Because nT is a function of s cannot integrate Eq. (9) directly as s varies with z in plug-flow reactors.
Nevertheless, application of equation developed in this section can give satisfactory results for design of fixed-bed immobilised-enzyme reactor.
t is the reactor residence time defined in equation ,t = 1/D = V /F , สมการใน continuous operation of mixed reactor x i is the biomass concentration at inlet - x f is the biomass concentration at outlet. The form of Eq. (10) is identical to that of equation สมการ 13.20 for batch recation.
In practice, batch processing is much preferred to PFTR systems because of the operating problems mentioned in Section packed bed.However, as discussed in Section packed bed, the total time for batch operation depends on the duration of downtime between batches as well as on the actual conversion time. Because the length of downtime varies considerably from system to system , cannot account for it here in a general way. Downtime between batches should be minimised as much as possible to maintain high overall production rate.
At the beginning of batch culture, rate of substrate conversion is generally low because relatively few cells are present; it takes some time for cells to accumulate and the rate to pick up. However, in CSTR operation, substrate entering the vessel is immediately exposed to a relatively high biomass concentration so that the rate of conversion is also high.
For example, according to Eq. (12), µmax and Ks can be determined from the slope and intercept of a plot of 1/D versus 1/s. The comments made in Part section about distortion of experimental error apply also to Eqs.(12)-(14)
quality of the steam must be sufficiently high to avoid contamination of the medium by metal ions or organics.
Ideally, all fluid entering the equipment at a particular instant should spend the same time in the sterilizer and exit the system at the same time ; unless this occurs we cannot fully control the time spent in the sterilizer by all fluid elements. No mixing should occur in the tube
Deviation from plug flow behavior is characterized by the degree of axial dispersion in the system, , i.e. the degree to which mixing occur along the length or axis of the pipe.
The membrane themselves must be sterilized before use, usually by stream
ก่อนสี่ Air-borne particles penetrate the bed to various depths before their passage through the filter is arrested;
ก่อนหนึ่ง Filters are also used to sterilize effluent gases leaving fermenters. In this application, the objective is to prevent release sole in the atmosphere of any microorganisms entrained in aerosols in the headspace of reactor.
Sterilization-Batch heat sterilization of liquid-Continuous heat sterilization of liquid-Filter sterilization of liquid-Sterilization of air
Continuous operation of a plug flow reactor Plug flow operation is….. • Alternative to mixed operation for continuous reactor. • No mixing occurs in ideal plug-flow reactor • This is achieve at high flow rate which minimize backmixing and variation in liquid velocity. • Plug-flow is most readily achieve in column or tubular reactor.
Plug-flow reactor• Operated in upflow or downflow mode or, in some case, horizontally.• Plug-flow tubular reactor are known by the abbreviation PFTR.• Liquid in PFTR flows at constant velocity.• Reaction in the vessel proceeds, concentration gradient of substrate and product develop in direction of flow.
• This exit concentration can be relate to the inlet condition and reactor residence time.
Enzyme reaction• To develop equation for plug flow enzyme reactor• Consider a small section of reactor of length ∆z as indicated in the figure.• Steady state balance on substrate around the section using mass balance equation
Enzyme reaction /mass-balance equation is : ( at steady state, left-hand side (1) of equation is 0. )•F = volumetric flow rate through the reactor•Fs│z = mass flow rate of substrate entering the system• s│z = Substrate concentration at z•Fs│z+ ∆z = mass flow rate of substrate leaving the section• vmax = maximum rate of enzyme reaction• Km = Michaelis constant•S = Substrate concentration•A∆z = section volume where A is the cross-sectional area of the reactor
Enzyme reaction• The volumetric flow rate (F ) divided by the section volume (A∆z) is equal to the vmax (maximum rate of enzyme reaction) divvied by Km (Michaelis constant): (2)• The volumetric flow rate (F )divided by the reactor cross-sectional area (A) is equal to the superficial velocity through the column ( u ) : (3)
Enzyme reaction• For F and A constant, u is also constant.• Eq.(3) is valid for any section in the reactor of thickness ∆z. For it to be valid at any point in the reactor, take the limit as ∆z 0: (4)• apply the definition of differential from equation : (differential equation for the (5) substrate concentration gradient through the length of plug-flow reactor)
Enzyme reaction• Assuming u and the kinetic parameter are constant,• Eq.(5) is ready for integration.• Separating variable and integrating with boundary condition s = si at z = 0• Gives an expression for the reactor length (L) require to achieve an outlet concentration of sf (6)
Enzyme reaction• Residence time (t) for plug-flow reactor parameter L and u : (7)• Therefore Eq 6. can written as (6) (8)• Eqs (6) and (8) allow to calculate the reactor length and residence time require to achieve conversion of substrate from concentration si to sf at flow rate u .
Enzyme reaction • Plug- flow operation is generally impractical for enzyme conversions unless the enzyme is immobilised and retained inside the vessel. • For immobilised enzyme reactions affected by diffusion, Eq.(5) must be modified to account for mass- transfer effect : (5) (9)nT is the total effectiveness factor representing internal and externalmass transfer limitation, s is bulk substrate concentration,and vmax and Km are intrinsic kinetic parameter.
Enzyme reaction• Plug-flow operation with immobilized enzyme is most likely to be approached in packed-bed reactor.• Packing in column can cause substantial backmixing and axial dispersion of liquid, thus interfering with ideal plug flow.
Cell culture• Analysis of plug-flow reactor for cell culture follows the same procedure as for enzyme reaction.• If the cell specific growth rate is constant , equal to µ max throughout the reactor and cell death can be neglected.• Equation for reactor residence time are analogous to those derived in section cell culture for batch fermentation, - t is the reactor residence time - x i is the biomass concentration at (10) inlet - x f is the biomass concentration at outlet.
Cell culture• Plug-flow operation is not suitable for cultivation of suspended cells unless the biomass is recycle or there is continuous inoculation of the vessel.• Plug flow operation with cell recycle is used for large scale wastewater treatment; however application are limited.• Even so, operating problems such as those mentioned in Section packed bed mean that PFTRs are rarely employed for industrial fermentation.
Comparison between major model of reactor operation• The relative performance of batch, CSTR and PFTR reactors can be consider from a theoretical point of view in term of the substrate conversion and product concentration obtained from vessel of the same size.• Because the total reactor volume is not fully utilized at all times during fed-batch operation,• It is difficult to include this mode of operation in a general comparison.
Comparison between major model ofreactor operation• Kinetic characteristics of PFTRs are the same as batch reactor; the residence time required for conversion in plug-flow reactor is the same as in a mixed vessel operated in batch.• The number of stages in a CSTR cascade increases, the conversion characteristics of the entire system approach those of an ideal plug-flow or mixed batch reactor.
• Concentration change in PFTR, single CSTR and multiple CSTR vessel. • smooth dashed curve represent the progressive decrease in substrate concentration with time spent in a PFTR or batch reactor; concentration is reduce from si at the inlet to sf at the outlet. • single well-mixed CSTR operated with the same inlet and outlet concentration, because condition in vessel are uniform In cascade of CSTRs, the concentration in uniform in each reactor but there is a step-wise drop in concentration between each stage.
Comparison between major model ofreactor operation• The benefits associated with particular reactor design or modes of operation depend on the kinetic characteristics of the reaction.• For zero-order reaction there is no difference between single batch, CSTR and PFTR reactor in term of overall conversion rate.• For most reaction including first-order and Michaelis-Menten conversions, rate of reaction decrease as the concentration of substrate decrease.
Comparison between major model ofreactor operation• Reaction rate is therefore high at the start of batch culture or at the enhance to plug-flow reactor because the substrate level is greatest.• Subsequently, the reaction velocity falls gradually as substrate is consumed.• In contrast, substrate entering CSTR is immediately diluted to the final or outlet steady-state concentration so that the rate of reaction is comparatively low for the entire reactor.• For first-order and Michaelis-Menten reaction, CSTRs achieve lower substrate conversion and lower product concentration than batch reactor or PFTRs of the same volume.
Comparison between major model ofreactor operation• The comparison between reactors yields a different result if the reaction is autocatalytic.• Catalyst is produce by reaction in fermentation processes; therefore, the volumetric rate of reaction increase as the conversion proceeds because the amount of catalyst builds up.• Volumetric reaction rate continues to increase until the substrate concentration becomes low, then it declines due to substrate depletion.
Comparison between major model of reactor operation• Rate of conversion in chemostats operated close to the optimum dilution rate for biomass productivity are greater than in PFTR or batch reactors.• For most fermentations, CSTRs offer significant theoretical advantage over other modes of reactor operation.• Despite productivity benefits associated with CSTRs, an overwhelming majority of commercial fermentations are conducted in batch. The reasons with the advantages associated with batch culture
Comparison between major model ofreactor operation• Batch processes have a lower risk of contamination than continuous-flow reactor; equipment and control failures during long term continuous operation are also potential problem.• Continuous fermentation is feasible only when the cells are genetically stable• In contrast freshly-produced inocula are used in batch fermentation giving closer control over the genetic characteristics of the culture.
Comparison between major model ofreactor operation• Continuous culture is not suitable for production of metabolites normally formed near stationary phase when the culture growth rate is low, but productivity in a batch reactor is likely to be greater under these conditions.• Continuous fermentation must be operated for lengthy periods to reap the full benefits of their high productivity.
Evaluation of kinetic and yieldparameters in chemostat culture• In steady-state chemostat with sterile feed and negligible cell death, the specific growth rate (µ) is equal to the dilution rate (D) .• This relationship is useful for determining kinetic and yield parameters in cell culture. If growth can be modeling using Monod kinetics, for chemostat culture, µmax = maximum specific growth rate Ks = substrate constant• (11) s = the steady-state substrate concentration in reactor
Evaluation of kinetic and yield parameters in chemostat culture• Eq. (11) gives the following linearised equation which can be used for Lineweaver-Burk, Edie-Hofstee and Langmir plots, respectively:• (12) (13)•• (14)
Evaluation of kinetic and yieldparameters in chemostat culture• Chemostat operation is convenient for determining true yields and maintanace coefficient for cell culture.• In chemostat culture with µ = D . (11) (15) Y xs = observed biomass yield from substrate Y xs= true biomass yield from substrate ms = maintenance coefficient
Graphical determination of maintenance coefficient ms and Yxs using data from chemostat culture .• Plot of 1/ Y xs Vs. 1/D gives a straight line with slope (ms ) and intercept 1/Y xs• In chemostat with sterile feed, the observed biomass yield from substrate Y xs is obtained as follws ; x = steady –state cell (16) s = substrate concentrations si = inlet substrate concentrations
Sterilization• The methods available for sterilization including ;o chemical treatment,o exposure to ultraviolet,o gamma and X -ray radiation,o sonication,o filtration and heating .• Aspect of fermentor design and construction for aseptic operation were considered in part section (aseptic operation and fermentation inoculation and sampling ).• In this section consider design of sterilization system for liquid and gasses.
SterilizationBatch heat sterilization of liquids• Liquid medium is most commonly sterilized in batch in the vessel.• Liquid is heated to sterilization temperature by introducing steam into the coils or jacket of the vessel or steam is bubbled directly into the medium, or the vessel is heated electrically.• If direct steam injection is used, allowance must be made for dilution of the medium by condensate which typically adds 10-20% to the liquid volume.
Sterilization Batch heat sterilization of liquids• Typical temperate-time profile for batch sterilization is shown in below figure(Variation of temperature with time for batch sterilization of liquid medium.) • Depending on the rate heat transfer from the steam or electrical element , • The holding or sterilization temperature is reached, temperature is held constant for a period of time t hd . Cooling water in the coils or jacket of the fermentor is then used or reduce the medium temperature to required value.
SterilizationBatch heat sterilization of liquids• Operation of batch sterilization systems, we must be able to estimate the holding time required to achieve the desired level of the cell destruction.• Destroying contaminant organisms, heat sterilization also destroys nutrients in the medium. To minimize this loss, holding time at the highest sterilization temperature should be kept as short.• Cell death occur at all times during batch sterilization, including the heating-up and cooling-down periods. The holding time t hd can be minimized by taking into account cell destruction during these periods.
Sterilization Batch heat sterilization of liquids (Reduction in number of viable cells during batch sterilization) The number of contaminants present in the raw medium No >>During heating period No is reduced to N1 . >>The end of the holding period, the cell number is N2 ; final number after cooling = Nf , >>Ideally Nf = 0; at the end of sterilization cycle we want to have no contaminants present.Normally, the target level of contamination is expressed as afraction of a cell , which is related to possibility of contamination
SterilizationBatch heat sterilization of liquids• Rate of heat sterilization is governed by the equation for thermal death outline .• In batch vessel where cell death is the only process affecting the number of viable cells : N = number of viable cells (17) t =time and kd = specific death constant .• Eq (17) applied to each stage of the batch sterilization cycle: heating, holding and cooling.
SterilizationBatch heat sterilization of liquids• kd is a strong function of temperature, direct integration of Eq.(17) is valid only when the temperature is constant, i.e. during the holding period. The result is: (18) (19) or thd = holding time N1 = number of viable cells at the start of holding N2 = number of viable cells at the end of holding • kd is evaluated as a function of temperature (20) using the Arrhenius equation: A = Arrhenius constant or frequency factor, Ed = activation energy for the thermal cell death, R = ideal gas constant T= absolute temperature
SterilizationBatch heat sterilization of liquids• To use Eq. (19) we must know N1 and N2.• These numbers are determined by considering the extent of cell death during the heating and cooling periods when the temperature is not constant.• Combining Eq. (17) and (20) gives: >> (21)• Integration of Eq. 21 gives for heating period: >>> (22) and for cooling period : >>> (23) t1 = time at the end of heating / t2 = time at the end of holding and tf = time at the end of cooling
Generalized temperature-time profile for the heatingand cooling stages of batchsterilization General equations for temperature as a function of time during heating and cooling periods of batch sterilization
SterilizationBatch heat sterilization of liquids • Applying an appropriate expression for T in Eq.(22) (22) • From table allows to evaluate the cell number N1 at the start of the holding period. • Similarly , substituting for T in Eq.(23) for cooling gives N2 at the end of the holding period. (23) • Use of the resulting values for N1 and N2 in Eq.(19) completes the holding-time calculation. (19)
SterilizationBatch heat sterilization of liquids• The design procedures outlined in this section apply to batch sterilization of medium when the temperature is uniform throughout the vessel.• However, the liquid contains contaminant particle in the form of flocs or pellets, temperature gradient may develop.• Cell death inside the particles is not as effective as in the liquid.• Longer holding times are require to treat solid- phase substrate and media containing particles.
Sterilization Batch heat sterilization of liquids• Heat sterilization is scale up to larger volumes,• Scale-up also affects the temperature-time profile for heating and cooling.• Heat-transfer characteristics depend on the equipment used; heating and cooling of large volumes usually take more time.• Sustained elevated temperature during heating and cooling are damaging to vitamins, proteins and sugar in nutrient solutions and reduce the quality of the medium.• Because it is necessary to hold large volume of medium for longer periods of time, this problem is exacerbated with scale-up.
SterilizationContinuous heat sterilization of liquids• Continuous sterilization, particularly a high- temperature, short-exposure-time process, can reduce damage to medium ingredients while achieving high level of cell destruction.• Improved steam economy and more reliable scale up.• Time require is significantly reduced because heating and cooling are virtually instantaneous.
SterilizationContinuous heat sterilization of liquids• Typical equipment configurations for continuous sterilization are shown in below figure
SterilizationContinuous heat sterilization of liquids• Heat-exchange systems are more expensive to construct than injection devices; fouling of the internal surfaces also reduces the efficiency of heat transfer between cleaning.• On the other hand, a disadvantage associated with steam injection is dilution of the medium by condensate; foaming from direct stream injection can also cause problem with operation of the flash cooler.• Important variable affecting performance of continuous sterilizers is the nature of fluid flow in the system.
Sterilization Continuous heat sterilization of liquids• The type of flow in pipes where there is neither mixing nor variation in fluid velocity is called plug flow
SterilizationContinuous heat sterilization of liquids• Deviation from plug flow behavior is characterized by the degree of axial dispersion in the system.• Axial dispersion is critical factor affecting design of continuous sterilizers.• The relative importance of axial dispersion and bulk flow in transfer of material through the pipe is represented by a dimensionless variable called the Peclet number.
Sterilization Continuous heat sterilization of liquids Pe = Peclet number, (24) u = average linear fluid velocity, L = pipe length Dz = axial- dispersion coefficient.• For perfect plug flow, Dz = 0,•Pe is infinitely ; in practice, Paclet number between3 and 600 are typical.•The value of Dz for a particular system depend on theReynolds number and pipe geometry.
SterilizationContinuous heat sterilization of liquidsThe extent of cell destruction insterilizer can be related to thespecific death constant kdN1 is the number of viable cellsentering the system,N2 is the number of cells leaving ,Pe is the Peclet number as definedby Eq.(24) andDa is another dimensionlessnumber called the Damkohler number kd = specific death constant, (25) L = the length of the holding pipe u i= average linear liquid velocity. The lower the value of N2/N1 the greateris the level of cell destruction
SterilizationContinuous heat sterilization of liquids• Heating and cooling in continuous sterilization are so rapid that in design calculation they are considered instantaneous.• While reducing nutrient deterioration, this feature of the process can cause problems if there are solids present in the medium.• It is important therefore that raw medium be clarified as much as possible before it enters a continuous sterilizer.
SterilizationFiltration sterilization of liquids• Sometimes, fermentation media or selected ingredients are sterilized by filtration rather than heat.• For example, media containing heat-labile components such as enzymes and serum are easily destroyed by heat and must be sterilized by other mean.• Membrane used for filter sterilization are made of cellulose esters or other polymers and have pores between 0.2 and 0.45 µm in diameter.
Sterilization Filtration sterilization of liquids• Bacteria and other particles with dimensions greater than the pore size are screened out and collect on the surface of the membrane.• To achieve high flow rates, large surface areas are required.• Liquid filtration is generally not as effective as heat sterilization. Viruses and mycoplasma are able to pass through membrane filters; care must also be taken to prevent holes or tears in the membrane.• Usually, filter-sterilized medium is incubated for a period of time before use to test its sterility.
SterilizationSterilization of air• The number of microbial cells in air is of the order 103 - 104 m-3.• Filtration is the most common method for sterilizing air in large scale bioprocesses.• Depth filters consisting of compacted beds or pads of fibrous material such as glass wool have been used widely in the fermentation industry.• Depth of the filter medium required to produce air of sufficient quality depends on the operating flow rate and the incoming level of contamination.
SterilizationSterilization of air• Cells are collected in depth filters by a combination of impaction, interception, electrostatic effects.• Depth filters do not perform well if there are large fluctuations is flow rate or if the air is wet; liquid condensing in the filter increase the pressure drop, cause channeling of the gas flow.• Cartridge filters, these filters use steam-sterilizable polymeric membrane which act as surface filter trapping contaminants as on a sieve.
SterilizationSterilization of air• Containment is particularly important when organisms used in fermentation are potentially harmful to plant personnel or the environment; companies operating fermentations with pathogenic or recombinant strains are require by regulatory authorities to prevent escape of the cells.
Summary reactor EngineeringThis chapter contains a variety of qualitative and quantitative information about design and operation of bioreactors. After studying this chapter, you should1 be able to assess in general terms the effect of reaction engineering on total production costs in bioprocessing2 be familiar with a range of bioreactor configurations in addition to the standard stirred tank including bubble column, airlift, packed-bed, fluidized- bed and trickle-bed designs;
3 understand the practical aspects of bioreactor construction, particularly those aimed at maintaining aseptic condition;4 be familiar with measurements used in fermentation monitoring and the problems associated with lack of online methods for important fermentation parameter;5 be familiar with established and modern approaches to fermentation control;6 be able to predict batch reaction time for enzyme and cells reaction;
7 be able to predict the performance of fed-batch reactors operated under quasi-steady-state conditions;8 be able to predict and compare the performance of continuous stirred-tank reactor and continuous plug flow reactor;9 know how to use steady-state chemostat data to determine kinetic and yield parameter for cell culture and10 know how batch and continuous system are designed for heat sterilization of liquid medium and methods for filter sterilization of fermentation gases.