LAB MANUAL

BT1355 BIOPROCESS II

III YEAR BIOTECHNOLOGY Section A

Facult inc!a"#e
S$Sa%a"uni&!a Be#u'( Seniou" Lectu"e"( Biotec!$ )e*t$( REC

CONTENT
1$ BATCH REACTOR E+PERIMENT ,$ FE) BATCH FERMENTATION OF PHOSPHOBACTERIUM 3$ ESTIMATION OF -La BY )YNAMIC GASSING METHO) .$ CALCULATION OF MASS TRANSFER COEFFICIENT AN) PO/ER NUMBER CORRELATION 5$ )ETERMINATION OF 0OLUMETRIC MASS TRANSFER COEFFICIENT BY SO)IUM SULPHITE O+I)ATION METHO) 1$ HEAT TRANSFER 2$ BATCH HEAT STERILI3ATION AN) THERMAL )EATH -INETICS 4$ RESIS)ENCE TIME )ISTRIBUTION

BIOREACTOR 5 CASE STU)Y

)EFINITION6 Fermenters are large quantities cylindrical tanks, which provide controlled environment for growth of microorganism or any cells to obtain a desired product. BO)Y CONSTRUCTION6 GLASS !. "rovide smooth, corrosion proof surface #. $t is non%to&ic '. (&amination of interior of the vessel is easy S)A$*L(SS S)((L !.$t limits corrosion. #.$ts combination with molybdenum improves resistance to halogen salts. '. +ontamination is less. ,.+ost effective S(AL$*G Sealing of top and bottom plates to the body of the fermantor can be done using % % % 3se )o maintain uniform environment throughout the vessel content, o&ygen transfer, heat transfer suspension of solid particles. )ypes 4isc turbines, vaned disc,open turbines of variable pitch, propellers 5(+6A*$S5 !. Agitator give rise to small air bubbles when air hits the disc. #. .ubble break up gives rise to gas filled cavities. '. Gas dispersion occurs from gas filled cavities. *etryl-.utyl rubbers Glass-metal sealing with compressible gasket /ring 0single-double1

AG$)A)/2

S)$22(4 GLASS A*4 .2(A7$*GS Stirring mechanism is ideal for animal cell culture. Four to eight baffles may be used according to the vessel. $t has similar action to agitator. .AFFL(S .affles are metal strips roughly !-!8th of vessel diameter Four to eight baffles may be used accordingly to the vessel. $t has a similar action to agitator. S"A2G(2 4evices used for producing air into the liquid in fermentor. )ypes "orous sparger, /rifice sparger, no99le sparger. 5(+6A*$S5 Air holes emit are present on the under surface of the tubes )hese holes emit air from tubes under a reduced pressure. AS(")$+ $*/+3LA)$/* "$"( Addition of innoculum, nutrient : other supplements are done with the aseptic inoculation pipe. "ositive pressure should be maintained in the pipe to prevent entry of microorganisms, dust etc. SA5"L$*G "/2) $t should be maintained asceptically with the help of steam. 2emoval of both and its transport should takes place contaminant condition. Sampling helps !8 moniters and control physiochemical parameters. S(*S/2S "2/.(S )o maintain and monitor physiochemical parameter insites. Glass probes with double ;/< ring seal are normally used. =AL3(S A*4 S)(A5 "2/.(S =alues are used to controlling the flow rate of liquid gases. )hey are also used to control backward flow of fluids.

)>.(S Gate values, globe values, piston values, needle valves, plug valves, ball valves, butterfluy valves, and pinch valves, diaphram valves. S)(A5 )2A" $t is used to remove steam condensate from the fermentors. $t consists of valve and seat assembly with operator on the basis of properties of fluid.

BATCH REACTOR E+PERIMENT

E+ NO6 )ATE6 AIM6 )o determine the specific growth rate and yield coefficient of the given microbial strain through the .atch reactor. INTRO)UCTION6 .atch culture system represents growth in a closed system. )hey use a flask or fermenter containing a suitable growth%supporting medium operated under optimum conditions of temperature, p6 and redo& potential. )he medium is introduced with the cells grown until some essential component of the medium is e&hausted or the environment changes because of the accumulation of a to&ic product, p6 change etc. $n general microbial growth is determined by cell dry weight measurement. )he growth of curve can be divided into three phases 1$ La# *!a&e 4uring this period the cell adapts to the new environment by synthesi9ing necessary en9ymes for the utili9ation of available substrates. ,$ E7*onential *!a&e )he cell constituents in this phase increases at a constant rate so that the cell population doubles and continues to double at regular intervals. 3$ Stationa" *!a&e6 $n this phase, cell death occurs because of depletion of essential nutrients, accumulation of to&ic etc. )he rate of cell death is balanced by the rate cell growth? hence there is no net growth or increase in cell number. )his is followed by a death phase. S*eci8ic G"o9t! "ate6 )he growth of the microbial cells is autocatalytic )he mass balance equation used is Cell :" 9ei#!t accu'ulation @ #"o9t! 5 cell "e'o;al 5 cell :eat! 6ere we neglect cell death and no cells are removed in a batch reactor. 6ence :+<:t = +( where is specific growth rate. /n integration we get, A @ A8 et,where A8 is innoculum @ 0$n &# B $n &!1-0t#%t!1 @ 0$n /4# B$n /4!1-0t#%t!1

Su%&t"ate utili>ation Substrates are consumed to provide the necessary carbon, energy and structural components for the cell growth as well as for the maintenance of cell viability. )he single substrate utili9ation can be determined from Substrate accumulation @ 0substrate feed B substrate consumed for growth B substrate for product synthesis B substrate consumed for maintenance B substrate removal1 ds-dt @ FS-= % CA->&-s B qpA->p-s B mA B F<S<-= Dhere, F and F< are flow rates of medium into and out of behavior respectively 0!-hr1 = is the volume of the culture 0litre1 S and S< are the substrates concentration going in and out of the culture 0g-l1 >&-s is the biomass yield coefficient >p-s is the product yield coefficient C is the specific growth rate 0time%!1 E is the product formation rate $n the batch reactor, with no feed or removal, the equation becomes ds-dt @ %CA->&-s B qpA->p-s B mA $n aerobic cultures if no products are formed and maintenance is negligible, the equation reduces to ds-dt @ % CA->&-s MATERIAL AN) METHO)S6 .$/2(A+)/2 0(&%situ type1 )he studies that will be reported here were conducted on Flit 0working volume '.8lit1 reactor. )his reactor is equipped with flat four%blade 2ushton turbine. 6eight #GFmm,diameter of tank !,Gmm, diameter of impeller FHmm, inter impeller distance I'mm, distance between last impeller and tank #!#mm. )he tank has two baffles with a width and height of !G and #Fmm. "re inoculums )ype !F%#8J glycerol stock 5edium L. medium +ulture /4 8.F =olume taken ! ml $noculums details =olume "6 )emp 2pm Final /4 '88 ml F.I #HK+ '88

5edia composition Glucose >east e&tract Ammonium *itrate

8.Fg 8.Fg 8.!g

Fermentation condition $nitial volume '.8 lit 5edium L '88 ml of $noculum p6 F.I ==5 Anaerobic )emp #H K+ 2pm '88 After inoculation, readings are taken every hour and tabulated. )he values of /4, residual glucose and dry weight were thus recorded. RESULT6 )he specific growth rate and yield coefficient of the given microbial strain are determined through the .atch reactor. INFERENCE6 For the batch reactor the following graphs are drawn. i =ariation of various parameters as a function of time. ii ln /4 =s )ime graph is plotted for the calculation of C iii .iomass =s residual substrate concentration graph is plotted for > &-s calculation iv 3sing pure culture the quality parameters of the reactor could be verified.

FE) BATCH FERMENTATION OF PHOSPHOBACTERIUM

E+PT NO6 )ATE6 AIM6 )o determine the feed rate0F1, Growth rate0C1 and biomass0&1 production rate of the culture Phospho bacterium INTRO)UCTION6 A fed batch culture is one in which one or more nutrients are fed into the fermentor during the fermentation period. $n this way nutrients can be added at the same rate as they are used up, so e&cess of nutrients and any inhibition resulting from this can be avoided. A classical e&ample is penicillin fermentation where the different nutritional requirements at different phases can be catered for feeding in particular nutrients at the time they are required. )wo basic approaches to the fed batch fermentation can be used !. Fi&ed volume fed batch #. =ariable volume fed batch =A2$A.L( =/L35( F(4 .A)+6 As the name implies, a variable volume fed batch is one in which the volume changes with the fermentation time due to the substrate feed. )he way this volume changes is dependent on the requirements, limitations and obMectives of the operator. )he feed can be provided according to one of the following options !. )he same medium used in the batch mode is added. #. A solution of the limiting substrate at the same concentration as that in the initial medium is added. =ariable volume fed batch can still be classified as !. 2epeated fed batch process or cyclic fed batch culture. #. Single fed batch process. 2("(A)(4 F(4 .A)+6 "2/+(SS $n this method once the fermentation reached a certain stage after which is not effective anymore, a quantity of culture is removed from the vessel and replaced by fresh nutrient medium. )he decrease in volume results in an increase in the specific growth rate, followed by a gradual decrease as the quasi steady state is established.

S$*GL( F(4 .A)+6 "2/+(SS $n this case, supplementary growth medium is added during the fermentation, but no culture is removed until the end of the batch. )his system presents a disadvantage over the fi&ed volume fed batch and the repeated fed batch process much of the fermentor volume is not utili9ed until the end of the batch and consequently, the duration of the batch are limited by the fermentor volume. A)0ANTAGES AN) )ISA)0ANTAGES OF THE FE) BATCH REACTORS6 A4=A*)AG(S !. "roduction of high cell densities due to the e&tension of working of working time. #. +ontrolled conditions in the provision of substrates during the fermentation, particularly regarding the concentration of specific substrates as for e&ample the carbon source. '. +ontrol over the production of byproducts or catabolite repression effects due to limited provision of substrates solely required for product formation. ,. Allows the replacement of water loss by evaporation. F. $ncrease of antibiotic marked plasmid stability by providing the correspondent antibiotic during the time span of the fermentation. 4$SA4=A*)AG(S !. $t requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity. #. $n a cyclic fed batch culture, care should be taken in the design of the process to ensure that to&ins do not accumulate to inhibitory levels and that nutrients other than those incorporated into the feed medium become limiting. Also if many cycles are run, the accumulation of non%producing or low producing variants may result. "35"S )here are two types of pumps which are suitable for the aseptic pumping of small volumes of culture media !. "eristaltic pump #. 4iaphragm dosing pump "(2$S)AL)$+ "35" A main body that comprises of both the derive motor and electrics and the rotating unit of rollers typically constitutes the peristaltic pump. )his unit if rollers occludes the tube which, as it recovers to its original si9e passes to the ne&t roller until it is e&pelled, as the moves around. )he flow rate can be varied by either the speed setting or by changing the diameter of the tube being used. 4$A"62AG5 4/S$*G "35" )he diaphragm%dosing pump consists of a main body and a detectable heat sterili9able head. )he fluid is sucked into the pump head. )he suction inlet tube then closes and the pressure discharge tube opens and forces the fluid out. )he suction and

pressure forces in the pump head are generated by the reciprocating action of both diaphragm plunger and the return spring. 5/4(L$*G F(4 .A)+6 F(25(*)A)$/* =A2$A.L( =/L35( F(4 .A)+6 $n a variable volume fed batch fermentation, an additional element should be considered, the feed. +onsequently, the volume of the medium in the fermentor varies because there is an inflow and no outflow. Again, the growth of the fermentation of the microorganism is limited by the concentration of one substrate. For the mathematical developments that will be presented, the assumptions are !. Specific growth rate is uniquely dependent on the concentration of the limiting substrate. #. )he concentration of the limiting substrate in feed is constant. '. )he feed is sterile. ,. )he yields are constant during the fermentation time. +onsidering the overall mass balance 0$nput1 @ 0output1 L 0accumulation1 F @ 8 L d=-dt F @ d=-dt 0!1 Dhere, = B the time volume of the fermentor ) B time F B the feed rate 0volume-time1 +onsidering now the balance to the biomass 0Accumulation1 @ 0in1 L 0produced1 B 0lost by cell death1 d0=&1-dt @ F. Ao L r&.= B rd.= 0#1 where, & B biomass concentration 0mass of biomass-volume1 Ao % biomass concentration in the feed 0mass of biomass in feed-volume1 r&% biomass production rate 0mass of biomass-volume. time1 rd B biomass death rate 0mass of biomass -volume. time1 As the feed is considered to be sterile, Ao amounts to 9ero. )herefore eqn 0#1 becomes d 0=&1-dt @ r&.= B rd.= =.d&-dt L &d=-dt @ r&.= B rd.= =.d&-dt L &F @ r&.= B rd.= 0using eqn !1 .ut, r&.= @ C.&.= and rd.= @ kd.&.= where, C @specific growth rate 0time%!1 k d @ specific death rate 0time%!1 )herefore =.d&-dt L &F @ C.&.=% kd.&.=

2earranging the equation d&-dt @ & 0C.=% kd.= % F1- = Specific growth rate is smaller so it can be neglected. +onsider now the balance to limit substrate 0accumulation1 @ 0in1 L 0consumed1 d0=.s1-dt @ F.so B rs.= Dhere, s is the substrate concentration in the fermentor 0mass of substrate - volume1 so is the substrate concentration in the feed 0mass of substrate in feed- volume1 rs is the consumption rate of substrate 0mass of substrate-volume. time1 =ds-dt L s.d=-dt @ F.so B rs. = $t should be noted that the consumption rate of substrate includes the specific consumption used for biomass production, product formation and maintenance of the cell. $ncluding now the concept of yield rs. = @ C. &. =->&-s Dhere, >&-s is the ratio between the mass of cells produced per mass of substrate consumed. Substituting rs. = @ C. &. =->&-s =ds-dt L s. F @ F.so B C. &. =->&-s 2earranging the equation, ds-dt @ F.0so B s1- = % C.&.->&-s )he table below summari9es the equations that have Must been developed 5ass balance for the main components for a fed batch reaction Co'*onent /verall .iomass Substrate "roduct Ma&& %alance e?uation F @ d=-dt d&-dt @ & 0C.=% kd.= % F1- = ds-dt @ F.0so B s1- = % C.&.->&-s dp-dt @ Ep.& B ".F-=

= is the volume of the fermentor ) is the time F is the feed rate 0=olume-time1 A is the biomass concentration 0mass of biomass - volume1 C is the specific growth rate. 0timeB!1 kd is the specific death rate 0time%!1 s is the substrate concentration in the fermentor 0mass of substrate - volume1 so is the substrate concentration in the feed 0mass of substrate -volume1 >&-s is the yield factor 0mass of biomass - mass of substrate1 N is product concentration 0mass of product - volume1 Ep is the specific production rate of product 0mass of product -mass of biomass. time1

MATERIALS AN) METHO)S6 .$/2(A+)/2 0(&%situ type1 )he studies that will be reported here were conducted on Flit 0working volume #.Flit1 reactor. )his reactor is equipped with flat four%blade 2ushton turbine. 6eight #GFmm, diameter of tank !,Gmm, diameter of impeller FHmm, inter impeller distance I'mm, distance between last impeller and tank #!#mm. )he tank has two baffles with a width and height of !G and #Fmm. )he organism used was "hosphobacterium. +3L)32( 5(4$A +/5"/S$)$/* g-lt .eef e&tract ' "eptone F *a+l F 4istilled water !888ml $*/+3L35 "2("A2A)$/* A single loop of "hosphobacterium culture was scrapped using a sterile inoculation loop and then aseptically transferred into a !Fml nutrient medium. )his starter culture was grown overnight. )his was mi&ed with '88ml of media 0having same composition as the reactor media i.e. nutrient medium1 and then inoculated into #.Flt reactor media to start fed batch fermentation. "A2A5()(2S )ime of sterili9ation )emperature "ressure 6old time $noculum )ime of feed I 88am !#!K+ !bar #8min '88ml # 88pm

F(4 .A)+6 F(25(*)A)$/* )he e&perimentation was conducted in a FL fermentor equipped with temperature, p6, airflow, 4/, pressure and agitation controllers. )he e&periment was conducted at '8 K+. 4/ was measured by a 4/ probe. )he culture media of the above composition was used. Dhen the substrate is completely consumed by the biomass, ds-dt @ 8 6ence the substrate balance equation reduces to FoS-= @C.&.>&-s F @ C. &. =-S8 >&-s RESULT6 6ence the feed rate 0F1, growth rate 0C1and biomass concentration0&1 are determined for the culture of "hospho bacterium. INFERENCE6

)hrough the fed batch method following graphs were determined for /4 =s A From this standard graph corresponding biomass concentration are determined with respect to /4 values. Feed rate =s )ime De obtain a graph of increasing order to the corresponding values of feed and time, which concludes the increase in the growth rate of the organism. Growth rate =s )ime From this graph we obtain a linear increase in growth corresponding to the time till the substrate is completely utili9ed, then the growth rate decreases. /4 =s time )here e&ists a linear increase in /4 with respect to time. ln /4 =s time )here e&ists a linear decrease in /4 with respect to time.

ESTIMATION OF -La BY )YNAMIC GASSING METHO)

E+PT NO )ATE 6 6

AIM6 )o estimate the volumetric mass transfer coefficient 07La1 by dynamic gassing method. INTRO)UCTION6 Gas liquid mass transfer is of paramount importance in bioprocess because of the requirement of o&ygen in aerobic fermentations. /ne of the most critical factors in the operation of a fermentor is the provision of adequate gas e&change. /&ygen is the most important gaseous substrate for microbial metabolism and carbon%dio&ide is the most important gaseous metabolic product. Dhen o&ygen is required as a microbial substrate, it is frequently a limiting factor in fermentation. .ecause of its low solubility, only 8.' m5 o&ygen, equivalent to I mg-ml, dissolves in one liter of water at #8K+ in an air- water mi&ture. 4ue to the influence of the culture nutrients, the ma&imal o&ygen content is actually lower than it would be in pure water. As temperature increases, solubility of o&ygen reduces. For e&ample, at ''K+ the solubility is O.# mg-l. 5ost aerobic microbial processes are o&ygen limited. )hat is the reason why the concept of gas Bliquid mass transfer in bioprocess is centered on o&ygen transfer even if other gases such as carbon dio&ide and ammonia can be involved. /A>G(* 5ASS )2A*SF(2 )he solubility of gases follows 6enry<s law in the gas pressure range over which the fermentors are operated. )his means that if the o&ygen concentration in the gas phase increases, the o&ygen proportion of the nutrient solution increases. +onsequently the highest o&ygen partial pressures are attained during aeration with pure o&ygen. +ompared to value in air 0I mg-l1, pure o&ygen has a solubility of ,' mg-l in water. 2(S$S)A*+(S )/ /A>G(* )2A*SF(2 For o&ygen to be transferred from a gas bubble to an individual cell, several independent partial resistances must be overcome. 4iffusion from the bulk gas%to%gas liquid interface.

5ovement through gas liquid interface. 4iffusion of o&ygen through relatively unmi&ed liquid region adMacent to the bubble into the well%mi&ed bulk liquid. )ransport of o&ygen through bulk liquid to a second relatively unmi&ed liquid region surrounding the cells. )ransport through second unmi&ed liquid region associated with the cells. 4iffusive transport through cellular floc. )ransport across the cell envelope and to intracellular reaction site /A>G(* )2A*SF(2 +/(FF$+$(*) )he mass transfer of o&ygen into liquid can be characteri9ed by o&ygen transfer coefficient 7La. )his has been thoroughly e&amined as a critical parameter for bioreactor function. $t depends upon the following parameters =essel geometry 5i&ing properties Aeration system *utrient solution 5icro organism Antifoam agent )emperature .affles produce a large planer liquid surface and a uniform flow pattern and also increases liquid hold up for a given fermentor volume. Surfactants such as antifoam agents reduce o&ygen transfer rate. 5icroorganisms act as a barrier for o&ygen transfer and hence reduce o&ygen transfer rate. )he gas bubbles are replenished in the locations of the bioreactor where there is negative pressure, such as behind the agitator blades. As aeration increases, various conditions can be characteri9ed. At low aeration rates, large bubbles form behind the turbine blades and smaller bubbles are spun off into the nutrient solution. As the aeration rate is increased, gas bubbles collect behind all the turbine blades and continue to accumulate. )he energy input is one%third less than that used for un aerated systems. $n this intermediate stirring range, gas dispersion is the best. At every high aeration rates, many large bubbles adhere to each other and the impeller is flooded with gas, resulting in sharply lowered gas dispersion. +2$)$+AL /A>G(* +/*+(*)2A)$/* )he critical o&ygen concentration is the term used to indicate the value of the o&ygen uptake rate or o&ygen absorption rate which permits respiration without hindrance. Generally the critical o&ygen concentrations are F%#FJ of the o&ygen saturation value in culture. At o&ygen absorption rates that are lower than the critical concentrations, respiration rate is correlated with the o&ygen concentration in the solution. Above this value, no dependence between respiration rate and dissolved o&ygen has been observed. $n yeast and bacterial fermentations, the critical o&ygen concentration

is constant and not affected by fermentation conditions. $n filamentous microorganisms, the critical o&ygen concentration has been shown to be dependant on fermentation conditions. (S)$5A)$/* /F 4$SS/L=(4 /A>G(* +/*+(*)2A)$/*S /A>G(* "2/.(S )he reduction of dissolved o&ygen at a noble metal surface negatively polari9ed with respect to reference electrode forms the basis of design and operation of o&ygen electrodes. 5embrane covered o&ygen probes measure o&ygen partial pressures because the electrode generates the current that is proportional to the amount of o&ygen through the membrane that is directly proportional to the o&ygen tension in the medium. )he two type of o&ygen electrodes that may be used are "olarographic electrodes. Galvanic electrodes. "/LA2/G2A"6$+ (L(+)2/4( A constant potential difference is maintained across a reference electrode 0Anode% +alomel or Silver - Silver +hloride1 and a platinum cathode in a suitable electrolyte like "otassium chloride. $n this design, a membrane that is permeable to o&ygen 0")F( or Silicon reinforced with steel mesh1 separates the fermentation fluid from the electrode. /&ygen diffuses through the membrane to the cathode where it reacts to produce a current between the anode and the cathode proportional to the o&ygen partial pressure in the fermentation broth. +athode /# L #6#/ L #e% 6#/# L #/6% 6#/# L #e% #/6% Anode Ag L +l% Ag+l L e% /verall reaction ,Ag L /# L #6#/ L ,+l% ,Ag+l L ,/6%

E+PERIMENTAL PROCE)URE6 )he Fl fermentor was aerated by air supplied from tanks through a compressor. Air was passed into the fermentor through a flow meter - regulating valve system and then through a sterili9ing filter. $t was introduced at the bottom of the filter through a sparger, which is an arrangement of pipe work perforated with small holes. .y shutting off the regulating valve, the air supply to the fermentor was stopped. )he fall in the 4/ concentration was noted down at regular intervals. After the 4/ level came to around #8J, the valve was opened to allow inflow of air. )he rise in 4/ concentration was noted at regular intervals till the value become constant. )ISCUSSION6 )his method for measuring 7La is based on unsteady state mass balance for o&ygen. At some time, the broth is deo&ygenated by stopping the airflow. 4/ concentration P4/Q drops during this period. Air is pumped to the broth at a constant flow rate and increase in P4/Q is monitored as a function of time. $t is important that the

level remains above the critical 4/ concentration so that the o&ygen uptake is independent of o&ygen level. Assuming the re%o&ygenation of the broth is fast relative to cell growth, the dissolved o&ygen level, will soon reach a steady state value P4/Q which reflects a balance between o&ygen supply and consumption in the system. 4uring the cut% off step, there is no mass transfer as the dissolved o&ygen is only used. 6ence, EoA @ dP4/Q-dt 4uring the re%o&ygenation step, the system is not at steady state, dP4/Q-dt @ 7La 0dP4/Q -dt L Eo#A1 - 0 P4/QR % P4/Q1 )he obtained 7La value is in the lower range that implies that the ability of the reactor to deliver o&ygen to the cells is limited. )he Eo#A is lower in the second case. )his might be because the cells were subMected to o&ygen concentrations below critical levels or might be because of reduction in impeller velocity. 7La can be used characteri9e o&ygen mass transfer capability of fermentors. 3nder steady state there can be no accumulation of o&ygen at any location in the fermentor. )herefore, the rate of o&ygen transfer from the bubbles must be equal to rate of o&ygen consumed by the cells. 7La 0P4/QR %P4/Q1 @ E/#A De can predict the response of fermentor to change in mass transfer conditions using the above equation RESULT6 )he volumetric mass transfer coefficient 07La1 determined by the dynamic gassing method INFERENCE 6 )he 7La obtained 08.88G S%!1 quite low. )his can be attributed to the following reasons. Dhen the readings were taken the cells were suspected to have entered the death phase. 0Since it had been Ghrs from the start of the reactor.1 4uring the gassing phase the 7La is limited by the mass transfer coefficient between air supplied over the medium. $t can be eliminated by increased aeration. .ecause of the increased residence time of /# bubbles in medium resulting in low 7La value. )he response of the probe may not be good. A thorough knowledge of the response time of the probe is essential

CALCULATION OF MASS TRANSFER COEFFICIENT AN) PO/ER NUMBER CORRELATION E+PT NO )ATE AIM6 )o calculate the volumetric mass transfer coefficient 07La 1 using power number correlation. INTRO)UCTION6 )he traditional method to determine mass transfer coefficient 07 La 1 for large antibiotic fermentor has been based upon log mean relationship between the inlet and e&it equilibrium dissolved o&ygen concentration and the actual dissolved o&ygen in the fermentor. )he resulting 7La is then related to power unit volume, agitator speed and superficial gas velocity. )his method has a number of disadvantages. )he dissolved o&ygen in the fermentor varies from top to bottom of the fermentor. )he 7La value can be determined on%line from /# analysis and a single dissolved o&ygen measurement at any stage of the fermentor. An aerobic cell will be able to utili9e a richer carbon source fully only if o&ygen can also be maintained at a higher concentration in the direct vicinity of the cell. )he overall mass transfer coefficient 7La is a measure of the ability of fermentor to supply o&ygen to cells. )he solubility of o&ygen in aqueous solutions under ! atm of air and near ambient temperature is of the order of !8ppm. /&ygen is sparingly soluble in aqueous solution and hence gas liquid mass transfer should be increased. Agitation enhances gas liquid mass transfer. 7La depends on power input and power consumption and quantity of air supplied. 7La is highly dependent on the culture conditions, with age, cell concentration and growth rate having a maMor effect. +onsistent 7La values can be obtained at the same point in each run. $n low viscosity liquids, 7La is a relatively strong function of gassing rate, with 7La and hold up increasing monotonically with power input and similar values are obtained regardless of agitator type. $ncreasing viscosity reduces 7La and at the highest viscosities. 7La is independent of the gassing rate, as is the power input. (&periment is conducted in water so that 7La is highly dependant on the physiochemical 6 6

nature of the solution in low viscosity systems, as shown by the results correlated by =an<t reit. For pure water he proposed 7La @ 8.8#G 0"-=18.,=s8.F And for strongly ionic solutions 7La @ 8.88# 0"-=18.O=s8.# )hese correlations show how 7La varies and also how the dependence of 7La on the power input and gassing rate varies with a change in ionic strength. )his is considered to be due to the change in bubble coalescing properties. $n pure water, bubbles coalesce easily forming bubbles of low surface area to volume ratio when ascend stabili9ed by reducing hold up and interfacial area. $n ionic solutions, the interface is stabili9ed by ions and coalescence is reduced. )his maintains smaller bubble si9es and leads to a higher hold up and therefore higher interfacial areas. )he power consumption in a bioreactor is reduced by aeration. "ower number is used to calculate the power required to rotate a given impeller at a given speed. )he power number depends on dimensions of tank and impeller, the distance of impeller from the tank floor, the liquid depth and the dimensions of the baffles, if these are used. )he number and arrangement of the baffles and the number of blades in the impeller must also be fi&ed. )he variables that enter the analysis are the important measurements of tank and impeller, the viscosity C and density S of the liquid, the speed n, and because the *ewton<s law applies, the dimensional constant g. )he various linear measurements can all be converted to dimensionless ratios, called Tshape factorsU, by dividing each of them by one of their number, which is arbitrarily chosen as basis. )he diameter of the impeller is taken as 4 a and of the tank as 4t and the shape factors are calculated by dividing each of the remaining measurements by the magnitude of 4 a or 4t. Let the shape factors, so defined, be denoted by S !, S#, S', VVV..Sn. Dhen the shape factors are temporarily ignored and the liquid is assumed T*ewtonianU, the power " is a function of the remaining variables, or " @ W 0n, 4a, gc, C, g, S1 Applying law of homogeneity, we get "gc-*'4aFS @ W 0n4aF S-C, n#4a-g1 where "gc-*'4aFS * 4a# S-C *#4a-g 2eynolds no Froude no. power no.

$n use turbulent regime, the power input is independent of 2eynolds no.

" X n'4aF *p is a constant. Laminar flow relation is given by " X n#4a' or *pX !-*2e )he proportionality constant in each case depends on impeller geometry. )he o&ygen transfer rate per unit volume is given by Eo#@ 7La 0+R % +1, where a interfacial area per liquid volume, 0+R % +1 /verall concentration driving force.

)he ratio of power requirement in aerated versus non%aerated vessels " g-" versus the aeration no *a can be correlated. *a @ Fg - *i 4M' ? Fg *a @ 0Fg-4M#1- *M4M air flow rate where *a@ superficial gas velocity - impeller tip velocity.

)he air - gas flow rate can be calculated from the ;vvm<. )he overall mass transfer coefficient can also be given by 7La @ k 0"-=17 =sY Dhere 0"-=1 is the power per unit volume =s is the superficial gas velocity. 7La @ # Z!8%' 0"-=18.O 0=s18.# S%!. (E3$"5(*) A*4 (A"(2$5(*) 5()6/4 )he studies that will be reported here were conducted on Fl 0working volume #.Flit1 reactor. )his reactor is equipped with flat four%blade 2ushton turbine. 6eight #GFmm, diameter of tank !,Gmm, diameter of impeller FHmm, inter impeller distance I'mm, distance between last impeller and tank #!#mm. )he tank has two baffles with a width and height of !G and #Fmm. )he impeller is run with IGD0#,=olt A, Amp1 motor. )he power input to the motor was measured by monitoring voltage and ampere on a multimeter. (ffective power was calculated by assuming the efficiency to be I8J. All studies were conducted with water. 3ngassed power number 0*p1 )he effective power number was calculated from the 2ushton power equation *p @ 0"eff1g- *'4a FS Gassed power number 0"g1

2atio of "g-" as a function of aeration number was plotted. "g -" increased with increase in liquid level below turbine. 4A)A 3S(4 4iameter of impeller @ FH mm 4ensity of water @ !888kgm%' =iscosity of water @ !8%'"a F/253LA( 3S(4 "ower, " @ [' =$ cos\ 0watts1, where, = @ =oltage, =olts $ @ current, Amps +os \ @ phase difference @ 8.I (ffective power "eff @ 8.I " watts. 2eynolds number *re @ 4#*S-C Dhere 4 @ diameter of impeller * @ 2evolution per second S @ 4ensity of water C @ =iscosity of water "ower number *p @ "eff - *'4FS RESULTS6 )he 7La values were determined and were found be For vvm @ 8 For vvm @ 8.F For vvm @ ! For vvm @ !.F ?7La @ ?7La @ ?7La @ ?7La @

2atio of "g-" as a function of aeration number was plotted. $t was found that both aeration number and "g- " ratio increase with impeller velocity. *o significant variation was observed in the correlation between * p and *2e for the gassed and ungassed cases. CONCLUSION6 $n a multiturbine fermentor, it would be desirable to be able to estimate the power input of each turbine in the fermentor. 3sing this information, a modeling approach can be used to relate stage wise mass transfer coefficients to stagewise power inputs. $t would then be possible to predict the a&ial dissolved o&ygen distribution of the fermentor and how the distribution reacts to changes in such process variables as pressure, airflow rate, the agitator speed. $t would also be possible to optimi9e the agitator configuration to obtain the most efficient use of power inputs.

)he aeration number was plotted against the ratio of gassed to ungassed power absorption. )he trend should show that the power drawn decreases to as much as F8J of ungassed power drop during aeration. )he ungassed power drawn is important in selecting the appropriate operating speed of agitators, especially for fi&ed motors. For instance, in certain plants, the nutrient medium is sterili9ed in the fermentor and as it is necessary to turn the air off during this operation, the impeller would draw ma&imum power. Also compressor failure causing loss of aeration during operation would increase the power draw to the ma&imum load at the operating speed. $n order to prevent over load of the agitator motors during these conditions, the operating speed must be specified with respect to the ungassed power for fi&ed speed motors. INFERENCE6 Aeration of a liquid decreases the power consumption during agitation because an aerated liquid is less dense than an unaerated one.

)ETERMINATION OF 0OLUMETRIC MASS TRANSFER COEFFICIENT BY SO)IUM SULPHITE O+I)ATION METHO)

E+PT$ NO )ATE AIM6 )o determine the 7La through the SodiumSulphite o&idation method. THEORY6 )he sodium sulphite combines with o&ygen to give sodium sulphate, with +uS/, as catalyst in the reactor. )he concentration of sodium sulphite at various time points is inversely proportional to the o&ygen transfer rate. *a#S/' L 8.F/# *a#S/, 6 6

)he kinetics of this reaction is independent of the sulphite concentration. )he reaction consumes o&ygen at a rate that is sufficiently fast so that transport of / # from gas to liquid through liquid film is the rate%limiting step. )he rate of the reaction is 9eroth order in *a#S/'. $f the reaction is not fast enough the reaction occurs in the liquid film around the gas bubbles. )his would decrease apparent film thickness and give incorrectly high values of 7La. +oncentrations of unreacted sulphite are determined by reacting the sulphite with e&cess iodine and then back titration of the iodine with thiosulphite. $t is important to note that the 4/ is 9ero through the reaction. S/'#% L $# S#/O#% L$# 7La @ P/#QR % P/#Q P/#Q @ 8 and P/#QR @ H.,'R!8%' g-l 5oles of /#R'# /)2 @ #$%L S/# #$% L#S/#

2ate of sulphite consumption

)ime R volume of reactor

/&ygen consumed @ ] P*a#S/'Qf % P*a#S/'Q$ ^< # Dhere =olume /f Standard *a#S/' @ 8.Fml MATERIALS RE@UIRE)6 8.F5 *a#S/' !J starch solution 8.!5 *a#S#/O 8.88'5 +uS/,, F6#/ Standard *a#S/' 08.!5 to 8.F51 8.!5 $odine solution 0#8 g-l 7$ L !#.O g-l $#1 5easuring cylinder 4istilled water )est tubes, eppendorfs etc.

PROCE)URE6 Sampling Fermentor is filled with 8.F5 *a#S/' and 8.88' 5 +uS/,. F6#/ 0#.F litre1. Agitation is given at ,88 rpm. After the aeration begins collect samples every !8 mins. (stimate the concentration of *a#S/' by titration. )itration .urette solution 8.! 5 *a#S#/O. Flask solution F88Cl of *a#S/' solution 0sample or standard1 L !F ml iodine solution L F8Cl starch. 4uring titration colour changes to straw yellow. Add the starch solution at this point only and not before. (stimate the concentration of *a#S/' in sample from standard curve. RESULT6 )hus the 7La through the SodiumSulphite o&idation method is determined INFERENCE6 )hus 7 La is calculated by sodium sulphite o&idation method. )he 7 La calculated is generally higher than that is obtained from other methods because of the following reasons.o&ygen supplied to the reactor has to transfer through the gas%liquid interface. since sodium sulphite is available in the interface itself it leads to the reaction and hence higher 7La values. 7La is greatly affected by aeration rate .6ence proper aeration must be supplied to the reactor.

HEAT TRANSFER E+PT NO )ATE AIM6 )o determine the overall heat transfer heat transfer coefficient ;3< from e&periment and conforming the values using correlation. THEORY6 4riving force for the heat transfer is the temperature difference between any two bodies. )wo bodies at different temperatures when in contact tend to attain the same temperature by heat transfer. )his phenomenon is used in reactors to heat or cool the medium to desired temperature. )o determine the flow rate temperature of the heating or cooling substances, such that the operation of heating or cooling is performed effectively and efficiently. $t is necessary to know the overall heat transfer coefficient ;3<. )he transferred is proportional to the temperature gradient and inversely proportional to the area. 6ence, E _ A `) 0or1 E @ 3 A `) 0!1 )hus, by knowing the overall rate of heat transfer 0E1 we can determine ;3< .y a simple thermodynamic relation E @ mf +p 0)o B )i 1 %%%%% 0#1 Dhere, mf B mass flow rate of heating or cooling substance, kg-m s +p B specific heat capacity of heat substance, a - kg 7 )o B the outlet temperature of substance for heating or cooling, 7 )i B the inlet temperature of substance for heating or cooling, 7 mf +p 0)o B )i 1 6 6

3@

0'1

A `) 6ow `) is defined in different ways. )he best would possibly be log mean temperature difference 0L5)41 )o B )i L5)4 @ 0,1

ln b0)F%)o1- )F%)ic Dhere )f is the fermentation temperature. Also, the value of 3 depends on the nature of material across heat is being transferred, the systems geometry, the nature of fluid etc., the dependence of ;3< on all of the above is quite comple& and is evaluated using empirically. +alculation ! @ ! L ! . do L Aw . do 3 ho hi di kw di where, ho B heat transfer coefficient of liquid 0film1 outside hi B heat transfer coefficient of liquid 0film1 inside. RESULT6 )he overall heat transfer coefficient ;3< was calculated using correlation n and e&periment 3 e&pt @

3 corr @

An increase in value of heat transfer coefficient was seen with increase in rpm INFERENCE An increase in rpm allows better transfer of heat all through the liquid and hence the overall heat transfer coefficient is seems to be increased Also the outer film transfer resistant 0!-ho1 decreases due to better and vigorous mi&ing )he correlation values are lesser than that by the e&periment because of the assumption of water constant at temperate #I87 )he diameter of the agitator and flow rate were also appro&imated which would have been the cause for difference in the e&perimental and correlation values

BATCH HEAT STERILI3ATION AN) THERMAL )EATH -INETICS

E+PT$NO )ATE AIM6 )o determine the holding time of reactor in batch kinetics. THEORY6 A fermentation product is produced by a culture of certain organisms in a nutrient medium. $f a foreign microorganism invades the fermentation, the following may be the consequences )here would be competition between the foreign microorganism and the production microorganism for the same nutrient medium, thus leading to loss of productivity. $f the fermentation is of continuous mode, the contaminant may outgrow the production microorganisms, and displace it from fermentation, thus leading to the loss of money, time and energy. Final product may be contaminated. (&traction of final product may be very difficult 4egradation of the desired product may take place due to metabolism of the contaminant. )his is especially true in the case of the production of antibiotics. $f the contaminant happens to be a bacterial strain, which is resistant to the action of the antibiotic, and this antibiotic also happens to be a part of the normal metabolic pathway of the contaminant, then it leads to a problem. (g. 4egradation of % lactam antibiotics by % lactamase producing bacteria. +ontamination of bacterial fermentation with bacteriophages could result in lysis of the bacterial cells. .ut this does not occur very often. Avoidance of contamination may be achieved by 3sing a pure inoculum to start out with. Sterili9ing the medium, fermentor vessel and all the materials used in the process. 6 6

5aintaining aseptic conditions during the fermentation.

Liquid medium is most commonly sterili9ed in batch mode in the same fermentor where it will be eventually used. )he liquid is heated to sterili9ation temperature by introducing steam into the coils or the Macket of the vessel. /ther ways of doing this could be heating the vessel electrically or by bubbling steam into the medium. $f direct steam inMection is used, allowance must be made for dilution of the medium by condensate, which typically adds !8%#8J to the liquid volume quality of the steam must also be high enough to avoid contamination of the medium by metal ions or organics. 4epending upon the rate of heat transfer from the steam or electrical element, raising the temperature of the medium in large fermentors can take a significant period of time. /nce the holding or sterili9ation temperature is reached, the temperature is held constant for the period of time thold. +ooling water in the coils or Macket of the fermentor is then used to maintain the temperature. For operation of batch sterili9ation systems, we must be able to estimate the holding time required to achieve the desired level of cell destruction. As well as destroying contaminants, heat sterili9ation may also destroy the nutrients in the medium. )o minimi9e this loss, holding times at the sterili9ation temperature must be kept as short as possible. +(LL 4(A)6 7$*()$+S $n a lethal environment, cells in a population do not all die at once. 4eactivation of the culture occurs over a finite period of time depending on the initial number of viable cells and the severity of conditions imposed. Loss of cell viability can be described mathematically as follows. +ell death is assumed to be a first order process rd @ %d*-dt @ kd * 0!1 Dhere rd is the cell death rate, * is the number of variable cells, and kd is the specific death rate. $nstead of *, we can also e&press the rate of cell death in terms of biomass 0A1 of variable cells. $f kd is constant we can integrate eqn, 0!1 as follows to derive an e&pression for * as a function of time.
* *o t

d 0!-*1 d* @ kd dt dt
o

0#1

)o get ln *t @ ln *o%kdt 0'1 0%k t1 *t @ *o e d 0,1 )hus, if first order kinetics applies, a plot of ln * t versus t gives a straight line with slope kd. 6owever, first order kinetics does not always hold, particularly for bacterial spores immediately after e&posure to heat. )he value of k d is not only species dependent, but also dependent on the physiology of the cell itself. For eg. Bacillus stearothermophilus is the most heat resistant bacteria known.

Like other kinetic constants, the specific death constant also depends on temperature, ). this effect can be described using the Arrhenius relationship 7d @ Ae0%(a-2)1 0F1

Dhere A is the Arrhenius constant or frequency factor, ( a is the activation energy for thermal cell death and 2 is the universal gas constant. 4(S$G* +/*S$4(2A)$/*S 4(L FA+)/2 +ombining eqns ' : F, we get ln 0*o - *t1 @ Ae 0%(a-2)1R t 0H1 )his term, ln 0*o-*t1, has been described as 6umphrey and 4eindoerfer as the 4el factor, , or the *ebla factor or the sterili9ation criterion. )hus, the 4el factor is a measure of the fractional reduction in viable organism count by a certain heat and time regime. 2earranging 0G1, we get ln t @ (-2) L ln 0-A1 0O1 )hus, a plot of ln t versus ) world yield a straight line of slope (-2 and y intercept ln 0-A1. )hus, the same degree of sterili9ation may be obtained by heating the substance at a high temperature for a short time, or a low temperature for a long time. *3)2$(*) 4(S)23+)$/* )wo types of reactions contribute to loss of nutrient quality during sterili9ation. $*)(2A+)$/*S .()D((* S/5( *3)2$(*) +/5"/*(*)S /F )6( 5(4$35 A common reaction during sterili9ation is the 5aillard type browning reaction. )his results in discoloration of the medium as well as loss of nutrient quality. )hese reactions are normally caused by the reaction of carbonyl groups, usually from reducing sugars with the amino acids of proteins. Sterili9ing the sugar separately and mi&ing it with the rest of the sterili9ed medium after cooling resolve this problem. 4(G2A4(4 6(A)% LA.$L( +/5"/*(*)S +ertain amino B acids and proteins may be degraded during a heat sterili9ation regime. )hermal destruction of nutrient components conforms with first order kinetics and may be degraded by a from similar to the Arrhenius equation 0At-Ao1 @ e 0%kt1 0H1 =alues of activation energy (a for thermal destruction of vitamins and amino acids are H, to I# ka-gmol, for proteins it is about !GF ka-gmol. As these values are a bit lower than that of microorganisms, raising the temperature has a greater effect on cell death than nutrient destruction. )his means that sterili9ation at higher temperatures 0and shorter times1 has the advantage of killing cells with limited destruction of medium components. )6( "2/.A.$L$)> /F +/*)A5$*A)$/*, *

Let *o denote the number of contaminants present in the raw medium . 4uring the heating period, the holding period and at the end of the cooling period, the final number is reduced to *. $deally * should be 9ero. .ut this is practically impossible to achieve, as absolute sterility would take infinitely long to reach. *ormally the target level of contamination is e&pressed as a probability of contamination, and this value is fi&ed at 8.88! i.e. one batch in every !888 is e&pected to be non%sterile. )o apply this kinetics, we need to know some of the thermal death characteristics of the bacteria present in the medium. )he assumption that Bacillus stearothermophilus one of the most heat resistant bacteria known, is the only microbial contaminant present, is a safe one0we cannot practically calculate the average thermal death kinetic values of every known ta&on that might constitute a contamination1.)hus a considerable safety factor can be built into the design by adopting B.stearothermophilus as the design organism. )he death characteristics of this organism are (a @ #H'888 a-mol 0I1 'O A @ I.F R !8 - min De must keep in mind that these values will vary considerably depending on the type of medium used. )his is particularly relevant when considering the sterili9ation of oils and fats 0common fermentation substrates1 where relative humidity may be relatively low 0spores of .. stearothermophilus are ten times more resistant when dry, then when wet1. 6/L4$*G )$5( 4(S$G* $f *o and * known. De can determine the holding time required to reduce the number of cells by considering the kinetics of cell death. total @ heating L holding L cooling total @ ln 0*o-*1 0!81 from eqn 0G1

De know that for the organism B.stearothermophilus, a good estimate of the number cells per unit volume of reactor would be @ !8G spores - mL )he reactor volume we had was @ OF8mL )hus, initial number of organisms we start out with @ *o @ !8G R OF8 @ O.F R !8H heating and cooling are collected as follows De know that heating @ k R t k@Ae 0%(a-2)1

from eqns 0F1 : 0G1 eqn 0F1

For the organism .. stearothermophilus (a and A are known. De also have a set of readings of ) 0temperature1 vs. t 0time1 for heating and for cooling. )hus for each value of ), we can get the corresponding value of k 0from eqn.F1 $f we now plotted k against t, we would obtain a graph whose A3+ 0or area under the curve1 is @ kR t, which in turn, is equal to . 4epending on which set of readings we took of ) vs. t 0whether the heating or the cooling1 we would get heating or cooling.

)hus, from eqn !8. we can calculate holding. De know that the holding temperature is going to be !#!e+. from this, we can calculate the corresponding value of k and t hence, the holding period for the sterili9ation. */* B 6/5/G(*/3S 5(4$A )hese design procedures apply to batch sterili9ation of medium when the temperature is uniform throught the vessel. 6owever, if the liquid contains contaminant particles in form of flocs or pellets, temperature gradients may develop. .ecause heat transfer within solid particles is slower than in the liquid, temperature at the center of the solid will be lower than that in the liquid for some portion of the sterili9ing time. As a result, cell death inside the particles is not as effective as in the liquid. Longer holding times are required to treat solid% phase substrates and media containing particles.

PROCE)URE6 )he e&perimental batch data is obtained using a #L batch fermentor. $t is filled to the OF8mL mark with water. )he reactor is heated using a heating finger, and temperature was noted down regularly. A point to remember at this stage the safety valve has to be removed during the heating stage to prevent reactor bursting. /nce the temperature has reached !#!e+, the heating element is disconnected, and cooling is begun. /nce again, temperature is noted down regularly. De must remember to plug safety valve back in again at this cooling stage to prevent a vacuum from developing inside the reactor, which would induce it to suck in 0non%sterile1 air from outside. )his is the first part of the e&periment. $n the second part, !8ml culture was e&posed to a temperature of G8 e+ for F min, !8 min and !F min. At the end of each time interval, ! mL of culture was withdrawn and plated in L. plates at dilutions ! !8#, ! !8,, and ! !8G. )he same procedure was repeated for H8K+. )he plates were incubated overnight and the colonies were counted the ne&t day.

RESULT6 )he holding time and thermal death rate constant 0kd1 of batch heat sterili9ation are determined INFERENCE6 )hus tholding can be considerably reduced by considering heating and cooling parts of cycles in the sterili9ation process. )he main aim of designing a process is to achieve the probability of obtaining sterility with minimum loss of nutritive quality. 6ence, e&posure of medium is kept to be

minimum. An important observation is that the del factor does not include the absolute number of contaminant concentration and concentration of survival. 5oreover , the holding time is kept as minimum as possible since heating sterili9ation may also destroy the medium nutrients. )hus by including heating and cooling cycles ,the holding times are kept minimum which minimi9es the loss

RESIS)ENCE TIME )ISTRIBUTION

E+PT NO )ATE AIM6 )o calculate the residence time of the given reactor by 0i1 pulse input 0ii1 step input. THEORY6 A fluid flowing in a reactor takes different times to pas through the reactor. )he distribution for these streams of liquid leaving the vessel is called (&it age distribution (, or the 2esidence )ime 4istribution, 2)4 of the fluid. ( has the unit time%!. )he amount of fluid coming out of the reactor in a given time interval is `* @ E.+`) Dhere, E @ =olumetric flow rate 0hr%!1 + @ concentration 0g-l1 `) @ total amount of substance 0g1 $f +o is the total amount of input, then the fraction e&iting *-*o @ E.+.)-*o )he 2)4 function can be e&pressed as ( 0t1 @ E.+-*o @ E.+- dE.+dt @ +-+o Dhere +o @ d+dt $t is convenient to e&press the area in such a way that the area under the curve is unity. d( 0t1. dt @ ! )his procedure is called normali9ing the distribution. )here should be no flow diffusion or up flow eddies at the entrance, or at the e&it of the reactor. )his is called the closed reactor boundary condition. 6 6

Dhich this representation, the fraction of e&it steam of age between t and t L dt is (.dt. )he fraction younger than t is d( 0t1 dt and the fraction of the stream older than t is ( 0t1 dt @ !%d( 0t1 dt )6( "3LS( (A"(2$5(*) )o find the ( curve for a vessel of volume =m' through which flows v m '-sec of fluid, instantaneously introduce into the fluid entering this reactor, 5 units of tracer 05oles or kg1. 2ecord the concentration%time profile to tracer leaving the reactor. )his is the + pulse curve. $n this case the tracer used was ! g-l of glucose. Area under the curve, A @ +dt @ +iti @ 5-v 5ean of the pulse curve, t @ f t+dt f +dt 4ividing the concentration readings by 5-v, we get, ( 0t1 @ +pulse 5-v Average time-resistance time )m @ d( 0t1 tdt 0( 0t1 dt @ !1 PROCE)URE6 )he e&periment aimed at calculating the resistance time of a '%liter reactor. )he reactor is filled with distilled water. /&alic acid 0!.8*1 was used as tracer and !8.8 ml of tracer was added 0"ulse input1 to the reactor. )he reactor was started. Samples were withdrawn at regular intervals. )he concentration of tracer in each sample was found out by!8.8 ml with 8.!* *a/6. )he + =s t profile was plotted. $nitial +oncentration of tracer +o @ +o@ RESULT6 )he residence time of the given reactor by 0i1 pulse input 0ii1 step input are determined. INFERENCE6 2)4 of a material flowing through the vessel, along with the state of aggregate of the following materials and their earliness or lateness of the mi&ing factor Dhich wake up the countacting or flow pattern. $n order to predict the behaviour of the vessel. )he pulse input e&periment using a nonreactive tracer was used )he graph of (0t 1 =s t and t (0t1 =s t are also plotted mg- lit