................25 Equilibrium Isotherms .............. . .......... ...... ..... ..25 Kinetics ......... .................. ........... ..............25 Resin Porosity & Area ..... ..... .................... .......... 26 Porosimetry ................................. .......... .....26 Scanning Electron Microscopy .................................26 Production ............ ...... .................................26 Batd'lGrowth.............. ..... ..... ..... ...... ..........26 Resin Recycle... ............................................. .27 iii Title m (RAPPER IV: was & DISCIBSICN .................................... 28 1. Batch Sorption Evaluation of Resins ........................... 28 Mechanical Strength ......................................... 30 mrtlier Evaluation .......................................... 32 2. Cell Sorption Equilibria ...................................... 36 Isotherns ................................................... 36 Ion Type .................................................... 39 SurfaceArea ........................... . ......... ..........39 Crosslinking ......... . ............... . ...................... 53 pa .................... . . . . ....... . .................... . . . .54 IonConcentration ............... ............. . ..... 57 3. Kinetics ................... . ....... . ........... . ..... ........60 Reaction Order ............................................. 62 Macroporcus Resins ......................................... 66 Mass Transfer .............................................. 70 Mass Transfer Model ........................................ 73 Effects of Diameter & Surface Area ......................... 74 4. Growth ........................................................ 78 Free Cells ................................................. 78 Total Cells ................................................ 80 Production ....................... . ....................... . . 82 5. Recycle ...................................................... 85 CHAPTER V: WISHING .............................................. 89 GIAPIER VI: was ......................................... 90 APPENDICES .......................................................... 91 Appendix 1: S.E.M .......................................... 91 Appendix 2: Preparation of Biological Samples . . . ......... 102 for S.E.M. Appendix 3: Assay fora-amylase....... ......... ...... 103 Appendix 4: Diameters ofMeshSizes 105 Appendix 5:EquilibriumSpreadsheet......................106 Appendix 6: Kinetic spreadsheet ................. . . ....... 116 Appendix 7: Equilibria Data .............................. 142 Appendix 8: Kinetic Data ................................. 167 Appendix 9: Growth&Production Data .............. .......195 Appendix 10: Recycle Data ........................ . ........ 198 LISP OF m ................................................. 201 iv _L1_Ti e m TAEIELRSJpportrequirements ......... .......... 2 MEI-.2: Wilizationmfllws ..OOOOOOOOOOIOOO. OOOOOOOOOOOOOOOOOO 2 ME 2.1.1: Features of supports for adsorption .................... 6 Table 2.11.1: Oxidative Activities of Substrates .................. . .13 with Free and Adsorbed Cells, pH = 7.0 (Hattori & Furuska, 1960) ME 2.12.1: Summary of Anion Exchangers used in ......... . ......... 17 Continuous Fermentations TABLE 2.14.1: Summary of Ion Ebrohange for 1;; coli ................... 19 ME 3.1.1: Physical Parameters L coli ........................... 20 'IABIE 3.1.2: Optimum Growth Conditions E_. coli ...................... 20 TABIE 3.1.3: Growth plate composition ............................... 21 TABLE 3.1.4: LB broth composition ................................... 21 ME 3.2.1: Linear equations for the calculation ................... 23 of concentration mart: 4.0.1: Intonation on Resins Studied" . . . . . . ...... . ............ 29 ME 4.1.1: Assumptions made for study ............................. 30 ME 4.1.2: Adsorption of cells by ion exchangers .................. 32 Initial Concentration: .00043 g cell/ml ME 4.1.3: Positive Resins after Initial EValuation ............... 34 128818 4.1.4: Adsorption of cells by anion exchangers ..... ...... 35 Initial Concentration: . 00017 g cell/ml ME 4.1.5: Adsorption of cells by anion exchangers ..... ....... 35 Initial Concentration: .00035 g cell/ml TAEIE 4.2.1: Equilibrium Constants K1 & K2 at ....................... 37 usrormrs pH = 7.5, Tris = [.05] V Title ME 4.2.2: ME 4.2.3: ME 4.2.4: ME 4.2.5: ME 4.2.6: ME 4.2.7: MB 4.2.8: ME 4.2.9: .& Equilibrium Constants K1 & K2 for Type I & II .......... 39 stronghaseanionexdaangersatpH=7.5, Tris = [.05] Adsorption Capacity and Surface Area . .................. 41 Percentage of unused surface area* . .................... 45 cell area = 1.5 x 10' Adsorption Capacity and Surface Area of differing ...... 54 crosslinking of 200-400 mesh gel spheres, Type I strong base anion exchanger Constants for the function of K]. on pH (Eqn. 5) ........ 55 Effect of pH upon K2 ................................... 57 Resin: IRA-938 .05 M Tris buffer Effect of pH upon K2 ................................... 57 Resin: XU-434-200.00 .05 M Tris buffer Effect of buffer concentration upon K2 ................. 59 1285111: XU-434-200.00 pH=7.5 ME 4.2.10: Effect of buffer concentration upon K2 ................ 60 m 4.3.1: mam 4.3.2: ME 4.3.3: ME 4.3.4: Resin: IRA-938 pH = 7.7 Constantsusedforkineticstudy... ........ . ........... 62 pH = 7.5, Conc. = .05 MTris Kinetic Constants for XU-434-200.00 . ..... . ............. 63 pH = 7.5, .05 M Tris buffer [C10 =1.04 x lo8 cells/ml, [R10 =4.667 x lo8 site/ml 10 ml cell soln., .5 g dry resm Reaction order of Cells ................................ 66 Resin: XU—434-200.00 pH = 7.5, .05 M Tris buffer [C10 =1.04 x lo8 cells/ml, [R10 are“; x lo8 site/ml 10 m1. cell soln., .5 g dry resin Adsorption constant and time for macroporous ........... 69 resin IRA-938. [C]0 = 2.45 x 108 cell/ml, [R19 =4.64 x 103 sites/ml 10 m1 cell soln., .5 g dry resin pH = 7.5, .05 M Tris buffer vi Title E9 m 4.3.5: Linear Regression of ka-D vs. Dl’2 .. ..................... 72 Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer ME 4.3.6: Linear Regression of ka-D vs. Cell Oonc. ..... . ......... 72 Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer M34.3.7: Predictedandexperimental initial ..... ..76 adsorption rate, ka, for macroporous IRA-938 vii {HUHJICX’PEGURES 11:19 Ease Fig. 2.4.1: Hypothetical dipolar character of cell. ...... . ..... ......9 Fig. 3.2.1: Calibration of Klett optical density for ........... .....24 Escherichia coli Fig'4.1.1: Batch Adsorption Of XURF0525’187-239. series Of ..........31 resins Init. cell conc. = 3.24 x 10"4 g cell/ml pH = 7.5, .05 Tris buffer Fig. 4.1.2: Comparison of kinetics of three resins ...... . ...... .....33 pH = 7.5, .05 M Tris buffer Fig. 4.1.3: Kinetics of XU-40091.00 ................................. 33 pH = 7.5, .05 M Tris buffer Fig. 4.1.4: Comparison of kinetics of two resins .................... 34 pH = 7.5, .05 M Tris buffer Fig. 4.2.1: Linearization of Langmuir Isotherm ...................... 38 Resin: XU-434-200.00 pH = 7.5, .05 M Tris buffer Fig. 4.2.2: langmuir Model of Adsorption .......................... ..38 Resin: XU-434-200.00 pH = 7.5, .05 M Tris buffer Fig. 4.2.3: Comparison of Type I & Type II Strong Base Anion ........40 Ebtchangers Resin: Type I: XUS-40187.00 Type II: XU-40170.00 pH = 7.5, .05 M Tris buffer Fig. 4.2.4: Surface of resin XU-434-200.00 ............. . ..... .......42 Fig. 4.2.5: Surface of resin IRA-938.......... ....................... 43 Fig. 4.2.6: Cells immobilized on the of the resin 44 XU-434-200.00 Fig. 4.2.7: Immobilized cells on strong base Type I anion ........... 46 exchanger IRA-938 sliced through middle. viii Title 2Q? Fig. 4.2.8: Immobilized cells on strong base Type I anion ............ 47 exchanger IRA-938 sliced through middle. Depth 0 - 26 mm Fig. 4.2.9: Immobilized cells on strong base Type I anion ............ 48 exchanger IRA-938 sliced through middle. Depth 24.5 - 76 mm Fig. 4.2.10: Immobilized cells on strong base Type I anion ........... 49 exchanger IRA-938 sliced through middle. Depth 74.5 - 126 mm Fig. 4.2.11: Immobilized cells on strong base Type I anion ........... 50 exchanger IRA-938 sliced through middle. DepthlZlum- center Fig. 4.2.12: Cell distribution for depth into Type I ...... 51 strong base macroporous anion exchanger IRA—938 Fig. 4.2.13: ‘Iype I strong base gel anion exchanger ................. 52 XU-434-200.00 with immobilized cells, entire exchange bead. Fig. 4.2.14: Type I strong base gel anion exchanger ................. 53 XU-434-200.00 with immobilized cells, surface of exchange bead . Fig. 4.2.15: K1 as a function of pH, Resin: XU-434-200.00 ........... 56 .05 M Tris buffer Fig. 4.2.16: K1 as a function of pH, Resin: IRA-938 ......... . ........ 56 .05 M Tris buffer Fig. 4.2.17: Resin XU-434-200.00 as function of buffer .............. 58 concentration. pH = 7.5 Fig. 4.2.18: Resin IRA-938 as a function of buffer .................. 59 concentration. pH = 7.7 Fig. 4.3.1: Linearization to calculate adsorption constant . . . . . ..... 64 Resin XU-434-200.00, pH = 7.5, .05 M Tris buffer [C10 =1.o4 x 103 cells/ml, [R10 =4.667 x 103 site/ml Fig. 4.3.2: Reaction models of cell adsorption ...... ..... 65 Resin XU-434-200.00, pH = 7.5, .05 M Tris buffer [C10 =1.o4 x 103 cells/ml, [R]0 =4.667 x 103 site/m1 Fig. 4.3.3: Reaction order of cells ....................... . ........ 67 Resin xu-434-2oo. 00, pH = 7. 5, .05 M Tris buffer [C10 =1. 04 x lo8 sails/ml, [R10 =4. 667 x 103 site/ml Title M Fig. 4.3.4: Kinetics of macroporous (IRA-938) and 68 gel (XU-434-200. 00) anion exchanger. pH= 7. 5, .05 M tris buffer, [010 =8. 21 x 107 cell/ml 0.500 g dry resin Fig. 4.3.5: Macroporous ,resin (IRA-938) using initial ............... 69 adsorption constant. 10 m1 cell soln., .5 g dry resin [C]0 = 2. 45 x lo8 cell/m1, [R]0 =4. 64 x lo8 sites/m1 pH = 7. 5, .05 M Tris buffer Fig. 4.3.6: Macroporous resin (IRA-938) adsorption using ............ 70 constants from Table 4.3.4.10 ml cell soln., .Sgdryreein, pH=7. 5, .05MTrisbuffer [C]0= 2. 45 x lo8 cell/m1, [R]0 =4. 64 x lo8 sites/ml Fig. 4.3.7: ka-D vs. 011 for Type I strong base ...................... 71 gel type anion exchanger. Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer Fig. 4.3.8: Linear model for ka-D vs. D11 for Type I ................. 74 strong base gel type anion exchanger. Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer Fig. 4.3.9: Resin diameters effect on ka for Type I ................. 75 strong base gel type anion exchanger. Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer Fig. 4.3.10: Surface area effect on ka for Type I ................ ...76 strong base gel type anion exchanger. Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer Fig. 4.4.1: Free cell concentration of production ........... . ...... 78 experiment14.35wt.%'1ypeIstrongbaseanion exchange resin, macroporous and gel type structures. Fig. 4.4.2: Free cell concentration of production 79 experiment24.35wt. %TypeIstrongbaseanion exchange resin, macroporousandgeltype structures. Fig. 4.4.3: Free cell concentration of production ..... ...... 79 experiment39.1wt. %'IypeIstrongbaseanion exchange resin, macroporous and gel type structures. Fig. 4.4.4: Total cell concentration of production ...... 81 experiment 1 4.35 wt. 95 Type I strong base anion exchange resin, macroporous and gel type structures. Title gags Fig; 4.4.5:‘Tctal cell concentration of production .......... . ....... 81 experiment 2 4.35 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Fig; 4.4.6: Total cell concentration of production .................. 82 experiment 3 9.1 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Fig; 4.4.7: Production of a-amylase, production experiment 1 ........ 83 4.35 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Fig; 4.4.8: Production of a-amylase, production experiment 2 ........ 84 4.35 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Fig. 4.4.9: Production of a-amylase, production experiment 3 ....... .84 9.1 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Fig. 4.5.1: Recycle experiment 1: 5 wt. % Type I strong base ........ 86 anion exchange resin, macroporous and gel type structures. Init. cell conc. = .00049 g cell/ml Solution vol. = 10 ml., pH = 7.5, .05 M Tris buffer Fig. 4.5.2: Recycle experiment 2: 5 wt. % Type I strong base ........ 87 anion exchange resin, macroporous and gel type structures. Init. cell conc. = .00049 g cell/ml Solution vol. = 10 ml., pH = 7.5, .05 M Tris buffer Fig. 4.5.3: Recycle experiment 3: 5 wt. % Type I strong base ........ 88 anion exchange resin, macroporous and gel type structures. Init. cell conc. = .00037 g cell/ml Solution vol. = 10 ml., pH = 7.5, .05 M Tris buffer xi (Iflfifliillk INHRDDUCEE]! The simple observation of algae covered rocks in a stream suggests that immobilization of cells has occurred as long as the combination of solids, cells and water. Industrially the use of immobilized cells has been used since 1823 where Acetobacter was adsorbed onto wood chips for the "quick" vinegar process. Waste water treatment is another process in which immobilized cells are used as a part of the process. The use of immobilized cells in industrial fermentations is a recent phenomena. The first article appeared in 1960 (Hattori and Furuska, 1960) and the first application for continuous production was with L—aspartic acid. (Chibata et a1., 1974; Sato et a1., 1975) The methods of cell immobilization are similar to methods used for enzyme immobilization. A definition for immobilized cells was given at the first Enzyme Engineering Conference in 1971. Immobilized cells are: "physically confined or localized in a certain defined region of space with retention of their catalytic activity and - if possible or even necessary - viability and which can be used repeatedly and continuously." The advantages of using immobilized cells in fermentations are: (1) a higher amount of cells per unit volume of reactor, (2) a lesser amount of free cells in the effluent, (3)higher dilution rates. The first reason allows for smaller reaction vessels or greater 2 productivity of existing vessels and the last two reasons directly affect the downstream separations needed for the bio-products. Reccmmerdations for the case of product expression or over production of cells are shown in Table 1.1 . ME 1.1: Support requirements 1 ) High carrier ability 2 ) Availability in quantity 3.) Low cost 4 ) Easy scale-up 5 ) Safety of material Three methods fitting these requirements are shown in Table 1.2. TABIE 1.2: Immobilization Methods Entrapment (moss-linking Carrier-binding Entrapment is the most widely studied method of cell immobilization. Cells are entramed in the matrices of various polymers. Matrices that have been studied are: collagen, gelatin, agar, alginate, carrageenan, cellulose triacetate, polystyrene and polyacrylamide. Polymer precursors are mixed with cells and the polymer is formed around the cells. Problems faced in the entrapment method are possible death of the cells caused by the polymerizing reagent and low rates of diffusion of substrate to the cell and product out of the matrix. In the cross-linking method, the cells are linked to one another by a suitable reagent (Chibata et a1, 1974) . The carrier-binding method 3 has the cells covalently bound or adsorbed onto insoluble matrices. Ion exchange resins have been used to separate microorganisms from solution. They have been used in clinical microbiology, waste and water treatment. The separation technique was empl'lasized in Daniel's review of the use of ion exchange resins with microorganisms (1972) . This review covered the separation of algae, diatoms, bacteria, fungi, protozoa, rickettsia and viruses from an aqueous solutions. Studies of ion exchange resins as cell supports in fermentations has been limited. As of yet no study has shown the qualitative effects that surface area, surface charge and resin morphology have on sorption and adsorption rate. The fowsofthisstudyistofindaionexchangeresinthat has good adsorption of E_. 9g]; and the adsorption equilibrium and the kinetics of the adsorption based on resin morphology. Other areas of interest are resin geometry, exchange group, resin recyclability and use of the resin in actual growth conditions. The gram-negative prokaryote Escherichia coli was chosen because the gene locations of the organism are well studied and there are a great many genetically engineered organisms available. GIAPI‘ERII: IlTERKIUREREVIBV Immobilized cells have several advantages. They allow a higher concentration of cells in the fermentor than possible for suspended cell culture, particularly for bacterial cultures. The higher cell mass can result in increased production of cells and products. If the immobilized cells do not release into the media there will be fewer cells in the reactor effluent and less downstream processing of the product stream is required. The increased cell and product concentration in the fermentor as compared to free cell is known as overproduction. Immobilization will also allow a greater dilution rate in a continuous fermentation (Kolot,1981) . If cells express an enzyme needed for a process, then cell immobilization has advantages over enzyme immobilization since enzyme purification is not needed, enzyme loss is held at a minimum, and the enzyme will be inherently more stable: it is in its natural environment (Durand & Navarro,l978). me to the large scope of cell immobilization and of cell adsorption, this chapter will cover the use of ion exchange resins for the overproduction of cells and enzymes in both categories. 2.1 WONT FER MOBILIZATION Cell adsorption is done at mild conditions as compared to the entrapment and crosslinking techniques. The cells just need to get in 5 contact with the carrier. Mild conditions and simplicity are the main advantages of adsorption used for cell immobilization (Tampion and Tampion, 1987; Klein 8 Wagner,1983: Klein 8 Vorlop,1985). Since adsorptiondoesrotpitmlcl'lstressmthesystem,thecellsare usually viable, unlike entrapment where the cells will care in contact with polymerizing agents. There are two types of ion exdlange resins: solid gels and macroporous. The solid gels will sorb cells only on their exterior surface while the porosity of macroporous resins will allow cells to sort on interior surfaces. Mass transfer of nutrients and products depends on the reactor system. mtrapped cells and cells in macroporous resins rely on mass transfer of nutrients and products into the interior of the particle. In sate entrapped gels, the yeast will grow only within 50 u of the surface (Ollis, 1989) . Entrapment gels can be broken by grwirg cells. Crosslinked resins are less susceptible to disruption (Ollis, 1989). Since adsorption is a surface rhenarena, mass transfer of substrates and products has little interference from the carrier, unlike entraprent and encapsulation (Kolot,1981). Since ion exchange resins can be regenerated, this carrier also has the possibility to be reused. The disadvantages of this method are the possibility of large amounts of cell loss frumthesurfaceardtleadverseeffects opron adsorption (Tampion 8 Tampion,1987) . The features sought for carriers used for adsorption are sham in Table 2.1.1 (Tampion 8 Tampion, 1987) . 2.2 It)! mam Ion exchange resins are polymeric beads that have a charged matrix with exdlangeable ions. The exchangers are divided into two principle categories: structural (the geaietr'y of the polymer matrix) and functional (the ion-active groups) . TABLE 2.1.1: Features of supports for adsorption Non-toxic High cell retention capacity High cell loading capacity Stable to heat sterilization Stable at appropriate pH values Resistant to microbial degradation Availability in appropriate shapes and sizes Cost ppropriate to application Density appropriate to reactor type Reusable Therearearangeoffunctionalgralpsoncarmercial ion exchange resins. Cation exchangers have a negatively charged matrix that exchanges a cation (positive) charge and anion exchangers have a positive charge that exchange an anionic (negative) charge (Wheaton 8 Seamster, 1966) . These are further divided by charge strength. The anion exchangers are divided into strong, intermediate and weak base. Strong base exchangers are highly ionized (dissociation into ions) amencaiparedtotheintemediateardweakbase. Strongbase exdlangersareoftwotypes: 0'13 + TYPE I _/—\_C}iz—-N—Ch3 L/ l C33 CH3 + TYPE II ____/"\ CH2-—-df-<}gr-—-C320H CH3 Type I exchangers have a smaller capacity than Type II but are more stable chemically. They also differ in their affinities for chloride and hydroxide ions (Applebamm, 1966). Type II exchangers are more readily regenerated due to their greater affinity for the hydroxide ion. Weak base exdiangers generally are a mixture of polyamine functional groups containing primary amine, -NHZ, secondary amine,- NHR, and tertiary amine, -NR2 (Wheaton 8 Seamster,1966). Intermediate baseexchangershaveamixtureof exchangegroupsfrombothstrongand weak base exchangers. Strong base anion exchangers have an operating range of 0-14 pH in both the salt and free-base form. Weak base anion exchanger are highly ionizable over pH of 7 and only in the salt form (Applebatmm, 1966) . These work best with strong acids such as HCl and H2804 (Dorfner, 1972). Cation exchangers are separated into strong and weak acid groups . Strong acid excl-angers have sulfonic acid, -SO3H, and weak acid exchangers have carboxylic acid, «DOB, as exchange groups. The structural portion of exchangers are divided into two categories, gel and macroporous (macroreticular) . Macroporous resins have pore sizes ranging up to several thousand angstroms with interconrecting pores. These resins swell very little in aqueous solutions. Gel type exchangers are polystyrene with divinylbenzene (DVB) crosslinks where porosity is inversely proportional to crosslinking. Gel type resins sham slight swelling in poor solvents 8 such as water. Exchangers are normally available as spheres. The specific gravity of exchangers is 1.1 -1.5 (Wheaton 8 Seamster, 1966) . 2.3 mam W Ion exchange involves exchanging one ion for another with a simple reversible reaction. As expressed in the follaving two equations: Cation Exchange: ZS-a+ + b+ —‘_‘ Zs-b+ + a+ where the ZS is the matrix, anion fixed site and a+ and b+ are the cations. Anion Exchange: As-c' + d“ :4 AS-d' + c" where As is the matrix and the cation fixed sight and c” and d’are the anions. 2.4 W OF IMDBILIZA‘I'ICN According to a hypothesis made by Daniels and Kempe (1966) bacteria are explained to be "macroscopic zwitterions". At pH's above the isoelectric point of their surfaces, they act as anions and at pl-I's below the isoelectric point of their surfaces, they act as cations, This hypothetical dipolar ion on the cell surface is shown in Figure 2.4.1 (Daniels,1972) . According to this explanation all bacteria should adsorb to all types of exchangers depending on the pH of solution. The adsorption is thought to be an electrostatic attraction between the cell surface and the carrier (Daniels 8 Kempe,1966). The cell surface 9 group, carboxyl gralps, electrostatically interacted with the quaternaryaminegroupson thestrongbaseanionexchangeresins studied (Zvaginstev and Gisev,1971). This was found to be the common group among cells with differing cell wall structures, gram-negative and gram-positive bacteria and yeast. H" or Cell Cell :'————‘———’ Cell n——t~—mr3+ n—clz—mf H—(l!-—-NHZ 2m. 3...- J...- Cation Dipolar Ion Anion Fig. 2.4.1: Hypothetical dipolar character of cell. 2.5 (111.. WOW The studies of adsorption of bacteria onto ion exchange resins have shown that the best exchangers are anion exchangers with preference to strong base type (Wood,1980) . The capacity of strong base type exchangers have been found to be in excess of 1010 cells per gram dry resin (Daniels 8 Kempe,1966; Wood,1980). Some studies have been sham that cation exchangers gather a fat cells, depending on the pH of solution (Gillisen et al,1961) . Gram negative organisis (i.e. L Q11) often have surface structures that have been thought to be ideal for adsorption with appendages such as flagellae and lipopolysaccharide chains. An adsorption study carparing mechanically de-flagellated and normal L _cgli shaved ro difference in the amt of cells adsorbed. A study done with stooth and Rough strains of Salmonellae mimurium and iii. m shared that lipopolysaccharides actually inhibit the 10 adsorption to anion exchangers (Wood,1980) . Since this research is conducted toward the use of immobilized cells for fermentation purposes the pH range considered for study will be closer to optimum physiological, 7.2, for L geiiwriel) . The amount onguadsorbedontoananionexchangerbetweeanofSardB shaved to distinct maximum level (Niehoff and Echols,1973). This observation agreed with the adsorption characteristics found for several species of Gram- negative bacteria onto DEAE cellulose over the same pH range (Wood, 1980). Cation exchangers have been studied for adsorption of bacteria and have been found to adsorb cells at low pH (Daniels 8 Kempe,1966). For strong acid cation exchangers, negligible adsorption was observed for higher pH, 4-10 (Wood,1980). Another factor involved with the cell environment is the amount of salts that are in solution. The effect of varying sodium and calcium chlorides concentrations, zero to 1.0 M, on the amount of L _cii adsorbed onto an anion exchanger at pH 6.5 was that the amount of adsorption did not change for polystyrene exchangers (Jarvis, Lach and WOOd: 1977) . Physical parameters of the resin will effect the amount of cells adsorbed. For Phase I Hamuus grtussis, cell adsorption was found tobeproportionaltotheinverseofthenumberofcrosslinksofDavex 1 (Kuwajima et al, 1957). The increased water content at lav crosslinking was thought to provide a more natural environment for the organism. According to Zvaginstev and Gusev (1971) only 0.3% of the amount ll of actual adsorption sites bind to cell for anion exchangers in the chlorine form, (this is about the small amount of exchange sights found on the resin surface). 2.6 mm The amount of cells adsorbed from solution depends on the amount of adsorbent that is present in solution (Daniels,1972; M. Barr, 1957). The adsorption isotherm for bacteria has been determined to be Langmuir in nature (Daniels,1972; Bar et al,1985; Grulke et al,1989). The observation of a Iangmuir isotherm suggests that the cells cover the resin in a monolayer. This has been observed by using Scanning Electron Microscopy, S.E.M., where the cells have been observed to layer the surface much like pins in a pin cushion (Daniels,1972) . It is thaight that some free cells found in immobilized systems occur from reproduction of the adsorbed cells instead of cell desorption (R. Hattori,1972) . The amount of cells that become free is independent of the initial amount of cells adsorbed since the cells divide on the surface until capacity is readied. 2.7 KINETICS The rate of adsorption has been given the model: A/Ao = kat + k'a Jt_ wereA, Aoareadsorbances (at420nm) attimestandOofthecell suspension. ka and k' a are experimentally derived constants that depend on solution conditions, cell and resin type (Daniels,l972) . 12 2.8 (ELL AGE In a study with W mobilis (Krug 8 Dougalis,1983) the age of the cells being immobilized had an effect on the amount of organism adsorbed. As the cells' age increased, the amount adsorbed decreased. This occurred for two strong base macroreticular anion exchangers. 2.9 W AREA Since adsorption is a surface effect, the amount of surface area plays a large factor in the amount of cells that can be adsorbed. This was shown by Wood (1980). As the sphere diameter increased, the amount of adsorption decreased. The surface area presented is dependent on the particle size. Daniels (1972) mentioned that better adsorbers were small spheres with large surface area. 2.10 mmsm The pores of macroretiallar ion exchange resins will have on cell adsorption shalld be taken into account. The pore size and distribution of pores has been studied for the effect it has on the immobilization of microbes (Messing 8 Opperman,l979). The research was separated into two categories, dependent upon the type of reproduction that occurred, fission and budding. Fororganisms that reproducebyfission, suchach_o;_i_, itwas found that the optimum pore size is one that is double the length of the organism. This diameter allows for cell reproduction with very little interference from the pore walls. To get a high accurmlation of cells it was famd that the carrier should have 70% of the pores 13 between one to five times the major physical dimension of the cells. This range of pore sizes would allw enalgh roam for two organisms directly across from on another to be able to reproduce, thus doubling their length, and allav passage for another cell through the pore. Pore sizes larger than this walld not have as much surface area available for adsorption. The particles used were fritted glass spheres between 18-25 mesh. 2.11 HIYSIOILIEIQL mess The effects that adsorption onto Danex 1, X-4, 100-200 mesh chloride form had on the activity of an organism, L coli Yamaquchi strain, was carpared to the free cells activity (Hattori and Funlska; 1960). The oxidation activity of the substrates, succinate, lactose, alanine and fumarate were compared. At a pH of 7 the oxidative activities of the adsorbed cells decreased from 7.2 % to 28.0 % of that of the free cells, Table 2.11.1. Table 2.1.1.1: Oxidative Activities of Substrates with Free and Adsorbed Cells, pH = 7.0 (Hattori 8 Furuska, 1960) Activity“ Remaining Substrate activity after free cell adsorbed cell adsorptionz) succinate 108 (pl) 17 (pl) 15.7 (9:) lactose 54 12 20. 2 alanine 43 12 28 . 0 fumarate 97 7 7 . 2 1) Activitywasexpressedtheamount of02 uptakeby 109 cells for an hour. 2) ((activity of adsorbed cell) / (activity of free cell)) x 100 14 {it shifts for immobilized enzymes have been observed. These shifts are thalght to occur because the support controls the solution environment near the enzymes. Activity of free cells for the aforementioned substrates shad a peak aramd a pH of 6-7. The peak of activity for the substrates of the adsorbed cellsoccursatapl-Ilevel oneunitgreaterthanthatofthe free cells. The warmers of having a peak of activity one pH unit greater for adsorbed cells is referred to as the B effect. The authors hypothesized that a cationic layer of molecules surrounds the anionic layer on the surface. This cationic layer is thought to be hydrogen ions. This would increase the H+ concentration thus decreasing the pH which the adsorbed cells are exposed. TherateofgravthofadsorbedngLiwasfamdtobefasterthan that of the free cells and has an optimmmm gravth rate approximately one pH unit higher than the free cells; this is similar to chemical activities B effect (R. Hattori et al, 1972) . This result is surprising since the cells presumably buffer their contents. The mechanism for this effect is not yet explained. Detached cells also show a faster rate ofgrowththan free cellsandalso sham lesser oxygenuptakethan that of free cells (R. Hattori et al,1972) . The detached cells shav a decreased lag time for enzyme induction than there is for free cells (R Hattori et al, 1972) . 2.12 MOBILIZH) PM“ The use of ion exchange resins as an immobilization technique for continuous fermentations is limited. Most of the studies involve ion exchangers as separation devices for microbiology and medicine (Rotman, 15 B., 1960: Puck, T.T. & Sagik, B. 1953; Zvaginstev 8 Gusev 1971: Daniels, 8. L.,1972, 1966). A potential advantage . of using ion exchange resins in fermentations is that these supports can be regenerated, sterilized and inoculated withalt leaving the reaction vessel. This is a great advantage when attempting to keep aseptic conditions (Groom et al,1988). Anion exchangers were chosen as the carrier for all tests in this study. No studies were found that incorporated cation exchange resins. Many types of reactors have been used: packed columns, stirred tank and airlift fermentors. The fermentation of ethanol using gyms—mas mobilis ATCC 29191 adsorbed onto a weak base anion exchanger, DEAE-cellulose, was studied using a stirred tank reactor (Bar, et al,1987) . During the study it was concluded that the use of the stirrer caused shear induced cell detachment. At the impeller speed of 190 rpm the adsorption of cells dropped from 41 mg/g resin to 31.5 mg/g resin after four minutes and eventually leveled off at 20.1 mg/g resin after 90 minutes. When the agitation was decreased to 70 rpm the adsorption increased to 34.5 mg/g resin after ten minutes. For the speeds of 500 and 1000 rpm the final adsorption decreased to 20.1 and 11.0 mg/g resin respectively. Bren at the lower speed of 70 rpm the adsorption never returned to the initial value of 41 mg/g resin. The method of calculating sorption was done by extracting the cell protein by boiling in an alkaline solution, followed by a protein assay. The effects of reactor carposition also was investigated (Bar et al,1987) . It was found that cell adsorption capacity decreased due to 16 effects of glucose, ethanol and other unknam components in the fenneTtation broth. This study concluded that envirormental effects in the reactor could change adsorption equilibrium from that observed in a static cultures. Another investigation using gyms mobilis ATCC 29191 was done using a packed column reactor for the continuous production of ethanol (Krug 8 Dougalis,1983). The carrier used was the Rohm and Haas macroreticular strong base anion exchanger IRA 938 . This reactor shared an increase in ethanol productivity, 89.8 g/L-hr to 377.4 g/L-hr, with an increase in dilution rate, 2.2 hr‘1 to 11.2 hr'l. Thaigh the productivity increased, the effluent concentration of ethanol decreased from 40.8 g/L to 33.7 g/L and the effluent cell concentration increased fram 1.01 g/L to 1.45 g/L, attributed to increased shear due to increased flat. The percentage of glucose used from the feed, 100 g/L, decreased from 97.4% to 79.6% with the increasing dilution rate. Aproblememcounteredwiththissystemwasthat it couldnotrun for long periods (greater than 200 hours) due to the reactor fouling because of filamentals growth occurring in the packed bed. One of the major uses for immobilized cells is to increase production of enzymes and/or specialty chemicals. One study used the organism Bacillus mloliwefaciens NRC 2147 ( National Research Council, Ottawa) with a packed bed reactor and stirred tank reactors, batch and continuals modes, with the Rohm and Haas large pore macroreticular anion exchange resin, Amberlite XE-352, for the production of a-amylase (Groom et al,1988). The results of this experiment showed that the highest concentrations and productivities were achieved with the packed bed reactor. system (18700 amylase 17 activity units per liter (18.7 kU/l) and 9.7 kU/L-hr respectively). Biofouling for this packed bed was avoided by periodically feeding the reactor with medium lacking in soluble starch and yeast extract. This systemhadagreaterproductivitythanbatchardcontinuousnodes ofa free cell stirred tank reactor but a smaller concentration of a- amylase than the batch reactor (150 kU/L) . Inastudyusingthethermophile, anorganismwithoptimnmgrowth between 55-75'C, Qcillus W Md: 29609 was used to produced-amylase'lhesupportusedwastheRdmmardHaas macroreticular strong base anion exchanger IRA 938. This resin was chosenbecause of its large surfaceareaascomparedtoasolidbead. This structure proved to be too fragile for a stirred tank reactor as the carrier fractured within minutes after agitation started. Due to its high oxygen transfer and low shear, an airlift fermentor was chosen (Grulke et al,1989). The study was done at an operating temperature of SS'C. The continuous production of a-amylase was greater for the immobilized cells than the free cell experiments. This study also shows a particular advantage of ion exchange resins having stability at higher temperatures . Most gels used for entrapment tend to degrade at ME 2.12.1: Summary of Anion Exchangers used in Continuous Fermentations -overproduction of products occurs ’ -environmental (i.e: impeller, product and substrate concentrations) effects will change the equilibrium adsorption of cells from those found from static cultures -packed bed reactors give a greater productivity than stirred tank reactors -packed bed reactors are susceptible to biofouling -are suitable carrier for thermophiles 18 temperatures suitable for thermophiles. A summary of the use of anion exchangers in fermentations is shown in Table 2.12.1. 2.13 REL'YCIE On experimentation with recovery of adsorbed cells, it was found with Staphylococcus auréyg that contacting supports with salt solutions can promote the cells to desorb (Wood;1980) . The amount of recovery of MlococcisLurgLsisdependentonthep-Iofthe solutionandonthe salt concentration, reaching a peak at 0.6 M NaCl. Concentrations of sodium and calcium chloride were varied and showed that conditions that may inhibit adsorption are not necessarily going to promote recovery. The same experiments were replicated for _E;._ coli and little promotion of recovery was found to occur (Wood, 1980) . 2.14 am The quality sought after is a large amount of cells adsorbed. The ion exchanger best suited for this are anion exchangers with a large surface area. The environmental effects of pH and salt concentration should have a negligible effect on the amount of E; coli adsorbed for optimum growth conditions. A summary of ion exchange for E; coli is given in Table 2.14.1. 19 ME 2.14.1: Smmary of Ion Exchange for E_. coli -is an electrostatic interaction with cell surface carboxyl groups -depends on surface area -has Iangmnir type adsorption isotherm -anion exchangers show best adsorption ~negligible change in capacity noticed for pH of 6-10 and for varying salt concentrations -will diange the optimum activity by (me pH 't um mm:mssn Methods needed for this study are growth plates of the organism, overnight cultures of organism, preparation of resins for adsorption and analysis of the adsorption. 3.1 ms: AND W 21mg The genetically engineered organism Escherichia coli 246 (EC246) , an organism created in Patrick J. Oriels laboratory (Michigan State University) was used for this study. It has a resistance to 10 x 10"6 g/ml solution of the antibiotic chloramphenicol and produces thermostable a-amylase than a wild type Bacillus stearothermophilug (Oriel). Other data used concerning L coli are in Tables 3.1.1 and 3.1.2. ME 3.1.1: Physical Parameters E; coli Mass: 9.5 x 10'13 g/cell Dimensions: length: 2 mm width: .5 pm ME 3.1.2: Qatimum Growth Conditions E; coli 7.2 37 20 21 m A grovth plate of the frozen L _co_li_ culture are made by the following technique. The growth support composition is shown in Table 3.1.3 (Oriel). The LB (Table 3.1.4), agar and H20 are mixed, taken to a pH 7.2 and autoclaved for 30-45 minutes. The solution is cooled to 50’C ME 3.1.3: Growth plate composition 20 g LB broth 20 g Bacto Agar 1000 g dHZO 10 mg. chloramphenicol ME 3.1.4: LB broth composition Ingredient wt. 95 Select Peptone 140 50 (Pancreatic digest of Casein) Select Yeast Extract 25 Sodium chloride 25 before adding of the chloramphenicol. Since chloramphenicol does not dissolve in water, a solution of 10 mg/ml chloramphenicol/ethanol solution is used to transfer the antibiotic into the plate solution. When the antibiotic is added, the solution should be gently agitated. The solution is poured into petri dishes that are resting on a level surface until a thickness 0.5-1.0 cm. is obtained. The dish is covered with its lid and allowed to cool to room temperature. At this time the solution is in a gel form and some condensation may occur on the inside of the cover. The plate is placed in a 37°C 22 oven until condensation dissipates. The plate is ready for inoculation. A thawed sample of the organism is required to inoculate the plate. A sterile inoculating loop (heated over a flame until red) is used to pick up a sample of the organism and spread a thin layer of it onto the growth plate. The plate is covered and placed in a 37°C oven so the organism can grow (24-48 hrs.). When the plate is grown it is stored at 4’C to help prevent further growth. This plate is sealed with Para-film and is used to obtain cells for overnight cultures. Overnight Cultures Overnight cultures were started with 100 ml. of sterile LB broth containing 10 x10’5 g/ml of chloramphenicol in a 250 ml. shaker flask. A heat sterilized inoculating loop was used to take a sample of the organism off the growth plate and place it into the 1.8 broth. When a few samples of organism had been placed in the broth, the shaker flask was covered and placed in a 37°C shaker at 180 rpm and left overnight. The growth plate was sealed and placed back into 4°C storage. 3.2 MEASURES CELL (INCENIRATICN grtical Deroity Optical density is used to measure the concentration of Escherichia $1; in solution. A K1ett-Summerson colorimeter with a green filter (400-450 x10"8 m wave length) was used. The concentration at low Klett readings are 1 x 106 cells/m1. solution per 1 Klett. (Oriel) Q_ett vLcell concentration calibration To get a Klett vs. concentration function, an overnight 23 culture was grown and the solution centrifuged at 8,000 rpm for ten minutes to separate cells from solution. The cells were then resuspended with .05 M Tris buffer at pH 7.5 to a Klett of 500. This solution was then diluted with a loom amount of Tris buffer solution and a Klett reading was taken after each dilution. This step was repeated until a Klett of 50 was obtained. This procedure was done twice on each sample. Knowing the amount of cells at the lowest Klett reading the amount of cells for the entire solution can be found by using: total # cells = (Klett) (1 x 106) (Vol. solution) Assuming a negligible amount of cells lost during the procedure the concentration to corresponding Klett reading can be found by: Klett = total # cells/ (volume) To obtain the cell mass multiply the above by 9.5 x 10‘13 g/cell. This calibration we is shown in Figure 3.2.1 and the equations used for calculating concentrations, found by least square linear regression, are found in Table 3.2.1. ME 3.2.1: Linear equations for the calculation of concentration Klett (x) Slope (m) Y-intercept (b) 0 - 125 9.500 x 10’7 0.0 126 - 300 1.242 x 10'6 -3.75 x 10’5 300 - 500 1.711 x 10"6 -1.80 x 10’4 The Klett meter should warm up for 10-15 minutes after being turned on. It should be zeroed with distilled water in the Klett test tube. After the zero is initially set take the test tube out and set thedialtoaKlettreadingofsixty.Reinsertthetesttubeandtake 24 Q d E 0.0006 > . '6 o . 0‘ 1 V c 0.0004 .9 fl 1 O b d C J 8 c 0.0002 0 o d 8 i — Calibration 00000 D pm - ° 0 100 200 300 400 500 Klett Reading Fig. 3.2.1: Calibration of Klett optical density for Escherichia _cLli thedial backtothezeroreading. Whenthearrowmatcheswiththe center line the machine has been zeroed. Any undue vibrations on the meter's foundation can lead to false readings. 3.3 ICN mom RESIN W 1:31 exchange resins need to be conditioned prior to use. The ion exchange resin was prepared in its chlorine form by the following procedureas suggestedtoPatrickJ. Oriel byRohmandHaas. This procedure is specific for anion exchangers. The hydroxide form is avoided as it can cause charges in pH. 1.) Contact the resin with a 10% NaCl solution for one hour at 37'C. 25 2.) Filter resin from NaCl solution and contact with a 4% H01 solution for one hour. 3.) Filter resin from the HCl solution and thoroughly rinse resin with distilled H20. 4.) let resin dry overnight at 55'C. ' 3.4 ABSORPTION Equilibrium isotherms were determined by the following procedures. 1.) Grow an overnight culture of BC246 in sterile 1.8 broth 37'C. 2.) Centrifuge cells of the overnight culture at 8,000 rpm for 10 minutes. 3.) Pour off supernate and resuspend cells in a Tris buffer solution of known molarity and pH until solution has a Klett reading of 450. 4.) Oontact two 0.4 9 samples of dry ion exchange resin with 9 ml of resuspended cell solution and put into a 37°C shaker at 180 rpm. 5.) Dilute resuspended cell solution to approximately Kletts Of 400, 350, 300, 250, 200, 150, 100, 50 and 0 and repeat step 4 for each different concentration. 6.) After 24 hr. take optical reading of each solution. 7.) Perform material balance on system to obtain adsorption of cells onto ion exchange resin. E ! . To acquire kinetic data prepare ion excharge resin as before and perform the following procedure. 1.) Repeat steps 1-3 of previous procedure. 2.) Oontact three 0.5 9 samples of dry resin with 10 ml of lmomm concentration resuspended cell solution. 3.) Take optical density readings of cell solutions at .25, .50, .75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 24 hours 4.) Perform material balance on system to obtain adsorption of cells onto ion exchange resin. ASml. syringewithafinemeshscreenwasusedtoseparatethe cell solution from the ion exchange resin. This prevented the large resin particles from interfering with the optical density. 26 3.5 RESIN PORDSITY &.AREA Harem The Micrcmetries Poresizer 9310 mercury poresemeter was used with 3 cc powder penetrameter to obtain surface area information. The data for this shady were pore sizes larger than 1 micron in diameter. No adsorption should occur in smaller diameters because the minimum dimensionocholiisfium. 3.6 SQNNIM; mm mm The Jeol T5330 scanning electron microscope was used with Polaroid type 52 blackandwhite filmwitha f-stop of 16wasusedtoexamine the resin and the cells sorbed to the resin particles. (Appendix 1) The pictures with the immobilized cells on the resin were produced with the help of the Electronic Imaging Center at Michigan State University. The steps used to prepare the immobilized cells are a series of ethanol drying steps and a critical drying step. (Appendix 2) 3.7 mm Batdl Growth The batch growth of the cells and a-amylase production were cmpared by the following procedure. 1.) Grow an overnight culture of E0246 at 37°C in sterile 18 broth. 2.) Prepare ion exchange resin in chloride form. 3.) After 24 hrs. put 5.0 g of ion exchange resin into 100 m1. of sterilized 18 media with corresponding amount of chloramphenicol and inoculate with 10 ml. of overnight culture. 4.) At periods of 24, 48 72, and 96 hours after inoculation take a Klett reading and take a small (approx. 11ml. ) sample of fluid, for all sutures. 5.) Spin cells out of fluid sample and keep refrigerated. 27 6.) Do an o-amylase assay on the fluid samples, Chmrioell and Manning as modified by Oriel and Schwacha (Appendix 3). 3.8 mm RECYCIE To check on the recyclability (the reuse of a resin already used for adsorption) of the ion exchange resins, the following procedure was followed: 1.) Prepare chloride form of resin as previously mentioned. Perform an equilibrium experiment on resin, as already described. When equilibrium experiment is through, separate the resin from the cell solution and rinse resin with 500 ml. distilled water. Prepare chloride form of resin as done in step one. Repeat step two. Compare cell loading for each recycle run. (IDEHEIKINEIRESUUES 8 DISCUSSION This study used anion exchangers and cation exchangers from Dow Chemical Company. This series contains a variety of particles sizes, morphologiesanddlarges. metoitsusebyotherresearohers for immobilization (Krug & Dougalis, 1983; Grulke et al, 1989) the Rohm and Haas anion exchange resin IRA-938 was studied also. The purpose was to determine how various resin properties affected sorption of cells. All resins investigated are shown in Table 4.0.1. 4.1 BATCH SORPTION’ENAIUEEICN’OFIRESINS All resins were evaluated in batch sorption tests to determine the sorption of cells per gram of resin, at typical pH for growing L 9g; of 7.5. To obtain the amount of cell adsorption onto the resin a cell balance was performed on the slurry. The final concentration of cells was subtracted from the initial concentration of cells, multiplied by the volume of solution and divided by the dry mass of resin in solution. (Oell mass on a wet basis). Ads = ( cyt- c )-v Ml Ads 2 amount of cells adsorbed (g cells/g dry resin) CO, C 5 concentration cells in solution (g cells/ml) , initial and time t V a volume of solution (ml) Mamassdryresin(g) The assumptions used for this adsorption study are listed in Table 4.1.1. 28 29 m3 4.0.1: Information on Resins Studied" Name (Codes) C/A S/I/W Type Structure, Mesh XU-40123.00 A w — Epoxy (14-40) XII-40091.00 A I — Epoxy (14-40) XU-40l96.0l A s I Gel 550 um x0-434-200.00 A s I Gel (200-400) x0-434-200.01 A s I Gel (100-200) xu—434-200.02 A s I Gel (50-100) XY-40032.00 c s — Macro. (16-45) XF-43356.00 A w — Macro. (16-50) HIS-43419.00 A w — Macro. (16-50) IRA-938“ A s I Macro. (20-50) XUS-40187.00 A s I Macro. (16-45) XII-40170.00 A s II Macro. (16-50) XUR-0525-187-239.00 A s I Macro. (20-50) XUR-0525-187-239.01 A 5 II Macro. (20-50) XUR-0525-187-239.02 A s — Macro. (20-50) XUR-0525-187-239.03 A w — Macro. (20-50) XUR-0525-187-239.04 A w —- Macro. (20-50) XUR-0525-187-239.05 A w —— Gel (20-50) XUR-0525-187-239. 06 N/A N/A N/A Acrylic (20-50) XOR-0525-187-239. 07 N/A N/A N/A Acrylic (20-50) XUR-0525-187-239. 08 N/A N/A N/A Acrylic (20-50) XUR-0525-187-239.09 N/A N/A N/A Acrylic (20-50) XUR-0525-187-239.10 N/A N/A N/A Acrylic (20-50) C= cation, A= anion, *All Dow resin except were noted, ** Rohm & Haas, N/A= not avail. S= strong, I= intermediate, W= weak 30 The first set of resins investigated were the XUR-0525-187-239 series.Thissetwaschcsenbecausetheywerethe first ME 4.1.1: Assumptions made for study - all cells viable - cells do not reproduce - negligible amount of cells adsorbed onto glass vial - negligible amount of cells and/or solution lost during separation for optical density readings - negligible amount of resin in solution while taking optical density - negligible amount of swelling occurred resins obtaired from Dow to study. As can be seen (Fig. 4.1.1) of the ten XUR-0525-187-239 resins samples .02, .03 and .04 show the best adsorption. Resin XUR-0525-187-239.00 poor adsorption may have been caused by the exchange groups being produced with a OH‘ group instead of the usual Cl' ion (Greenberg, 1989) . lbdenical m A more thorough kiretic study was dore on XU—43419.00, XF- 43356.00, XU-40123.00, XU-40091.00, HIS-40187.00 and XU-40l70.00. A "negative" sorption is due to the presence of small pieces of resin sorbing in the light path. As shown in Figure 4.1.2 the resins XU- 43419.00 and XF-43356.00 have a "negative" adsorption. XU-43419.00 started to fracture and continue to fracture throughout the trial and XF-43356.00 had good adsorption and then fractured. XU-40123.00 initially gave a "negative" adsorption then the cells sorbed onto the support with time, suggesting that it initially fractured a small amount and then stopped. XF-43356.00 suggests that resins should be agitated for some time to 31 .0021 ------—1 ‘Q 0‘ 3' ‘ .0010 - a 3 hr. .0015 “—— 0-...-- .0012 .0009 .0006 .0003 Adsorption (g cell/g dry resin) \X\\\\X % 1' J .00 .01 .02 .03 .04 .05 .06 .07 .03 .09 .10 XUR-OSZS-L87-239. -.0003 Fig 4.1.1: Batch Adsorption of XUR-0525-187-239. series of resins Init. cell conc. = 3.24 x 10'4 g cell/ml pH = 7.5, .05 Tris buffer determire wether they have good mechanical strength. When these solutions were observed under a light microscope small portions of resin were found floating. This verified that resin fragments result in an increase in light adsorption. The resins XU-43419.00 and XF-43356.00 are both macroporous slggestingthatsaremecroporulsresirsaremtstrongenwghforthe agitation that may be encountered. XU-40091.00 also gave an initial "negative" adsorption (Fig.4.1.3). It was concluded that the‘resins were too fragile at the conditions used. The epoxy resins XU-40123.00 and XU-40091.00 have a tendency to degrade in basic conditions (Greenberg, 1989) . 32 Further Evaluatim Figure 4.1.4 Show the resins, XUS-40187.00 and XU-40170.00, compared during a kinetic study, show a positive adsorption thus good mechanical strength. The resins XU-434-200.00, XU-40196.01, XY-40032.00 and IRA-938 were screered by comparing the samples after contact for twenty-four hours with cell solution at a typical cell concentration of L _gql_i under normal conditions (Table 4.1.2). TABEE 4.1.2: Adsorption of cells by ion exchangers Initial Concentration: .00043 g cell/ml Resin Adsorption (a cell/q drv resin) IRA-938 0.00855 XU-434-200 . 00 0 . 00590 XU-40196 . 00 0 . 00195 XY-40032 . 00 0 . 00197 To further study a resin it was decided that a positive adsorption greaterthan .001gcell/gdryresinwasrequiredandinthecaseof the XUR-0525-187-239. series the largest sorptions were required. From this initial screening period the resins suitable for further study are sham in Table 4.1.3. There is a possibility of missing good resins by this evaluation technique since the equilibrium isotherm is not known. Adsorption (g cell/g dry resin) 33 0.001 - -.00‘I k-__ -.002 “ ~~ \ Rosin: ““~~~-~\ . o-e xu-40123.00 “ o—e XUS-43419.00 o—o XF-43356.00 Adsorption (g cell/g dry resin) -.003 0 10 20 Time (hr.) Fig. 4.1.2: Carparison of kinetics of three resins pH = 7.5, .05 M Tris buffer 0.0002 0.0000 /_,— // i I' -.0006 -.0008--u -.0010s...-.ss ... I U ‘ 0 s 10 ' I; 20 ' ' Time (hr.) Fig. 4.1.3: Kiretics of xu-40091.00 pH = 7.5, .05 M Tris buffer 34 0.002 0.001 Adsorption (g cell/g dry resin) 0.000 /14, ‘ L // .'..U ”in: no EU-40170.0 o—o 05-40187. 0 0 5 10 15 2'0 35 Time (hr.) Fig. 4.1.4: Comparison of kinetics of two resins pH = 7.5, .05 M Tris buffer ME 4.1.3: Positive Resins after initial Evaluation Resin C/A S/I/W Type Structure XUS-40187.00 A s I Macro. XU—40170.00 A 3 II Macro. xu-40196.00 A s I Gel XY-40032.00 c s Macro. mm-05250137-239.02 A s I Macro. XUR-05250187-239.03 A w Macro. XUR-05250187-239.04 A w Macro. XU-434-200.00 A s I Gel IRA-938 A s I Macro. Table 4.1.3 shows that gel and macroporous resins have good sorption while the acrylic resins had poor adsorption of L coli. All but one resin giving good adsorption were anion exchangers. For further evaluation, samples of the resins XY-40032.00, XUR- 35 0525-187-239.02, .03 and .04 were contacted with a cell solution for twenty-four hours and compared with XU-434-200.00 and IRA-938 (Table 4.1.4 8 Table 4.1.5). From Tables 4.1.4 and 4.1.5 it is shown that the resin XII-434-200.00 has a much better sorption than the resins XY- 40032.00, XUR90525-187-239.02, .03 and .04, from 1.76 - ¢>times better sorption. The resin XUR-0525-187-239.03 looks like it might not be mechanically strong after noticing the decrease in adsorption during the initial batch adsorption (Fig. 4.1.1) . When all the resins that had positive cell adsorption during these batch adsorption tests are compared IRA7938 had the best adsorption. The resins XUS-40187.00 and XII-40170.00 were kept after initial screening to observe the differences between Type I and Type II strong base anion exchangers. TABLE 4.1.4: Adsorption of cells by anion exchangers Initial Concentration: .00017 g cell/m1 Resin Adsorption (q oelléarratlarsun___. IRAP938 0.00300 XU‘434-200.00 0.00214 XURr0525-187-239.02 0.00121 XUR90525-187-239.04 0.00000 TABLE 4.1.5: Adsorption of cells by anion exchangers Initial Concentration: .00035 g cell/ml Resin Adsorption (aioeLLaIrhaLlrsdmu___ IRA-938 0.00799 xu-434-200.00 0.00525 XURr0525-187-239.03 0.00299 XY-40032.00 0.00110 36 4.2 (ELL mm mm Resins with high cell sorption were tested over a range of conditions to determine the effects of p-I, slrface charges, buffer concentration, surface area, cross-linking and porosity on cell sorption. Comparison of pairs of resins will allow us to determine the effects of each variable on sorption. Isotherns As discussed in Chapter 2, the Langmuir Isotherm fits the cell sorption data for this system (Eqn. 1). Ads = Kl-c (l) Ads 3 amount of cells adsorbed (g cells/g dry resin) C a concentration cells in solution (g cells/ml) K1 5 resin capacity (g cells/g dry resin) K2 5 inverse of equilibrium constant (9 cells/ml) K1andK2canbedeterminedbyseveralmethods.Thecell concentration times the inverse of equation 1 is linear (Eqn 2). A least squares linear regression was dore to calculate the slope and Y- intercept. __C_ = .9. + __I<2 (2) K1 AsshowninFigure 4.2.1thedatadoescomeouttobelinearand the calculated K1 and K2 of the Iangmuir equilibrium provided good fit to the data (Fig. 4.2.2). K1 gives the capacity of the resin when the -cellconcentrationsinthemediumarehigh. I<2istheinverseofthe equilibrium constant this parameter is better when it is small because the resin will reach its cell capacity at a lower cell concentrations and at lower K2 the equilibrium constant will increase thus favoring 37 conditions for the immrbilized cells. A low IQ suggests a stronger interaction between the cells and resin surface. The reason for an equilibrium between the cells and the binding sitesoftleionexchangeresintoexistarenotloown.aewould suspect that the cells would bind with all the available sites until few sites are left. Possible causes of the e'quilibria phenomena are thought to be the resin binding with lipopolysaccharides that may have care off the cells and put in solution or the cells hindering adjacent binding sites. Thepurposeofmrstudyistofindresinshavingthelargest amount of cells sorbing onto the resin. This parameter, Kl, will be the center of this section since it provides a good comparison of resin capacity. The results of the equilibrium tests for the various resins are shown in Table 4.2.1 for pH of 7.5 and .05 M Tris buffer solution. From this test it is shown that the best resins for adsorption, have the largest K1, are XU-434-200.00 and IRA 938. Both of the best adsorbers are Type I strong base anion exchangers. TABLE 4.2.1: Equilibrium Constants 1C1 & IQ at pH = 7.5, Tris = [.05] K1 IQ r RESIN (g glllg d_ry resin) (g cellml) XUS-40187 . 00 . 004 664 . 0004187 . 920 XU-40170 . 00 . 000961 . 0000089 . 946 IRA-938 . 008819 . 0000039 . 995 XU—434-200 . 00 . 005050 . 0000008 . 998 38 0.030 0.020 / 0.010 / / 0.000 0.0000 0.00004 ' 0.00008 —- Linear Regression o Experimental 0.00012 ' 0.00016 0.00020 Concentration (g cell/ml) Cone/Adsorption (g dry resin/ml) T T Fig. 4.2.1: Linearization of Iangmuir Isotherm Rain: XU-434-200.00 pH = 7.5, .05 M Tris buffer 0.010 A c .6 O 0A0 41 D L 0.008 a 0 2‘ D 1 Q 0.006 B d 0 o 3 0.004 c .9 a 8 0.002 c m 2 ‘1- Experimental 0.000 . g , . , ' . j 1 1 1 ' TLan'gm‘mrjMo'el 0.00000 0.00005 0.00010 0.00015 0.00020 Concentration (g cell/ml) Fig. 4.2.2: Iangmuir Model of Adsorption Resin: XU-434-200.00 pH = 7.5, .05 M Tris buffer 39 Jen—119.9 'Ihe resins HIS-40187.00 arri XII-40170.00 differ only in their ecchangegrolps; TypeIandTypeII. Theybothhavesimilardlarge densities. Table 4.2.2 and Fig. 4.2.3 show that the Type I strong base animexd'langersarebetterthantheTypeIIstrolgbaseanion exchangers. The Type II exctlanger is "saturated" at coverage of .001 g cells/g dry resin at solution concentrations below a typical level of .0003 g cell/ml. Therefore, a Type II support mild not add significant amomrt of cells to a reactor at normal cell loadings. However Type II support might be good at low cell concentrations due to it having a smaller IQ. These two resins are a good corparison since they aremade with the same polymer matrix and are of the same mesh sizes, hence similar surface areas. ME 4.2.2: Equilibrium Constants K1 & IQ for Type I & II strong base anion exchangers at pH = 7.5, Tris = [.05] K1 IQ RESIN Type (a cell/q dry resin) (01 cellgml) XUS-4 0187 . 00 I . 004664 . 0004187 XU-4 0170 . 00 II . 000961 . 0000089 Surface Area Since Langmuir type equilibrium suggests monolayer adsorption, the amount of surface area available is of great importance to the amount of adsorption that may occur. Surface areas of the resin were measured using mercury porosimetry. The surface areas (mZ/g) are reported for pore sizes greater than 1 mm diameter. The cells cannot fit into of smaller pores. The XU-434-200.00 studied for surface area has a different adsorption capacity than reported in section 4.2.1 because 40 0.003 ’E '6 % Q) "1 L. I”’ E‘ ,4," '° 0.002 ,6” O5 ’I’ E ,z’ 3 12/9 0 O l/ O / UT ,’ V l/. ‘ ‘ CC) 0.001 1” J 5 0 Type II Exp. (0 a Type I Exp. 2 —- Type 11 Iso. 0.000 " Type '5“ Concentration (g cell/ml) Fig. 4.2.3: Corparison of Type I & Type II Strong Base Anion Exchangers Resin: Type I: XUS-40187.00 Type II: XU-40170.00 pH = 7.5, .05 M Tris buffer the resin studied forsurface area came from a different stock bottle of material and may of had a different surface area. Spheres this small, mesh size 220-400, are currently state of the art and could have some variances due to production (Greenberg, 1988) . Differing stocks of sphere (mesh) sizes of the anion exchanger XUS-434-200 were available in 50-100 mesh, 100-200 mesh and 200-400 mesh. As the mesh sizes increasettediameterofthespheresdecreasethus, density is constant, the surface area per mass of resin should increase. 0.0000 1 0.0001 1 0.0002 ‘ 0.0003 1 0.0004 1 0.0005 0.0006 assumingthe 41 TABLE 4.2.3: Adsorption capacity and Surface Area K1 K2 AREA POROSITY RESIN ILcell/q) (q cellgml) 11112qu (cc/q) IRA-9301 .008819 .0000039 .4279 1.1435 xu-434-200.00 .008867 .0000081 .0922 0.6787 xu-434-200.01 .006051 .0000082 .0549 0.6406 xu-434-200.02 .002660 .0000025 .0269 0.5917 Table 4.2.3 shows that IRA-938 has the greatest amount of surface areaoftheresirscteckedandthatitisalsohasthelargest porosity. The adsorption capacity, K1, shows to depend on the amount of surface area present and the inverse equilibrium constant, IQ, does not show any trend with either the porosity or the surface area available. As shown in Figures 4.2.4 and 4.2.5 that the surface of the XU—434- 200.00 is srooth with no visible pores and that the IRA-938 is very convoluted and has many pores. By looking at the cells on the surface of a resin (Fig. 4.2.6) the cell dimensions were found to be 3 mm x .5 mm. By taking the cell area, cell width times length, and multiplying it by the number of cells adsorbed onto the resin the amount of area can be obtained (Eqn. 3). Areausedbycells= (area (ml-z-[celln-Kug celng dry resin) (3) 9.5 x10‘ g/cell The dimensions used for this study, cell length and width, are for the minimum cell packing on the resin. The cell diameter, 0.5 mm, would be for the maximum cell packing. 1 The reported surface area and porosity for IRA-938 are for pores greater than 1 am in diameter. The absolute values are higher but not accessible to cells. 42 10KU X1,868 r—‘* .71, Fig. 4.2.4: Surface of resin XU-434-200.00 43 Fig. 4.2.5: Surface of resin IRA-938 44 Fig. 4.2.6: Cells immobilized on the surface of the resin XU-434-200.00 The percentage of surface area used by the cells is found by multiplying the surface area used by cells by 100 and dividing by the surface area of the resin (Eqn. 4). a; surface area used = Area used (ma/q drv resinl-loo (4) area of resin (mZ/g dry resin) Table 4.2.4 shows that the macroporous resin shows a greater percentage of unused area than the gel type resin. This is probably due to the cells reedjng to diffuse into the pores to use the available surface area, and causing possible fouling of the pores, preventing further use of the pore. This shows that the macroporous resin uses the 45 least amount of available slrface area of the resins studied. This is hypothesized to be due to the cells having to diffuse into the pores TABLE 4.2.4: Percentage of unused surface area* cell area = 1.50 x 10’12 RESIN % unused surface area IRA-938 96.75 140-434-200 . 00 84 . 82 XU—434-200 . 01 82 . 60 XU-434—200 . 02 84 . 39 *Areatakenforporesgreaterthanlmmdiameter. and consequently interfering with a free path into the pores due to the immobilized cells acomnllating at the surface of the resin and the entrance of the pores or the cells are not able to diffuse deeply into the pores. Figures 4.2.7 show the macroporous IRA-938 Type I strong base anion exchanger cut through the middle and that the majority of the cells are on the outer surfaces of the resin. Figures 4.2.8 - 4.2.11 show a closer view of the cells on the resin surface at various depths into the resin. As can be seen in Figure 4.2.8 the majority of the cells are in the first 30 m depth of the resin. A cell distribution according to resin depth is shown in Figure 4.2.12 were, using the photographs, the cells were counted on the resin surface and the depth measured. This distribution seems to agree with the calculated amount of area used (Table 4.2.4). The predicted amount of surface area (Table 4.2.4) used by the gel resins, z 15%, does not agree with the Figures 4.2.13 and 4.2.14 which show complete coverage of the surface. A possible cause of this may be that the minimum pore diameter used, 1.0 pm, from the porosity data may 46 have been too small thus giving a larger surface area than what is available to the cells. Fig. 4.2.7: Immobilized cells on strong base Type I anion exchanger IRA-938 sliced through middle. ‘ 47 Fig. 4. 2. 8: Immobilized cells on strong base Type I anion exchanger IRA—938 sliced through middl Depth 0_— 26 um 48 Fig. 4.2.9: Imrrobilized cells on strong base Type I anion exchanger IRA-938 sliced through middle. Depth 24.5 - 76 um 49 Fig. 4.2.10: Immobilized cells on strong base Type I anion exchanger IRA-938 sliced through middle. Depth 74.5 - 126 um 50 Fig. 4.2.11: Immobilized cells on strong base Type I anion exchanger IRA-938 sliced through middle. Depth 121 pm - center 51 Resin: IRA-938 a 3 1: s a s : ‘: 3, 5 7 3’ ‘ O 8 a g g ! WINCH-10M) Fig. 4.2.12: Cell distribution for depth into Type I strong base macroporous anion exchanger IRA-938 . 52 Fig. 4.2.13: Type I strong base gel anion exchanger XU—434-200.00 with immobilized cells, entire exchange bead. 53 Fig. 4.2.14: Type I strong base gel anion exchanger XU—434-200.00 with immobilized cells, surface of exchange bead. Crosslm' ' The effect that cross-linking of the polymer matrix was investigated with 200-400 mesh Type I strong base anion exchangers. As shown in Table 4.2.5 the adsorption capacity, K1, decreases with increasing cross-linking. This observation agrees with Kuwajima et a1 (1957) observation for Haemophilus mg. The cross-linking of the polymer is the cause of this effect since the surface areas are similar. 54 No trends were fomd for the effect that cross-linking has upon the inverse equilibrium constant. As the cross-linking of a resin increases the water retention of the polymer decreases thus the permeability into the resin decreases (Greenberg, 1989) . This decreased permeability ME 4.2.5: Adsorption Capacity and Surface Area of differing crosslinking of 200-400 mesh gel spheres, TypeIstrongbaseanionexchanger K1 K2 Percent (g cell) (g cell) Area* Porosity RESIN Crosslink (g dry resin) (ml) JmZ/q) (cc/q) XU-434-200.00 2 .008867 .0000081 .0912 .6707 XMFlO62-1824 4 .004545 .0000112 .0804 .6574 XY-40013.00 8 .001918 .0000057 .1257 .6263 * Area taken for pores greater than 1mm diameter. suggest that the cell has to actually reach into the resin in order to anchor itself to the resin. This decrease in the permeability is shown in Table 4.2.5 by the decrease in the porosity as the percent cross- linking increases. Macroporous resins usually have fourteen percent cross-linking (Greenberg, 1989) . This suggests that the amount of volume inside a resin could play a large factor in the amount cells adsorbed. EH The solutions pH was studied on its effect on the adsorption capacity of XU-434-200.00 and IRA-938. The pH of solution was kept :1 pH unit from optimal pH, 7.2 for L coli, so as to replicate variances that could be found in an actual fermentation system. Figures 4.2.15 and 4.2.16 show that around a neutral pH adsorption capacity is at its 55 lowest and increases as the pH gets away from neutrality. Theadsorption capacity increases faster when the solutions get more basic. The change in adsorption capacity from lowest to highest observed was 420%; and the highest capacity occurred at pH of 8.2. This increase of adsorption away from neutrality might be caused by the cells becoming a stronger ion. This wolld depend on the isoelectric point for L c_o_l_i. This observation is in conflict with that found by Niehoff and Edlols (1973) for L go_l_i were it was shown that adsorption was not effected within the pH of 6-10. K1 can be fit with a quadratic formula with a function of pH (Eqn. 5). 1 0.0084 1 i + 8 0.0082 -: C1 '4 . v '_ 0.0080 1 X ‘ i 0.0078 . 0.0076 . . s . . e e r T T . 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 Molarity [M] Fig. 4.2.18: Resin IRA-938: Kl as a function of buffer concentration. pa = 7.7 The ion concentration does not show any trend for effecting the inverse equilibrium constant, IQ, for IRA-938 and XU-434-200.00 (TABLES 4.2.9 & 4.2.10). ME 4.2.9: Effect of buffer concentration upon IQ Resin: XU-434-200.00 phz= 7.5 Tris [M1 IQ (q cell/uh .025 .0000044 .050 .0000057 .075 .0000078 . 100 . 0000004 60 ME 4.2.10: Effect of buffer concentration upon IQ Resin: IRA-938 pH = 7.7 Tris I’M] K2 (0 cell[1m_l) .025 .0000104 .050 .0000164 .075 .0000026 .100 .0000083 4.3 KINETICS Using the simple reaction used for ion exchange and modifying the reactants used to our system a simple second order reaction, as shown below, was assumed to take place. Reaction: ka Cells + Resin Cells-Resin Rd WIRE: [C], [C10 5 # cells in solution (# cells/m1 soln.) [R], [R]0 E # sites available for cells in solution (# sites/ml soln.) [R-C] E # sites occupied on resin (# sites occupied/ml soln.) ka 2 adsorption constant (ml/# sites(or cells) ~hr) kd E desorption constant (1/hr.) [R]o K1 (g cellig d_ry resin) omass d_l;y resin (g) (vol. solution (ml.))-9.5 x 10' g/cell [C]o -=- (Init. conc. cell(g cell(ml)) (mass cell (9.5 x 10" g/cell)) Assume all cells lost from solution are gained by resin then: Cell balance: . [C] [C10 - [R3C] Open site balance: [R] [Rio - [R'C] Occupied site balance: [R-C] = [C10 - [C] 61 Reaction Equation: Irreversible: d[R-C] = ka [C] [R] dt Substiolting and separating the irreversible reaction the equation becomes: ka-t =01“: dIR-C] ([Rio " [R'C])([C]o " [R'CD Integrating: ka '1: = - R C [Rio ’1[C]o ([Rlo ' C'[R C1) [C10 Reversible: d[R-C] = ka [C] [R] - kd [R-C] dt At equilibrium: Keq = ka/kd kd = ka-IQ/(9.5 x 10‘13 g/cell) then: Substituting and separating the reversible reaction equation becomes: k t - JR C] QJt[R]o[C]0' [R-c111c10+ [810+ 95 x IVE/mums]?- let: a = [R]o [C]0 = 1([R10 + [Clo + l/KZ) q = 4ac - b2 Ifq>0then: ka-t= 2 tan'1 2ch-01+b _ _.2_ tan’1 _b /q Jq 62 If q<0then: ka-t = 1 ln 2ch-01 + b - f-E ./-q 2c[R-C] + b + ,/-q —-_q_ln[b+/-q Table 4.3.1 shows the adsorption capacity, K1, and the equilibrium constant, IQ, used for the kinetic study as found from the equilibrium isotherm. TABLE 4.3.1: Constants used for kinetic suldy {ii = 7.5, Conc. = .05 M Tris K1 K2 _Resin (q cell/q) (q cell/ml) r IRA-938 . 008819 . 0000039 . 999 XU-434-200 . 00 . 0088671 . 0000081 . 989 XII-434-200 . 01 . 0060509 . 0000082 . 998 XU—434-200 . 02 . 00266 . 0000025 . 994 Reaction Order Figure 4 .3. 1 shows that the irreversible kinetic model gave a straight line for the XU-434-200.00 and that irreversible and reversible reactions have differing calculated adsorption constants, ka (Table 4.3.2). This also shows that second order overall kinetics is occurring for this system. The desorption constant, kd, is seven orders of magnitude greater than the adsorption constant. Figure 4.3.2 shows that the irreversible kinetics is a better model than the reversible when compared to the experimental data. This suggests that little desorption of cells is occurring and that the irreversible kireties is 63 the better model for this system. For the following example : Init. cell conc. .0000988 9 cell/ml Solution Vbl. = 10 ml Tris buffer ==.051M pH = 7.5 Resin: XU‘434-200.00 Mass Resin = .500 From Table 4.3.1 : K]. = .0088671 g cell/g dry resin From the equations already given for [R]o and [C]o it is folnd: [R10 = .0088671 01 cell] resin- .500 resin 10 ml.-9.5 x 10’ g/cell = 4.667 x 108 sites/ml [Clo = .0000988 g celle 9.5 x 10' g/cell 1.04 x 108 cells/ml TABLE 4.3.2: Kinetic Constants for XU—434-200.00 pH = 7.5, .05 M Tris buffer [010 =1.04 x 103 cells/ml, [R10 =4.667 x 108 site/ml 10 ml cell soln., .5 gdry resin Reaction Type ka x 108 kd r Irreversible 1 . 068 -—— . 998 Reversible 1.396 .11905 .998 f71uh\ T_____.._-- _-_.. .4 :; 1.2E—08 - - —- — 4)- ? U 1 1 1r 3 y g //o / 1 1 1 1 ° / ° 0 ‘3 8.0E—09 ‘ f r-e O O U E. ‘ /’/ o v / | / . .J l//8 03 405-09 ———/4;-- —— .7 - _— m U 0 / l 1 \—.I 4 I : L_.J 4 /’6’ . ! / l C. .1 / 1 / l 0.0'¢: If 1 f I 1 0.0 0.4 0.8 1.2 Time (hr.) Fig. 4.3.1: Linearization to calculate adsorption constant Resin XU-434-200.00, pH = 7.5, .05 M Tris buffer [010 =1.04 x 103 cells/ml, [R10 =4.667 x 108 site/ml 0020 — 0 1r , ____________ } _____ itfl re «1 ’9 i ' ‘171 1 ,5 ' ‘8 00016 J/ i l o 1 I >‘ '4 ' i L .0 4 | 1 .1 i 51‘ 0.0012 s . —_ 1 i I Q) 1 u ‘ i “ l 3 0.0008 C 1 O 4 1:2 4 9' 0.0004 . 8 ‘ 0 Experimental 2 ‘ --- Reversible Rxn. -— 'bl R . 00000; e - . e have? L n 0 1 2 3 4 5 Time (hr.) Fig. 4.3.2: Reaction models of cell adsorption Resin XU-434-200.00, pH = 7.5, .05 M Tris buffer [C]0 =1.04 x 108 cells/ml, [R]0 =4.667 x 108 site/ml To find the reaction order for the individual reactants for a binary reaction the amount of one reactant sholld be much greater than the other. The initial adsorption sites available on the ion exchange resin were approximately four times greater than the cell concentration. Using irreversible kinetics: d[C] = ka-[CJA-[RJB dt Taking the natural log of the above equation the following is used to find the reaction order of the reactants. ln(-(d[C]/dt)) = A-ln([C]) + 100(3) + B-ln([R]) Knowing the reaction is second order overall then: A + B = 2. 66 ln(ka) + B-ln([R]) is considered a constant because ka is a constant and [R] is coreidered a constant, since [R] >> [C]. Then the above equation becomes a linear equation where A is the slope. Figure 4.3.3 and Table 4.3.3 show a straight line for ln(-(d[C]/dt)) vs. ln([C]) using least square linear regression a slope of #1.0 was found. This indicates that the kineties are first order for both reactants. TABLE 4.3.3: Reaction order of Cells Resin: XU-434-200.00 pH = 7.5, .05 M Tris buffer [C10 =1.04 x 108 cells/ml, [R10 =4.667 x 108 site/m1 10 ml. cell soln., .5 g dry resin Slope (A) Y-int. (ln(ka) + b ln([RJ)) r 1.0526 0.0269 .9996 1.0956 -0.79068 .9960 1.1187 -1.01935 .9900 Ibrarrrmrarilrrzuas The macroporols resin, IRA-938, shows slower kinetics than the gel type resin, XU-434-200.00, for the same initial concentration of cells (Fig. 4.3.4). This suggests that the cells have to diffuse into the pores of the resin to reach unocolpied surface. The kinetic model was basedonthegeltyperesin, whichhasporestosmall forthecells, anddidnottake intoaccount changesduetopore falling. Themodel for the gel, which takes into account the initial mass transfer, is compared to data for the macroporols resin IRA-938 in Figure 4.3.5. Thisslggeststhattheapparentadsorptionconstant changesovertime. This is a typical result for systers in which the rate-controlling step can change from extra to intra particle mass transfer. 67 20 19 C ‘U 18 / \ / $6 1 I V 17 /6/ s / // . /7 15 Regression 14 15 16 17 Experimental 18 19 ln(Concentration (g cell/ml)) Fig. 4.3.3: Reaction order of cells Resin XU-434-200.00, [ii = 7.5, .05 M Tris buffer [C10 =1.04 x 108 cells/ml, [R10 =4.667 x 103 site/ml This is further shown by the change in the adsorption constant as time progressed. Table 4.3.4 shows that as time increased the ka decreased. This indicates that the cells must diffuse into the pores aroastimeincreasestheadsorptionoonstantdecreasesduetothe cells 68 ----------- P---_-1----a Adsorption (g cell/g dry resin) 0.0004 I-I RA-938 -4 4- 0. 0.0000 o—o 1 US 3 23' 00 0 5 10 15 20 25 Time (hr.) Fig. 4.3.4: Kireties of macroporous (IRA-938) and gel (XU-434-200.00) anion exchanger. pH = 7.5, .05 M tris buffer, [010 =8.21 x 107 cell/ml 0.500 g dry resin 69 0.005 A .5 . / ‘” Z L 0.004 a / “D -l ‘ 3 0.003 / o _ 1 % fi—o‘ 1 o o l ‘7‘ e v 0.002 o ..- C o .9 . g? ”8'- O 0.001 -0—- i 0‘) '0 < - O xperiinental 0.000 . . - , 4 . - . ' _r 111124330 st. 0.0 2.0 4.0 6.0 8.010.012014.016.018.020.022.024.0 Time (hr.) Fig. 4.3.5: Macroporous resin (IRA-938) using initial adsorption constant. 10 ml cell soln., .5 g dry resin [010 = 2.45 x 108 cell/ml, [R]0 =4.64 x 108 sites/ml pH = 7.5, .05 M Tris buffer TABLE 4.3.4: Adsorption constant and time for macroporous resin IRA-938. [010 = 2.45 x 108 cell/m1, [R10 =4.64 x 108 sites/ml 10 ml cell soln., .5 gdry resin pH = 7.5, .05 M Tris buffer Time (hr.) ka (m1./# cells-hr ) x 109 0 - 0.25 1.965 0.25 - 1.5 0.7031 1.5 - 3.0 0.5600 3.0 - 00 0.3400 having to get past cells that are falling the pores to contact unoccupied surface. Theseconstantswereusedtomakeakineticcurveand iscampared to he experimental data and show a close fit (Fig.4.3.6) . 70 0.005 ”c? "(7) .1 Q) / ...— 3' t 0.004 2,,- E E‘ . U . V/ 1 CD / > 0.003 c '6 U 0 0'1 V 0.002- C .9 +4 9 8 0.001 — 2 O Expe imenlol 0.000 . — Mo'crlpiro Js Model Time (hr.) . . 4 . . T . . . , 0.0 2.0 4.0 6.0 8.010.012.014.016.018.020.022.024.0 Fig. 4.3.6: Macroporous resin (IRA-938) adsorption using constants from Table 4.3.4. 10 ml cell soln., .5 g dry resin, EH = 7.5, .05 M Tris buffer [C10 = 2.45 x 10 cell/ml, [R10 =4.64 x 108 sites/ml Mass Transfer The effects that resin size and initial concentration have upon the adsorption constant were studied. Various sphere sizes of a gel Type I anion exchange resins XU-434-200.00, .01 and .02 were used. The equation below, modified from Eqn. 21.2-25 from Bird, Stewart and Lightfoot, was used for constant surface composition and small mass transfer rates . Cf’DABf 1/2 1/3 _lca-JL = 2.0 + 0.60 M): _u_ “f PDAB By multiplying the above equation by of DABf the equation 71 becomes: 1 1/2 1/3 ka-D = 2°0’Cf'DABf+ 0.60-D‘ _mmf _p__ 'Cf‘DABf Hf ’DAB f Assuming Cf, DABf and everything in the brackets of the above equation tobeconstanttheequationbecomeslinearinform, asshowninthe equation below. ka-D = b + m-Dl1 Were ka is the adsorption constant and D is the particle diameter. Figure 4.3.7 shows ka-D vs. D11 is linear and that ka-D decreases with increasing cell concentration. This shows that known mass transfer 1.500E-12 _f A J E 1.3005-12 1 .35 l a 75 1.1005—12 ° 1 3 9.0008-13 o E . _g 7.0005-13 .- u E . a V 5 CODE-13 . T . D ' 6 ' a no. 6 ‘ 4 0004117 9 . 4‘ 10005-13 - e .--_ 1 o .0001 49 10008—1: s 4 e s - I _r . _. 1'. 0200?}! 0.007 0.009 0.01 1 0.015 0.015 Square Root of Diameter (m’.5) Fig. 4.3.7: ka-D vs. 035 for Type I strong base gel type anion exchanger Resins: XU-434-200.00, .01 8.02 pH = 7.5, .05 M Tris buffer f0 72 phenolena can explain this system. Table 4.3.5 shows the constants folnd for a least squares linear regression done on the equation for each cell concentration studied. At cell concentrations greater than .00025 g cells/ml the ka-D decreases little. This point of cell concentration may be a mare 4.3.5: Linear Regression of ka-D vs. 0‘5 Resins: xu-434-200.00, .01 a .02 pH = 7.5, .05 M Tris buffer Cell 0611c. m x 1011 b x 1013 r q cell/m1 nil-03mg cell-hr) 1111-11111 cen-hr) .0000988 11.90 -3.00 .995 .0001649 9.30 -2.30 .982 .0002544 9.35 -4.40 .998 .0004028 8.13 -3.40 .984 critical concentration were the free path and the number of collisions do not change. Observing that concentration has an effect on the adsorption a least squares lirear regression was performed on the effect that initial cell concentration has on the ka-D for cell concentrations up to .0002544 g cells/ml for the various 011 (Table 4.3.6). BIBLE 4.3.6: Lirear Regression of ka-D vs. Cell Conc. Resins: x0-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer 1 D" 1111 x 109 b x 1013 r m5 ml°D§((fl cell-hr) ml-D/(# cell-hr) .01493 -3.39 18.00 -.980 .01045 -2.46 11.20 -.982 .00806 -2.36 9.37 -.997 73 Mass Transfer Model Sinceboththeinitial cell concentratianandthesquarerootof theresindiameterseemtohavealireareffectmtheka-Dalinear modelwasproposed forthesecolbinedeffects. Bydoingaleastsquares linear regression on the slopes and Y-interoepts of the lines of cell concentrations effect on the ka-D for each of the D11 the following was found: For the slopes of the concentrations: m = -1.567 x 10'7 (le-m‘i/(g cells-11 cell-hr)) b -9.875 x 10'10 (mlz-m/(g cells-11 cellohr)) r = -.996 and for the Y-intercepts of the concentrations: 111 = 1.290 x 10"10 (ml°m1‘/(# cells-hr)) b = -l.541 x 10'13 (ml-m/(# cells-hr)) r = .989 This gives the equation for ka-D as a function of the resin diameter and cell concentration as: ka-D(dia, conc) = (-1.567 x 10'7o(dia.)11 - 9.875 x 10‘10)-(conc.) + (1.29 x 10"10-(dia.)11 - 1.541 x 10’13) The use of the above equation with concentrations greater than .000255 9 cell/m1 is done by keeping the concentration at .000255 9 cell/ml. The fit of the above equation to the esperimental data is shown in Fig. 4.3.8. 74 1.500E-12 +:-—-1 A «1 ’3 1.3008-12 / .C i. 1 //fl 7, 1.1008-12 / ° 1 // 4 ‘5 900015-11 / j V o V / E J / // E 1 2’ 50008-1: V J 6 A K/ -- Model 9‘ 3.0005-13 .1 I: .000140 1.0005-13 . . s Isl - 1 4 34's rBL 0.007 0.009 0.011 - 0.013 0.015 Square Root of Diameter (m’.5) Fig. 4.3.8: Linear model for ka-D vs. D11 for Type I strong base gel type anion exchanger. Resins: XU-434-200.00, .01 & .02 pH = 7.5, .05 M Tris buffer Effects of Diameter 6 Surface Area Figure 4.3.9 shows that the particle diameter hasan effect on the adsorption constant. As the diameter of the particle increases the adsorption corstant decreases. This effect looks like the amount of surface area exposed to the solution is the cause for the decrease because, as the sphere diameter increases, the surface area should decrease for the same mass of resin, assnming constant density. Figure 4.3.10 shows that the ka increases with increasing available surface area. This increase is lirear with the surface area. The effect that the initial cell concentration has upon the adsorption constant is shown also in Fig. 4.3.9 and 4.3.10. As the cell concentrations increase the ka decreases until a concentration of 75 .000255 9 cell/ml is obtained, at this concentration the adsorption constant steadies at around 5 x 10"9 m1/# cell-hr. This increase of ka with smaller cell concentrations can be attributed to the cells having a longer free path and less collisions with one another at lower cell concentrations. This occurred for all three resin areas studied. ka (ml/If cellthr.) LIE-08 ‘ Canc ntraliavi .0000 8 105-09 \ , 8.0E-09 . \ \-\ '\ 7.05-09 \ \‘ 6.0E-09 - \\\ I \\o 5.0E-09- db . 4.05-09 #— | I V‘ ' fi ' ' t ' 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 Sphere Diameter (mm) Fig. 4.3.9: Resin diameters effect an ka for Type I strong base gel type anion exchanger. Resins: XU-434-200.00, .01 £1 .02 pH = 7.5, .05 M Tris buffer 1.1E-08 76 nos-031 9.05-09- 8.0E-09 ‘ H .000090 Cane. (g Eon/m1) T—T'IUDUTFS H .0002544 // / 7.0E-09 / / / / ka (ml/fl cellthr.) 6.0E-09 5.0E-09 I” 4.0E-09 / u/ / f__________...- 0.02 0.04 J v fl j 0.06 0.08 Surface Area (maZ/Q) _T Fig. 4.3.10: mrface area effect on ka for Type I strong base gel type anion exchanger Resins: XU-434-200.00, pH = 7.5, .05 M Tris buffer .01 81 .02 0.10 The linear model for ka was used on the macroporous resin IRA-938 for the initial Re and can only take into account its external surface area. As shown in Table 4.3.6 the fit is good with low concentrations and poor with the large using the resin diameter of .00042 m. ME 4.3.7: Predicted and experimental initial adsorption rate, ka, for macroporous IRA-938 Initial Conc. ka (ml/# cell-hr) x 109 (g cell/ml) Predicted Ehrperimental . 00023 3 . 918 1 . 965 . 00008 4 . 818 5 . 372 77 Table 4.3.7 indicates that the porosity of the resin must effect the initial ka of the macroporms resin since at large cell coroentrations the Ra is much slower than that predicted. This could neanthattheexternalsurfaceareatakenintoaccamtforthat dianetercouldbelargerforthemecroporousTypeIstrongbaseanion exchanger IRA-938. Itislmownthattheadsorpticnconstantdiangeswithtine for macroporous resins and that the predictive equation of Ra for gel type resins does not work well for macroporous resins. Tb create a nodel for ka for macroporous resins is beyond the scope of this study. 78 4.4 mm Fla & Abatdigrowthofcellswasperformedtocmparetheamountof free cells produced, total cells produced and the amount of extracellular a-amylase produced in a shaker flask. Three runs of this experiment were performed with a control batda, ro ion exchange resin in solution, and a 4.35 weight peroent solution of the anion exchange resins XU-434-200.00 and IRA-938 with the initial solutions at the same pH. Figures 4.4.1, 4.4.2 and 4.4.3 show that the control batch has a greater amount of free cells than both flasks with ion exchange resins. For all three experiments the IRA-938 had the smallest amount of free cells. The smaller amount of free cells in solution will allow less domstream processing to occur for the separation of cells. -°°°° g a... 1:0 434-20000 § IRA-938 1%. § Free Cell' Gone. (9 cell/ml) E .0004 ...o. / / —— Z"— .0002 l l fl "‘ 24 4a 90 Time (hr.) Fig. 4.4.1: Free cell concentration of production experiment} 4.35wt. %TypeIstrongbaseanionexchangeresm, macroporous and gel type structures. 79 .0007 E Central g g xu 434-20000 £30006 IRA-938 é E10005 __ .0004 __ 3 fl V / / / / E0003 5 —J -— é a r 000 / . 2 £4 4'0 712 96 Time (hr.) Fig. 4.4.2: Free cell concentration of production experiment 2 4.35 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. M. a w WM .0009d % J .0004 —— " 7' / 3 1 £000“ / /' 3m- é __ M1 r- OJ :4 a 72 Time (hr.) Fig. 4.4.3: Free cell concentration of production experinent 3 9.1wt. %TypeIstrongbaseanionexc:hangeresin, macroporms and gel type structures. 80 Total gigs Using the adsorption constants for XII-434-200.00 and IRA-938 (Table 4.2.3) the total amount of cells adsorbed onto the resin was fordarribyaddingthistotreamomrtoffreecellsthetotalamnmt of cells in the flasks were estimated for the three experiments (Fig. 4.4.4 - 4.4.6). The total cell concentration was greater for the resins than for the sample for the last two growth experiments (Fig. 4.4.5 & 4.4.6). This shows that a greater amount of cells can be aocurmlated in a reactor vessel than that without ion exchange resin. The first growth experiment (Fig. 4.4.4) shows a greater amount of cells for the imtobilized systems at twenty-four hours but a larger total cell concentration was noticed for the control, no ion exchange resin, after forty-eight hours. No explanation is knom of why this would occur. The total cell concentrations were calculated to be higher for the immobilized cells even though ro new nutrients were added. The immobilized cell systems may have depleted the nutrients scorer than the suspended cell system since the cell concentrations seemed to decrease or level off quicker than the suspended cells. These calculations of total cell concentration were assumed to follow the adsorption that was studied for a .05 M Tris buffer concentration at z pH 7.2. 81 as}? 3 .28 ...w ...— Fig. 4.4.4: Total cell concentration of production experiment 1 4.35 wt. % Type I strong base anion exdiange resin, macroporous and gel type structures. a can woo-200.00 \\ lax-950 momma“ Time (hr.) ilan/////.0V//// u e e cE\__8 8 5:8 =8 .08 Fig. 4.4.5: Total cell concentration of production experiment 2 4.35wt.%TypeIstrongbaseanionexchangeresin, macropormsandgeltypestructures. : a? S § \ \\\\\ \\ Tat. Cell Cane. (g cell/ml) E \\\\\\\\ / .000!” .0004- / .0000-J --— 24 a 12 Time (hr.) Fig. 4.4.6: Total cell concentration of production experiment 3 9.1 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Production The production of the enzyme az-amylase from ,B_. coli was studied (Fig. 4.4.7 - 4.4.9). In Fig. 4.4.7 and Fig. 4.4.8 after 24 hours the XU-434-200.00 gave a slightly larger amount of extracellular a-amylase. Thiswasnotthecasefortimesgreaterthan24 hours. At48hoursthe XU—434-200.00 gave the lowest concentration of enzyme for Fig. 4.4.? and 4.4.8. These differences in the enzyme concentration could be an effect of the immobilized cells grwth rates and differences in the structuresofthetworesireused. mringthefirstgrwthexperiment the concentration of enzyme dropped drastically for the IRA-938 between 48 and 96 hours. It was hypothesized that due to large porosity of this resinthattheenzymecouldhavediffused inootheporesandbeen adsorbedduetothegreatersurfacearea. Thiswasrotthecaseforthe 83 second growth experiment (Fig. 4.4.8) The amount of car-amylase for the second growth experiment was higher for the immobilized cultures for thefreecells. mentheamountof resinwas increasedtoz9%byweight (Fig. 4.4.9) the amount of enzyme for the xu-434-200.00 was the lowest ofthethreeoilturesarotheIRA-938wasaslargeasthefreecells for the twenty-four and seventy-two hour periods. the to the scatter of the data for the az-amylase between all experiments, it is concluded that the analytical methods used for the a-amylase concentration should be reviewed. Therefore, ro conclusions for what is better for the production of this enzyme can be made. 90 a Centre! xu. 434-20000 IRA-938 O O \l O Alpha-amylase (gactwlty units/ml) I 3 \ / l \ \ \ l I 1 4a 96 Time (hr.) a 9-\\ I Fig. 4.4.7: Production of a-amylase, production experiment 1 4.35 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. 84 400 C E } 9:300 C :1 {0 > . :3 r 33200 V \ :1 \1 1 ‘\ O \ ° \ 100 -- i. / § .C s < .J . 24 40 72 Time (hr.) E Central xu. -434-200.00 § IRA-938 Fig. 4.4.8: Production of a-amylase, production experiment 2 4.35 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. § § Alpha-amylase (activity unite/ ml) 5 § O l— a«\ Time (hr.) T 72 a anew 20.1-434400.00 § ”-939 Fig. 4.4.9: Production of a-amylase, production experiment 3 9.1wt. %TypeIstrongbaseanionexchangeresin, macroporous and gel type structures. 85 4.5 mam One potential advantage for the use ion exchange resins for the use of whole cell immobilization is the possibility of re-use. Three separate experiments were performed for multiple adsorption of whole cells. The initial concentration of cells was kept constant for all trials. A rormal resin preparation was performed between sorptions by contacting them with acid and salt soluticms. Figures 4.5.1 - 4.5.3 shw the amount of adsorption obtained for the samples of resin recycled. For all three experiments, the second cycle of the resins gave lesser amount of adsorption than the original use of the sample. The macroporous IRA-938 gave a better adsorption than the gel XU-434- 200.00. For the two cases were a third cycle was done (Fig. 4.5.1; 4.5.3) both showed better adsorption than the second cycle. The poorer performances of the recycled resins could be caused by the cells leaving proteins or polysaccharides on the resin surface that could not be removed by the regular acid and salt contact, thus causing some fouling. The better recyclability of the macroporous resin could be from the resin having a smaller percentage of its surface being used and more surface area left unaffected by the cell attachment. The cases when the third cycle had a better adsorption than the initial adsorption could be due to the effects the age of the cells being used. Younger cells might adsorb better than older cells. The age of the cellswasrottakenirrtoaccolmtduringthesenms. This set experiments show that normal resin preparation steps may not be sufficient to clean resin surfaces. Further study should be done tofindwhatfurtherstepsareneededtocleantheresinforre—use. 86 .01 O O m Adsorption (g cell/g dry resin) C .o C O 01 7 // ' r/A 2 3 Recycle # .002 \\\\\\\‘\\\\‘ g IRA-938 xu -434-200.00 Fig. 4.5.1: Recycle experiment 1:5 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Init. cell conc. = .00049 g cell/ml Solution vol. = 10 ml., pH = 7.5, .05 M Tris buffer 87 .0061 E “.93. 9. xu -434-200.00 '9 .0071 .0066 o o Y.’ 1 \ I é __ i 2 Recycle I} \W 1 .0056 Adsorption (g cell/g dry resin) L8. .0046 Fig. 4.5.2: Recycle experiment 2:5 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Init. cell conc. = .00049 g cell/m1 Solution vol. = 10 ml., [:18 = 7.5, .05 M Tris buffer 88 .006 a IRA-930 ' xu -434-200.00 .007 \ \\ 000 , —-1 // :1 8 00 \\ Adsorption (g cell/g dry resin) 2 ' 3 Recycle I} ‘\\\\\\ Fig. 4.5.3: Recycle experiment 3:5 wt. % Type I strong base anion exchange resin, macroporous and gel type structures. Init. cell conc. = .00037 g cell/ml Solution vol. = 10 ml., p-I = 7.5, .05 M Tris buffer 1.) 2.) 3.) 4.) 5.) 6.) 7.) 8.) 9.) WV: MICE Agreaterperoentageofsurfaceareaisusedbygeltyperesins verethereisalargeexternalsurfacearea. Adsorption of cells decrease with increasing crosslinking of the ion exchange resin. Little diffusion of cells into macroporous resin occurs. Equilibrium of cells and resin follows a Iangmuir isotherm. The adsorption reaction is second order overall reaction which follows irreversible kinetics with the reaction order of the cells and resin being first order. Masstransport ofthecellsontotheresincanbeexplainedby normal mass transport phenomena for surfaces with constant composition. The rate of adsorption decreases with increasing cell concentration and reaches a minimum around .000255 9 cells/ml. The rate of adsorption increases with increasing external surface area. 10.) The pH of solution has a parabolic effect on the amount of cells adsorbed, with minimum adsorption occurring around pH 7. 11.) As the amount of Tris buffer in solution increases the amount of cells adsorbed decreases. 12.) The use of ion exchange resins in batch fermentations will give lesseramotmtsof freecellsandgreateramounttotalcells in solution. 13.) Ian exchange can be reused for adsorbing cells, though their capacity is slightly diminished. 89 l.) 2.) 3.) 4.) 5.) 5.) 7.) 9.) GIAPIERVI: W106 Investigate what carpamds are left on the resin surface after and during the adsorption of cells. Further modelling of the rate of adsorption onto macroporous resins should be investigated with the effects of extra and intra particle diffusion. Investigate the effects of various porosities and pore size distribution have on the adsorption capacities of macroporous resins. Review the analytical methods for the estimation of a-amylase concentration. Further investigatetheuseofgeltypeandmacroporous resinsin actual fermentations, batch and continuous. Investigate cleaning methods of the used ion exchange resin for possible resin reuse. Investigate ion exchange resins for the use of mammalian whole cell immobilization. Further investigate how resin and solution properties effect the equilibrium constant of cell adsorption. 9O AM‘ 11mm 1: S.E.M. 91 The intent of this instruction manual is to provide the novice with a set of abreviated instructions that can be usedto begin observation of a specimen in the 31301:. T—330 SEN. It should be emphasized that this document does not attempt to make the prospective SEM user an expert in the techniques of electron microscopy. Rather, this document presents a set of instructions on how to obtain an image using the Secondary Electron Imaging mode on the T-330. It shold be noted that this particlar 8m an perform backseattered electron imaging also. In addition prospective users should consult with me to determine more on the following accessories which are not discussed herein: GAIVMA: Used for enhancing the image when extremes in contrast and brightnees occur. Objective Lens Operatures: Used for selecting higher resulution or greater depth of field. DFU (Dynamic Focusing Unit): Used to bring into focus the entire field when specimen is tilted. Working Distance: Used for selecting higher resolution or greater depth of filed. The Composite Materials and Structures Center has several texts and manuscripts that discuss in detail electrom microscopy theory and techniques. These contain information that should be reviewed by prospective SEM users so that they can operate the unit at its greatest potential for the specific sample that is to be observed. 92 1. Start Up. This sequence of steps should be followed to power—up the JOEL T-330 if the unit has been turned off. 1. Turn on the Neslab recirculating water chiller located behind the SEN. 2. Insert key into sea. This key is spring loaded and operates the same as an ignition key of an automobile. To turn on the power, turn key to the START position momentarily and then allow the key to return to the ON position. Power is now supplied to the SE14. 3. Place CHFIJKER in the PWR position. The ammeter needle of the CHECKER should be at 0.5, perfectly vertical. If not, the incoming voltage must be regulated using the VARIAC transformer located on the wall adjacent to the outlet supplying voltage to tie SEN. Adjust the transformer so that the CHFCKER ameter reads 0.5. The SEM should now be fully powered. It will take 15—20 minutes for the diffusion pump oil to reach operating temperature. During this period full vacuum in the column can not be achieved and thus it is not poesible to view a sample. The operator should observe tie SEQUENCE panel .to determine when the column is fully evacuated. The SEQUENCE panel provides the following information when the adjacent red- light is illuminated: POWER The sad is on. DP Diffusion Pump oil is leated: DP is now ready. PREEVAC Tleoolmhasbeenrougtedpmped:tleDPwillnow engage. EVAC The column has been fully evacuated. Hr READY Column has been fully evacuated and High Tension (high voltage) can now be applied-to the filament. HT The High Tension is on. FILAMENT Voltage is being applied to the filament and the filament is engaged. When the SEQUENCE light adjacent to HT READY is illuminated it is now possible to begin observation of the sample. If there is no sample on the SEN stage, proceed to section 2, SPEXSDIEN INTRODUCTION AND EXCHANGE. If a sample has been introduced into the column and the column has been evacuated, proceed to tie next section entitled SPECIMEN OBSERVATION. 2. 3. 93 WWWANDW. 1. Turn PW knob to zero. 2. Turn off the high tension by depressing the red HT button. 3., WAIT 5 mums to allow filament to cool down. 4. Depress VENT. The sample chamber and column will be returned to ambient pressure. Failure to wait for the filament to cool as directed in step 3 will cause the filament to burn upon exposure to oxygen. 5. When the PRE EVAC light is extinguished the stage may be pulled open. Always wear lint-free nylon gloves when opening chamber. 6. Remove sample mounting pedestal. 7. Insert new specimen. Use squeeze bulb to blow away dust on specimen or in chamber. Close the stage. 8. Depress PUMP DOWN. Observe the lights on the SEQUENCE parel which will indicate when chamber has been fully evacuated so that imaging may proceed. 9. Proceed to section 3 SPECIMEN OBSERVATION. SPECIMEN WATION. This section describes specimen observation using the SEI mode (Secondary Electron Imaging). The DET‘ (Detector) selector light should indicate that the sen is in the SEI mode. In addition tie SEI button should be depressed on the 1143 (Image Selection) panel. A series of adjustments must be executed to obtain high resolution images of. the specimen. These adjustments are grouped into 5 categories: 1. Adjustment of filament current and filament alignment. 2. Adjustment of contrast and brightness. 3. Focusing 4. Astigmatism correction. 5. Objective lens alignment. 3.1. 94 Adjustment of filament current and filament alignment. 1. Select the desired amount of AOCELERATING VOLTAGE. Do not exceed 20 RV. Consult Mike Rich for determining an appropiate initial level of accelerating voltage. 2. Set SPOT SIZE to the 2 O'Clock position. 3. Adjust BRIGHTNESS to the 12 O'Clock position. 4. Deprees the red HT button and observe that the red light react to HT on the SEQUENCE panel is illuminated. 5. Put cream in the LD CUR. (Load Current) position. 6. Slowly turn the FILAMENT knob clockwise to the 2:30 position while observing the CHECKER ammeter. Do not allow the needle to deflect beyond 0.6 mamps. If amperage exceeds 0.6 mamps prior to attaining the 2:30 position on the FIIAMENTlmob, tteGUNBIASmustbedecr-eased. TheGUNBIAS control knob is located in the tabletop to the immediate righthand side of the column. To decrease the GUN BIAS amperage turn the knob clockwise. 7. With FILAMENT knob at the 2:30 position, adjust GUN BIAS so that the altimeter reads 0.5 mamps, perfectly vertical. 8. Select LSP (Line ScanProfile) on tl'eMDEpanel and EXP. (Exposure) on the SPEED panel. 9 . Use BRIGHTNES knob to place waveform at center of CRT. Use CONTRAST to set the peak to valley amplitude of waveform so that it is approximately 1 inch on CRT. 10. While observing the waveform slowly turn the FILAMENT knob counterclockwise. As soon as the waveform begins to travel down the CRT stop turning the filament knob. Now slowly turn the FILAMENT knob clockwise the minimum amount necessary to set the waveform so that it is loacated at the highest point on the CRT. Do noteturn tte FILMIENT knob past the 3 O'Clock position. See Mike Rich should you encounter difficulty in adjusting the waveform to its peak height. 95 11. After. the waveform has been adjusted the filament alignment must be checked. Filament alignment is- controlled by the 2 pair of black knobs located atop the electron beam column. To align the filament, simultaneously turn in opposite directions (one clockwise, the other counterclockwise) a pair of knobs located across from each other while observing the waveform. Adjust the waveform so that a peak is achieved. Repeat this process with the remaining pair iof knobs. Normally little adjustment if any will be needed. With new filaments, alignment should be checked every hour for the first 5 tours of filament time. Adjustment of contrast and brightness. Before contrast and brightness can be adjusted the specimen must be located and placed into the path of the electron beam. The following sequence is helpful in locating your sample within the chamber. Remember, never remove the cap of the viewing port while the FILAMENT 1. Select PIC on moms and TV on SPEED panels. 2. Select minimum magnification level. 3. At this point you may have to decrease the CONTRAST to obtainaclear imageontheCRT. 4. Use the 14-? stage controls to locate the sample. 5. Usetl’eUP/DOWNARRCNSofFOCUSpanel tobringtheimage into focus 7. Select SLOW 1 on SPEED panel and proceed to the next sections on contrast and brightnees adjustment Contrast and brightness may be adjusted manually or automatically by the However the automatic mode may not be used during picture Because of the necessity of performing manual contrast and brightness adjustment wten photodocumenting the specimen, automatic contrast and brightness adjustment will not be presented here. See Mike Rich should you desire more information on this operation. unit itself. Manual Adjustment of Contrast and Brightness. 1. Select EXP on SPEED panel and PIC on MDDE panel. 96 2. Use'the CONTRAST knob to achieve 3-bars on tre'topl'at as shown in Fig 2. 3. Use the BRIGHTNESS knob to center the 3 bars of the tophat as shown in Fig 1. . .r ' ' ‘¥_.__. \ / D Insufficient A Low contrast brightness A __ _. c cptmum’ \ : _ h -J’_ ‘ “ ‘ ‘ \../ contrast —-—---’—— \- 8 high contrast and E Excessive brightness brightness Figure 1 . Contrast and Brightness Adjtstment . The contrast and. brightness will require adjustment whenever the magnification is changed and immediately prior to taking a photograph of the image. 3.3. Focusing. The JOEL T-330 has both automatic as well as manual focusing. The AFD (Automatic Focusing Device) does not mrk well if insufficient contrast exists. At high magnifications manual focusing is superior to automatic focusing. Thus automatic focusing is not discussed here. See Mike Rich should you desire more information on this operation. mammal Focusing. 1. Depress the RESET button on the FUNCTION panel. 2. To adjust the coarse focus depress either the up-arrow or dowrr-arrow located on the FOCUS panel while observing the image on tie CRT. 3. To adjust the fine focus turn the FINE focusing control lamb on the rows panel. It may be necessary to adjust the contrast and brightness during the focusing sequence. 3 . 4 . Astigmatism Correction. 97 Astigmatism correction can be performed automatically using ASD (Automatic astigmatism correction device) or marmally. Generally the manual adjustment is superioras well as quicker than ASD. Due to these limitations ASD is not discussed here. See Mike Rich should you desire more information on the ASD. ' Mammal Astigmatism Correction. 1. SelectSWlontheSPEEDpanel. 2. Adjust focus at a sufficiently high magnification. Astigmatism corrections are best made at magnifications of 5000 X or greater. Adjust the focus so that it is centered, which means that the focus is set to the middle of the blurring direction. 3. Turn X control of STIGMATOR while observing image. Adjust the X control to achieve the best focus. 4. Repeat step 3 using the Y control of STIGMATOR. 5. Refocus the specimen wing thefine focusing controls. 6. If difficulty. is encountered in adjusting the astigmatism. set both the X and Y STIGMATOR controls to their mid-positions and strart again with step 1.- It may be necessary to repeat the astigmatism correction operation several times to obtain the highest quality image. 3.5. Objective Lens Alignement. The objective lens should be aligned at the beginning of every session. It should also be adjusted if the accelerating voltage or if the aperature size is charged during the sen seesion. Aperature #2 should be used for most work. 1. Switch to TV MODE and manually adjust the CONTRAST and BRIGHTNESStoachieveagood imageontheCRT. 2. Depress ALIGN on the FUNCTION panel. Notice that the image on the CRT is wobbling. 3. To align the objective lens turn the 2 knobs located on the Objective Lens Selector which is situated on the lefthand side of the column. The intent is to adjust the image so that it is not moving horizontally or vertically as 4. 98 it wobbles. Use the end knob to adjust the horizontal movement, and the side knob to adjust the vertical movement. When perfectly alinged the image will seem to be moving in and out of the CRT and have the appearance of a- fast beating heart. 4 . When aligned depress the ALIGN button to stop the image from wobbling. At this time it may be necessary to again correct for astigmatism as described in section 3.4. Photography . 1. Using SLOWI on SPEED panel, obtain a well focused image at the desired magnification. Generally it is best to focus at a higher magnification and then lower the magnification to the desired level without further focusing. ' 2. Adjust mm and BRIGHTNI‘SS as described in section 2.2. 3. Select exposure marker by depressing ON/OFF-DATA. The exposure marker will label the photograph with the‘ accelerating voltage, magnification. magnification bar. and 6 digit identification number.There are 2 choices on the way this intonation is displayed on the micrograph: 1)on the photo itself when IMAGE BASE is selected. or 2) en a darkened backgrourrl at the base of the micrograph when BLANK is ' selected. . 4. Use ttmmbwteels of COUNTER FIIM MJMBER to select the desired 6 digit identification number. To engage automatic incrementing of identification number. depress ON/OFF of the COUNTER FILM NUMBER and note that the last 2 digits of number become illuminated on panel. 5. Select exposure duration. Use QUICK for faster processing time. Use NOR (Normal) for longer exposure and higher resolution micrographs. 6. Load film. 7. Depress 8mm to initiate exposure. Specimen image will reappear on CRT upon completion of exposure. 8. Process the film. 9. Recommended f stop setting: Filfiijpe ASA f-s_§>_p 55 50 5.6 52 400 16 53 800 22 99 5. WHAT TO DO WHEN FINISHED. 1. Turn FILAMENT knob fully counterclockwise and turn off the red HT button. . 2. Remove the sample from the SEM. See Section 2 for instructions. 3. Fully evacuate the column by depressing PIMP DCMN 4. Increase magnification to its highest level: 200 000x. 5. SelectSIDWZontheSPEEDPANEL. 100 WWW 1. Lift top to insert specimen to be coated. Place specimen in center of chamber. 2. Turn the collar lock atop chamber to release the target locking mechanism. Adjust the target so that it is'appromcimately 50 mm (2 in) from specimen surface. Note: the target is the gold ring located inside the top of the chamber. Do not ever touch the target. 3. Close top of chamber and open valve on argon cylinder. Adjust flow to 5 psi on the regulator. 4 . Turn OPERATION SWITCH to PUMP. The rotary pump will begin to evacuate the chamber. 5. When 0.1 mbar vacuum achieved,'open LEAK valve about 3 rotations and allow pressure to rise to about 0.5 mbar. Flush chamber with argon for 30 seconds, then close LEAK valve. 6. Repeat step 5. 7. Allow system to pump down to between .04 and .03 mbar. This may take up to 10 minutes. 8. Set OPERATION SWI‘ICH to SET HT. 9. While observing the ammeter, slowly increase the VOLTAGE knob to about 2.5 KV. If amperage exceeds 30 mamps decrease the voltage. A purple plasra discharge should now be evident coming from the target . Keep increasing VOLTAGE till 25 mamps or 2.5 kV is attained. 10. The vacuum will begin to degrade and amperage decline as the plasma is generated. Thus to maintain 25 mamps at 2.5 W a small amount of argon is required to be added to the chamber. Slowly open LEAK valve while observing the ammmeter. Adjust the LEAK valve so that 25 mamps is maintained at 2.5 kV. 11. The thickness of the deposited gold film can be estimated by using the empirically derived relationship: '1' = 7.51t where T is thickness inAngstramunits, I iscurrent inmamps, andt is time in minutes. This relationship is valid only for a specimen to 101 targetdistanceofSOmmandat 2.5 kV. Thusaiminuteexposuretothe plasma at 25 mamps, 2.5 kV. with a target to specimen distance of 50 mm, a thickness of approximately 200 Angstroms will be deposited on the sample surface. ' 12. After subjecting the sample to the plasma for the desired amount of time. return VOLTAGE to the zero position. 13. Turn OPERATION SWITCH to OFF. 14. Open VENT valve to return chamber to ambient pressure. 15. Remove specimen from chamber. 16. Close VENT and LEAK valve on sputter coater and close argon cylinder valve. 17. Clean the inside of the glass chamber using a Kim-wipe wetted with ethanol. W 2: Preparation of Biological Samples for S.E.M. 102 APPENDIX Biological Specimen Prwaratim for S.E.M. Thetissleard/orcellsareplaoedinasolutimwithafixative, usually 5% glutaraldehyde. The fixative is used in a buffer solution which maintains constant pH, usually 7.2. This is allowed to sit for aetoboolrmrrsatroamtslperatureorinanicebucket. ‘Ihesamplesarethenwashedwiththebufferonceortwiceto removethefixative. The sample is next dehydrated with ethyl alcohol . The sample is contacted with gradually increasing graduatiore of ethanol, 25%, 50%, 75%, 95% and 100%. About fifteenminutes of contact is requiredat each graduation. ‘1he100% ethamlstepisrepeatedtomakeslrethatthe cellsarefullydehydrated. Tielaststepisthecriticalpointdryingstep, ideallythis stmld occur directly afterthe ethanol drying step. Critical drying prevents the cells fran shriveling ordistorting because of the cells aremtallowed reacttothesurfacetension forcescausedbythe tissue water. Critical step drying is done with carbon dioxide at 700- 900psig. ‘mecontactwiththecnzallwstheeflanolinthesampleto bereplacedwithcnz. 'missoaJdngwithliquidcnz isrepeatedafew tilnestobesuretheetlanolisremoved. 'melastcontactingofliquid m2 istlenleatedarrithevaporremovedframthesamplecorrtairer until the critical point, 1120 psi and 32'C, is reached. Afterthis the sampleshouldbecalpletelydryardreadytobesputtercoatedforthe S.E.M. O m 3: Assay for a-amylase 103 AHYLASE ASSAY-STARCH IODINE METHOD REAGENTS: 0.2M acetate buffer (6.56 g NaAc + 1.2 gXNAc, adjust to 500 ml with 820, adjust pH to 5.25 with NaOR) 0.5M CaC12 12 starch (Difco soluable starch) solution in H20 1N HCL 32 KI/O.3Z I2 REACTION COCKTAIL: ml 0.2H NaAc ml CaClZ ml starch solution m1 dd H20 Ut—t-U! REACTION: 1) To 0.8 ml of reaction cocktail, add enzyme solution (about 25 microliters of a 1. 5 unit/ml solution gives a reasonable change in optical density in 5 minutes) 2) Stop reaction at desired time by immersing tube in ice, add 1 m1 of 1N HCl 3) Add 200 microliters of 32 KI/O.3Z 12 solution for color rxn A) After tubes have warmed to room temp. (about 15 min.) read on spectrophotometer at 620 nm (iodine binding is temperature dependent) Zero time points should be made by adding enzyme directly before iodine solution with no exposure to assay temperature. Blanks are made by substituting dd N20 for enzyme solutions. Standard assay temperature for B. stearOthermOPhilus amylase is 70 deg C. One unit of activity is defined as the amount of enzyme required to release 1 micromole of maltose/minute. We assume that all of the starch can be quantitatively converted into maltose. Thus, the subsequent loss of optical density is directly preportional to the amount of maltose released, which in turn is directly proportional to the enzyme activity. The assay is significantly non-linear with time and enzyme concentration. One should set up the assay such that replicates of several time paints are used, and that changes in optical density between times points are small. Beware of the source of the starch; there are great differences in‘iodine binding capacity with different suppliers. Blanks containing water«dnstead of enzyme should have an optical density of between 1.0 to 1.3 ref. Manning and Campbell 1961. J. Biol. Chem. 335:2952-2957. 104 a—amylaseAssay [ms—e1 = FsJEJ'Efl-Edw So,st=spectrmeterreadingattime0,attilnet V =- sample of enzyme solution used in assay (pl) t = time enzyme allowed to react (min.) :1 = dilution of original enzyme solution am 4: Diameters of Mesh Size 105 0.8 Sieve and Tyler Equivalents (ASIM-E-ll-Gl) Tyler equivalent Diameter Mesh (mm. ) 2.5 8.000 3.0 6.730 3.5 5.660 4 4.760 5 4.000 6 3.360 7 2.830 8 2.380 9 2.000 10 1.680 12 1.410 14 1.190 16 ' 1.000 20 0.841 24 0.707 28 0.595 32 0.500 35 0.420 42 0.354 48 0.297 60 0.250 65 0.210 80 0.177 100 0.149 115 0.125 150 0.105 170 0.088 200 0.074 250 0.063 270 0.053 325 0.044 400 0.037 APPENDIX 5: Equilibrium Spreadsheet 106 Equilibrium Spreadsheet A1 = "Resin: XU—434-200.00 F1 P= ”Int./Fin. A2 = ”Date: 82 = "7/13/88 02 P= "Solution pH F2 = "7.9/7.7 62 P= " K1= H2 P= A17 03 P= "Tris buffer F3 = .05 63 P= "M K2= H3 P= A18 (34 P= " r= H4 P= AH26 AA4 P= "Conc./Ads=(1/kl>¥Conc. + k2/k1 AIS P= .1 . C6 P= "Volume of Sample (ml) 05 P= "Sample Volume (m1) F6 = 9 AAb P= "Comm/Ads. vs. Cont. A16 P= 0 AH7 P: "Coeff. 1 A17 P= 1/AH23 CB P= " Resin E8 P= "Initial F8 P= " Final Klett 08 P= "Initial GB P= ” Adsorbed AAB P= "Exp AHB P= "Coefi. 2 A18 = A17XAH24 C9 P= " Mass (9) E9 P= "Klett F9 P= " (24 hrs.) J9 P= "Init. L9 P= "Cone. N9 P= "Conc. 09 P= "Cone. O9 P= " (g cell/g resin) 59 P= ”Exp. T9 P= "L.R. U9 P= " Conc. (g CEIIS/ml) Z9 P= ”Exp. AA9 P: "Langmuir 2 A89 P== ” X 0H9 F’= " r"2= A19 P= AH26“2 A10 P= " Sample # C10 = " #1 010 = " #2 F10 P= " #1 610 P= “ #2 FOR EDUCATIONAL USE ONLY 107 Equilibrium Spreadsheet J10 P= "Conc. L10 P= “ #1 N10 P= ” #2 010 P= "(9 cell/ml) 010 P= " #1 R10 P= " #2 810 P= "Average T10 P: "(L.R.) U10 P= ” #1 V10 P= " #2 NIO P= " Ave. Y10 P: "Cont. 210 P= ”Adsorb AAIO P= "Conc./Ads. A810 P= "Conc. ACIO P: 20 A01 P: ”x“2 AEIO = "v“2 PFIO P= “ xty A110 P= ”Mdl A11 P= 1 C11 = .407 011 = .395 E11 = 435 F11 = 269 611 = 243 111 P: IF(Ell<300,l.242E-6¥Ell-3.75E-5,1.711E-6I‘E11-l.81E-4) J11 P- IF(E11<125,9.SE-7*E11,111) K11 = IF A011 P: A811“2 A611 = AA11‘2 AF11 P= ABIIaAAll A111 P= AI7tY11/(A18+Y11) AJ11 P= AH23¥A811+AH24 A12 P: A11+1 C12 = .411 FOR EDUCATIONAL USE ONLY 108 Equilibrium Spreadsheet 012 512 F12 612 112 312 K12 L12 M12 N12 012 012 R12 812 T12 U12 V12 w12 Y12 212 AA12 A812 AC12 A012 AE12 AF12 A112 AJ12 A13 C13 013 $13 F13 '613 113 J13 K13 L13 M13 N13 013 013 R13 513 713 U13 V13 w13 Y13 Z13 AA13 A813 FFnuWFWWRTFRTFWTFTuuu. FFRFuYY FTFRWWRFFTPnFTFFTWuuuuu .408 382 151 155 IF J14 xFe/C14 xFo/Dl4 (014+R14)/2 U264014+U27 L14 N14 (014+v141/2 IF(U14<=0,0,U14) IF(F14<=0,0,014) 1F(F14<=0,0,Y14/Zl4) IF(AA14=0,0,Y14) IF(Y14<=O,ACl3-1,AC13) A814‘2 4414c2 A814¥AA14 A17*Y14/(A18+Y14) AH23¥A814+AH24 A14+1 .401 .403 210 4 s IF J15 (J15-L15llFb/C15 (JIS-lelth/DIS (015+RlS)/2 FOR EDUCATIONAL use ONLY 110 Equi librium Spreadsheet T15 P= U26XOIS+U27 015 P= L15 v15 P= N15 WIS P= (U15+V15)/2 v15 P= 1F(U15<=0,0,U15) 215 P= IF(F15<=0,0,015) AA15 P= 1F(F15<=0,0,Y15/215) A815 P= 1F(AA15=0,0,Y15) AC15 P: IF 016 = J16 016 = (Jlb-Lloleb/Clo R16 P: (J16—N16)#F6/016 516 = (016+RIb)/2 716 = 026¥016+U27 016 P: L16 v16 = N16 Nlb = (Ulb+V16)/2 v16 = 1F<016<=o,0,016) 216 = 1F 1F J17 (J17-L171xF6/C17 (J17-N17)¥F6/017 (017+Ri7)/2 026*017+U27 L17 N17 (U17+V17)/2 IF(Ul7<=0,0,Ul7) 1F(F17<=0,0,017) 1Fxr67018 (018+R18)/2 U26*018+U27 L18 N18 (U18+V18)/2 1F(UIB<=0,0,U18) 1F 1F(F19<125,9.5E-7KF19,K19) 1F(Gl9<300,1.2428-6tGl9-3.75E-5,1.711E-thl9-1.81E-4) 1F(Gl9<125,9.5E-7¥Gl9,fl19) J19 (J19-L19)#Fo/C19 (J19-N19ltFo/Dl9 (019+R191/2 ‘ U26¥019+U27 L19 N19 (U19+V19)/2 1F(U19<=0,0,U19) IF(F19<=0,0,019) 1F(F19<=0,0,Y19/Zl9) IF A031 P: AC30 A031 1:: SU“)(A011:A030) £31 = smbhr. .S XS+WB 015 N15 015 P15 AH9 AM9 AFB-Alatwa AG4-(AF4—AF9) (AF4—AF9)!W6*9.SE-l3/w5 (T11XAF9XAG9) AKB—ANBth AL4-(AK4-AK9) (AK4-AK9)¥wb¥9.5E-13/w5 U11tAK9¥AL9-U13¥(AK4-AK9) ” Sample # "ADSORBANCE “Conc. (g cell/ml) "Adsorbance (9 cell/ 9 resin) "Eq. fctr: 1 "Adsorbance6hr. .5 X8+w8 015 N15 015 P15 AH9 AM9 AFB-AIBXWB AG4- (AF4-AF9)¥th9.5E-l3/w5 (TlltAF9XAG9) AKB-ANBIWB AL4-(AK4-AK9) (AK4-AK9)¥N6*9.5E-13/N5 U1ltAK9¥AL9-U13*(AK4—AK9) " Sample 3 "ADSORBANCE "Conc. (g cell/ml) ”Adsorbance (9 cell/ 9 resin) "Eq. fctr: l "Adsorhance(g cell/g dry resin) x9+w8 AHlO AMIO AF9-A19xw8 AG4-(AF4-AF10) FOR EDUCATIONRL USE ONLY 119 Kinetic Spreadsheet AH10 A110 AKlO ALIO AMIOV AN10 C11 311 111 N11 011 811 T11 U11 V11 X11 AC11 ADll AF11 A611 AH11 A111 AKll AL11 AM11 ANll A12 812 F12 N12 V12 X12 ACIZ A012 AF12 A512 AH12 A112 AK12 ALIZ AMI? ANIZ A13 813 C13 013 613 113 1(13 N13 013 P13 111....1.11111111111"111111111111 (AF4—AF10)*w6t9.5E-13/w5 (TIIXAFloxABIO) AK9-AN9XWB AL4-(AK4-AK10) (AK4-AK10)xw6*9.5E-13/N5 U1liAKlOtALIO—Ul3t(AK4-AK10) ”Klett ‘ "(9 cell/ 9 resin) "Average " Experimental "Average "Ra: F53 J53 "Adsorpance vs. Time (Model) X10+WB AHII AMII AFIO-AIIOIWB AG4-(AF4-AF11) (AF4-AF11)XW6X9.SE-13/w5 (TlltnFlltAGII) AKIO-ANIOXNB AL4-(AK4-AK11) (AK4-AK11)#W6*9.5E-13/w5 UIIXAKI1*AL11-U13*(AK4-AK11) ”Time (hr.) "Time (hr.) x11+w8 AHlZ AMIZ AFll-Allltwe AG4-(AF4-AF12) (AF4-AF12)¥W6*9.5E-13/w5 (TIIXAFIZXAGIE) AKll-ANlltwe AL4-(AK4—AK12) (fiKfi-AKIZ)#W6#9.SE-13/N5 U11*AK121AL12-U131(AK4-AK12) "(hr.) M H U ..a (.4 N H U N FOR EDUCATIONAL USE ONLY 120 Kinetic Spreadsheet 013 513 T13 U13 X13 AC13 A013 AF13 A513 AH13 A113 AK13 13 AM13 AN13 A14 814 C14 014 E15 514 H14 114 J14 K14 N14 014 P14 014 X14 Yl4 214 AA14 A814 AC14 A014 AF14 A514 AH14 A114 AK14 AL14 AM14 AN14 A15 815 C15 015 F15 515 H15 115 1111.. .. .. ..1111111111111111111111111.. .. .. ..111111111111111 ”Ave. ”k0 = O J53/(1/(TS/9.SE-13)) x12+WB AHIS AM13 AFiZ—AIIwaB AG4-(AF4-AF13) (AF4-AF13)¥w619.5E-13/N5 (TlleFIZXAGIS) AKIZ-ANlZXUB AL4-(Ak4-AK13) (AK4-AK133wat9.SE-13/w5 U1liAKlStAL13-U131(AK4-AK13) 0 DB DB 08 IF(814<3£(51.242E-6tal4-3.75E-5.1.711E-6t814-1.81E-4; IF(814<125,9.5E-7IBI4,F14) IF(C14<300,l.242£-6*C14-3.7SE—5,1.7118-6XC14-1.BiE-4) IFW4¥W6,0,U24) X23+W8 018 N18 018 P18 AH24 AM24 AF23~A123*w8 ABA-(AF4-AF24) FOR EDUCATIONAL USE ONLY 126 Kinetic Spreadsheet AH24 A124 AK24 AL24 AM24 AN24 T25 U25 V25 X25 AC25 A025 AF25 A625 AH25 A125 AK25 AL25 AM25 AN25 F26 K26 X26 AC26 A026 AF26 A626 AH26 A126 AK26 AL26 AM26 AN26 A27 F27 627 H27 127 27 K27 L27 M27 X27 AC27 A027 AF27 A627 AH27 A127 AK27 AL27 AM27 111.111111111111111111 .11111111 ‘? D (AF4-AF24)¥w6¥9.5E-13/w5 (TlliAF24tA624) AK23-AN23XW8 AL4-(AK4-AK24) (AK4-AK24)!N6¥9.5E-13/w5 U11*AK24tAL24-U13t(AK4-AK24) (-T19-(T19“244*T18*T201‘.5)/<2*T18) (w4-(9.5E-13¥T25))¥W6/w5 IFw4wa,o,u25) X24+N8 AH25 AM25 AF24-A124tw8 A64-(AF4-AF25) (AF4-AF25)¥N6*9.5E-13/w5 (T11#AF25¥A625) AK24-AN24XW8 AL4-(AK4-AK25) (AK4-AK25)*W6#9.5€-13/W5 U11*AK25XAL25-U13i(AK4-AK25) ”Calculating ka Irr. Rxn. 2nd Order "Calculating ka Rev. Rxn. 2nd Order X25+W8 AH26 AM26 AF25—A125Xw8 AG4-(AF4-AF26) (AF4-AF26)*N619.5E-13/w5 (TIIXAF261A626) AK25-AN251w8 AL4-(AK4-AK26) (AK4-AK26)#w6X9.5E-13/w5 U11*AK26XAL26-U13x(AK4-AK26) "Calculating Rxn. Order Cells " BO= T4XC6/((9.5E-13)¥D7) T4106/((9.5E-13)*D7) T4¥E6/((9.5€-13)*D7) " a= 628:627 H28¥H27 128t127 X26+N8 AH27 AM27 AF26-A126tw8 AS4-(AF4—AF27) (AF4-AF27)*w6#9.5E-13/WS (TlltAF27XA627) AK26-AN26tw8 AL4-(AK4—AK27) (AK4-AK27)XN6#9.5E-13/w5 FOR EDUCATIONAL USE ONLY 127 Kinetic Spreadsheet AN27 P= U11¥A<27tAL27-U13¥(AK4-¢¥<27) A118 P: "dC/dt 828 P: ”C ave C28 = "C F28 = "A0 = 828 P= G14/9.5E-13 H28 P: I14/9.$-13 128 P: 514/9.$"13 .328 P: "b = K28 P= -(GZ7+628+(1/(1/(T5/9.5E-13)))) L28 P: ~(H27+HZB+(1/(1/(T5/9.5E-13)5)) P128 P: -(127+12‘8+(1/(1/(T5/9.5E-13>))) x28 P: 127 AC28 9: 96.28 A1328 P: N12-‘8 AF28 P: AF27-A127th A628 P:- AG4-(AF4—AF28) AHZS P: (Ara—AF‘zeuwbxmi-u/ufi A128 P: (TIHAFZBIKAGZB) AK28 F: A<27—ANZ7#WB AL28 = AL4-(Al‘;4-AK28) @128 = (M4—AK28HN61955-13/k6 ANZS P: U111M28tk28—U13HM4-AK28) A29 = (K15-K14)/(9.$-13HA15-Al4)) 829 = (1.14%;153/(219.SE-13) C29 P: «.14/9.5E-13 E29 P: 4.1130 (329 P: (1/(827—628H H29 P: (UH-1274428)) 129 = \‘1/(127—128H J29 P: “q = K29 P: 4kK27-K28"2 L29 = 41L27-L28"2 M29 = 4*P127-M28‘2 x29 P= X28+w8 AC29 = AHZ9 AD29 = #1429 AF29 P= AF28-AIZBWB A1329 P: AG4-(AF4-A-729) AHZ9 P: (AF4-AF29)!N6¥9.5E-13/h5 A129 P: (T111AF29XA629) AK29 P: M28—AN28WB AL29 P: k4-(Ax4-Ax’29) Al‘129 P: .m.4-A<29)#W6*¢.5E-13/h5 FW29 P= unmemm-msxmm-mzm A30 P= (K16-K15)/(9.5E-13IHA16-AlS)i 830 P= (K15+K16)/(2#9.$-13) C30 P= K15/9.5E-13 E30 P= 1 F730 = A14 530 = LN<((627-(814-814)/9.5E-13)t(328)/((528-(814-{3141/95’6—1 323627))1629 128 Kinetic Spreadsheet H30 130 K30 L30 M30 X30 A630 A030 AF3O A130 AK30 AL30 AM30 A31 831 631 E31 F31 631 H31 P4 ul H K3 L31 M31 X31 A631 A031 AF3I A631 AH31 A131 AK31 AL31 AM31 AN31 A32 832 C32 P: p: p: P: p: LN(((H27-(114-114)/9.58-13)*H28)/((H28-(114-114)/9.55-13) *H27))¥H29 LN(((127-(K14-K14)/9.5E-13)X128)/((128-(K14-K14)/9.5E-13) #127))*129 (1/(-K29)“.5)*(LN((2*(614—614)/9.56-13+K28-(-K29)“.5)/(2* (614-614)/9.56-13+K28+(-K29)“.5))-K37) (1/(-L29)“.5)*(LN((2*<114-114)/9.5E-13+L28-(-L29)“.5)/(2* (114-I14)/9.SE-13+L28+(-L29)‘.5))-L37) (l/(-M29)“.5)¥(LN((21(K14-K14)/9.SE-l3+M28-(-M29)“.5)/(2* (K14-Kl4)/9.56-13+M28+(-M29)“.5))-M37) X29+NB AHBO AM30 AF29-A129XN8 A64-(AF4-AF30) (AF4-AF30)*N6*9.5E-13/W5 (T111AF30XA630) AK29-AN29XN6 AL4-(AK4-AK30) (AK4-AK30)¥W6#9.SE-l3/N5 Ul1XAK30XAL30-U13X(AK4DAK3O) (K17-K16)/(9.5€-13¥(A17-A16)) (K16+K17)/(2t9.5E-l3) K16/9.5E-13 E30+1 A15 LN(((627-(614-615)/9.SE-13)X628)/((628-(614-615)/9.5E-13) $627))X629 LN(( F32“2 F32#632 F32tH32 F32t132 F321K32 F32¥L32 F321 ’2 X1? 219 AA19 A819 AC1? A019 x42+w8 AH43 AM43 AF42—A1421w8 AG4-(AF4—AF43) (AF4—AF43)#w6#9.SE-13/w5 (T11#AF43#AG43) AK42-AN42IN8 AL4-(AK4-AK43)