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Chap 8 Bioreactors

Introduction Applications of bioreactors: for production of vaccines, proteins, organics acids, amino acids and antibiotics; enzymatic or microbial biotransformations; bioremediation, etc. A production facility usually has a train of bioreactors ranging from 20 L to 250,000 L. he bioreactors are arranged in the series of increasing sizes, starting from small cultures to the final production culture.

I. Bioreactor configurations
Stirred tank reactors !eatures: "icrobial reactors generally have # baffles from the $alls to prevent vorte%ing of the fluid, the baffle $idth is &'&0 or &'&2 of the tan( diameter. he vorte% and circular flo$ result in little mi%ing bet$een fluids at different heights. At high speeds the vorte% may reach do$n to the impeller so that gas from the surrounding is dra$n into the li)uidhigh mechanical stress in the stirrer shaft, bearings and seal. *ioreactors for animal cell cultures usually do not have baffles +especially for small scale reactors, to reduce turbulence. he aspect ratio +height-to-diameter ratio, of the vessel is .-5 for microbial cultures but is normally less than 2 for animal cell culture. /parger: gas is sparged at the bottom using a perforated pipe ring sparger. 0umber of impellers depends on the aspect ratio. he bottom impeller is located at a distance about &'. of the tan( diameter above the bottom of the tan(. Additional impellers are spaced appro%imately & to 2 impeller diameter +d, distances apart. &'. 1
&-2 d

&

he superficial aeration velocity +the volume flo$ rates of gas divided by the crosssectional area of the vessel, in stirred vessel must be lo$er than that can flood the impeller +an impeller is flooded $hen it receives more gas than it can effectively disperse, other$ise the mi%ing is poor. /uperficial aeration velocities generally do not e%ceed 0.05 m's.

2mpellers: choice often depends on the viscosity of the li)uid and sensitivity of the cells to mechanical shear. 3ushton +4-flat-blade, disc turbine +a, and concave bladed impeller +b,: impeller diameter is about &'. of the vessel diameter and is often used for bacterial cultures. 3ushton turbine is most commonly used in fermentation technology. 5ydrofoil impeller +c,: diameter is about 0.5 to 0.4 times the tan( diameter and is an effective mi%er for highly viscous mycelial broths. "arine impeller +d,: usually single, large diameter, lo$ shear, used for animal cell culture.

!lo$ pattern of 3ushton turbine 2mpeller speed:

!lo$ pattern of marine impeller +promotes a%ial flo$,

6sually 7&20 rpm for animal cell cultures even for vessels 850 liters. 5igher stirring speeds can be used for microbial cultures. he impeller tip speed +..&# 9 impeller diameter 9 speed of rotation, is usually less

than :.4 m's for filamentous fungi.

;o$er re)uirement: critical for large-scale bioreactors /maller for sparged reactors because +&, gas bubble decreases the li)uid density; +2, gas-filled cavities develop behind the stirred blades, $hich reduce the resistance to fluid flo$ and decrease the drag coefficient of the impeller.

P0 = N p l N i Di5
.

for ungassed 0e$tonian fluid

;0: po$er input +<, l: li)uid density; 0i: rotational speed +&'s,; 1i: diameter of impeller 0p: po$er number +related to 3eynolds number,

Pg P0
fluid

= 0.&0 +

Fg NiV

0.25

N i2 Di# 0.20 + , gWiV 2 ' .

for gassed 0e$tonian

;g: po$er consumption $ith sparging; !g: volumetric gas flo$ rate; =: li)uid volume; g: gravitational acceleration; <i: impeller blade $idth Bubble columns

Air

6sually the height-to-diameter ratio is #-4. >as is sparged at the base through perforated pipes or plates or metal porous spargers. ?2 transfer, mi%ing and other performance factors are influenced mainly by gas flo$ rate and rheological properties of the fluid. "i%ing and mass transfer can be improved by placing perforated plates or vertical baffles in the vessel.

Airlift bioreactors

/eparated as t$o zones: the sparged zone is called the riser, and the zone that receives no gas is the do$ncomer.
the do$ncomer,.

he bul( density in the riser region is lo$er than that in the

do$ncomer region, causing the circulation +so circulation is enhanced if there is little or no gas in !or optimal mass transfer, the riser to do$ncomer cross-sectional area ratio should be bet$een &.@ and #... 5ighly energy efficient and productivities are comparable to those of stirred tan( bioreactors.

he rate of li)uid circulation increases $ith the s)uare root of the height of the airlift device. Aonse)uently, the reactors are designed $ith high aspect ratios. A gas-li)uid separator in the head-zone can reduce the gas carry-over to the do$ncomer and hence increase the li)uid circulation.

Fluidized bed reactors /uited for reactions involving a fluid-suspended particulate biocatalyst such as immobilized enzyme and cell particles /imilar to a bubble column e%cept that the top section is e%panded to reduce the superficial velocity of the fluidizing li)uid to a level belo$ that needed to (eep the solids in suspension. Aonse)uently, the solids sediment in the e%panded zone and drop bac(, hence the solids are retained in the reactor $hereas the li)uid flo$s out. Packed bed bioreactor A bed of particles are confined in the reactor. particles. A fluid containing nutrients flo$s through the bed to provide the needs of the immobilized biocatalyst. "etabolites and products are released into the fluid and removed in the outflo$. he flo$ can be up$ard or do$n$ard. 2f up$ard fluid is used, the velocity can not e%ceed the minimum fluidization velocity. he biocatalyst +or

cell, is immobilized on the solids $hich may be rigid or macroporous

II. Bioreactor design features

*io!lo 4000B /terilizable-2n-;lace !ermentor-*ioreactor (50 - 130 Liters) *io!lo &&0, &.. to &# liters

=ertical sight glass and ports for p5, temperature and 1? sensors +4,. Aonnections for acid and al(ali +for p5 control,, antifoam agents and inoculum are located above the li)uid level in the reactor vessel +&4,. ?2 and other gases +A?2 or 05. for p5 control; 02 for ?2 control, can be introduced through a sparger at the bottom +@,. !oam brea(ers +22, are used $hen antifoam is ineffective or the antifoam interferes $ith do$nstream processing +antifoam tends to foul the membrane during filtration,. Aan be sterilized in-place using saturated steam +&#, at a minimum absolute pressure of 2&2

(;a. ?verCpressure protection is provided by a rupture disc +2#, on the top of the reactor, $hich crac(s to relieve the pressure to avoid e%plosion. he vessel should have as fe$ internals as possible and should be free of stagnant areas $here poc(ets of solids or li)uids may accumulate. "a%imum allo$able $or(ing pressure is .::-#&2 (;a +absolute,&, allo$able temperature is usually &50-&@0A +8&2&A for sterilization,. he vessel should $ithstand

full vacuum or it could collapse $hile cooling after sterilization. 6sually made in ype .&4L stainless steel, $hile the less e%pensive ype .0# +or .0#L, is used for the Dac(et. $elds. he L grades contain less than 0.0.E carbon, $hich reduces chromium carbide formation during $elding and lo$ers the potential corrosion at the

III.

Design for sterile operation

Sterilization-in-place A bioreactor must be sterilized before inoculation because contamination is a common cause of process failure. !or large bioreactors, in situ sterilization is common. he components should be able to be sterilized during he filters are rated for independently

fermentation if re)uired. he aeration and e%haust groups must also be sterilized. removing particles do$n to 0.22 m or even 0.& m. ?ften the gas streams re)uire t$o filter cartridges in series, $ith the first serving to protect the second filter.

&

& atmF&.0&&05 ;aF&0& (;a

Clean-in-place (CIP) considerations

2ndustrial bioreactors should be cleaned in-place using automated methods, so as to ensure consistency and reduce do$n-time. o remove solid particles and avoid sedimentation, a flo$ velocity of 2 m's is preferred. Also, the piping should be free of dead space as much as possible. !or thorough cleaning, the A2; solutions are sprayed through a spray ball. !or cleaning $ith Det spray, pressures of .0@ to .:: (;a +absolute, are optimal.

he piping for air inlet and e%haust should also be cleaned. ;rocedures: &. 2. .. #. ;re-rinse for 5 min $ith deionized $ater +sufficient for bacteria, yeast and animal cell cultures,. Airculate &E +$'v, 0a?5 at :5-@0A through all product contact surfaces for &5-20 min. 1iscard the solution after$ards. 3inse at 25-.5A $ith deionized $ater to remove all al(ali. !inal $ash $ith hot G$ater-for-inDectionH grade $ater.

!or stirred tan( bioreactors, it is recommended to fill the vessel and agitate at 3eynolds numbers of &0@-&0@.5 during pre-rinse, al(ali recirculation and the final rinse for 2-. min +should be sufficient to dislodge adhering dirt or soil,.

ote! 1isposable bioreactors +e.g. <ave bioreactor, *elloAell, are gaining increasing interest due to smaller capital investment, easier operation and elimination of the A2; process.

*elloAell 500 A; <ave *ioreactor +&00-500 L, >I 5ealthcare, http:''$$$.$avebiotech.com', <ater for 2nDection +<!2,: high )uality $ater subDected to the follo$ing treatment: membrane filter +5-20 m, +Aesco *ioengineering,
http:''$$$.cescobio.com.t$'products.phpJtidF&

Activated carbon +organic and other contaminants are adsorbed, 2on e%change +cation L anion,

d2 $ater +purified $ater,

3everse osmosis

distillation

<!2

<!2

0ote: &. 2on e%changer need to be regenerated regularly by 5Al and by 0a?5 2. 3everse osmosis removes viruses, microorganisms, pyrogen and virtually all inorganic impurities.

Appendix IV.

ass transfer steps


2n bioreaction processes, substrates are consumed for the conversion. acids, and electron acceptors +e.g. ?2,. ypical substrates include carbon sources +e.g. sugar and oil,, nitrogen sources +e.g. ammonia and amino

"ffects of transfer limitations #$o effects if one step is slo$er than the ke% kinetic reaction step! &. he overall reaction rate is belo$ the theoretical ma%imum, and the process output is slo$er than desired. 3eversible effect: for the production of gluconic acid + , from glucose by Gluconobacter oxydans, the ?2 transfer is limiting. ?nce ?2 limitation is relieved, there is no irreversible effect on this microorganism. 2. 2rreversible effect: for the production of penicillin, ?2 limitation imposes an irreversible damage to the biosynthetic capacity of the cell. he selectivity of the reaction is altered. I%: ?2 serves as an electron acceptor in the formation of ba(erMs yeast from glucose. 2n the absence of ?2 the e- $ill be directed to pyruvate resulting in the formation of ethanol and A?2.

&0

ransfer of o%ygen involves a chain of mass transfer steps from a gas bubble.

he

slo$est one is the rate-limiting step and determine $hether the mass transfer rate $ould affect the overall process performance. <hen cells are $ell dispersed in the li)uid and the bul( li)uid is $ell mi%ed, step +iii, is the limiting step. #ransfer across the cell en&elope ransport across the cell envelope +may include cell $all and cytoplasmic membrane, can be limited. hree typical mechanisms: &. 2. .. !ree diffusion: passive transport do$n a concentration gradient !acilitated diffusion: as above but speeded up by a carrier protein Active transport: transport by a carrier protein $ith input of free energy

he diameter of microbial cell itself is small +usually &-5 m, so diffusion inside the cell is more rapid and not a limiting factor. he transport barrier imposed by the membranes of intracellular organelles in eucaryotic cells usually does not limit the overall transport rate.

V.

ass !ransfer "#uations

Fundamentals! !ic(Ms I)n. NF-1dA'd% +at steady state, N: molar mass flu% +mol'm2s, 1: diffusion coefficient +m2's, A: concentration of the substance to be transported

&&

%: distance Aonsidering transport in solid phase, 1 is the effective diffusion coefficient $hich lumps the diffusion coefficient, the porosity of the solid, and the shape of the channels. !or a flat plate $ith thic(ness d in a stationary fluid NF1A'd <here 1'd represents the mass transfer coefficient, $hile d'1 can be interpreted as the resistance against transport. !or unsteady state:

2 C C D = 2 t x
hese e)uations consider the diffusion process only but not convection. 2n reality, convection is often encountered and flo$ pattern is not (no$n, thus the mass transport often relies on empirical approach. 'ass transfer bet$een l-s phase or l-( phase (t$o film theory) !or gas film transport:
J g = k g + P Pi ,
+mass transfer rate, not flu%,

Li)uid film transport:


J l = k L a +C i C ,
+the main resistance to o%ygen transfer,

(L: li)uid phase mass transfer coefficient +m'h, ;i Ai but can be correlated by 5enry coefficient 5 +barm.'mol,: Pi=HCi !rom here, the volumetric mass transfer rate +N, mg'h'l,, can be derived
J = k L a +C O C ,

a +m2'm.,: the gas-li)uid interfacial area per unit li)uid volume, or area per unit gross vessel volume in the bioreactor.

&2

<hen dealing $ith the transfer of ?2 from gas to li)uid, N is called the ? 3 +o%ygen transfer rate, (La +&'h,: volumetric transfer coefficient, because a and kL are difficult to evaluate separately, (La is often e%pressed together. =arious e%pressions of (La can be found in literature. he value is typically 0.020.25 s-& AO: saturated 1? +in the case of o%ygen transfer, concentration +g'l,, i.e. the solubility of ?2 A: actual 1? concentration in the broth 0ote: mole fraction of ?2 in air is 0.20KK, so the partial pressure of ?2 at & atm air pressure is 0.20KK atm. *ased on 5enryMs la$, the solubility of o%ygen in $ater under & atm air pressure is 0.20KK times that under & atm pure ?2. +g'l,
o

?%ygen upta(e rate:


!" =
"

x +g'lh,

: specific o%ygen upta(e rate +g'gs,,

%: cell concentration

varies $ith cell species and nutritional environment


o

!or A8 Acrit +critical o%ygen concentration,, !or A7 Acrit ,


o

is a constant ma%imum

+usually 5-&0E air saturation, is appro%imately linearly

dependent on A. At steady state,


k L a +C O C , =
o

his e)uation can be used to predict the response of fermenter to changes in mass transfer operating conditions, for e%ample, if (La is raised by increasing stirrer speedA must rise. 'easurement of k)a &. ?%ygen-balance method: +based on steady state measurement, at s.s.
J = & ++ Fg C , i + Fg C , o , VL

$here l.h.s: rate of o%ygen transfer from gas to li)uid r.h.s.: difference in o%ygen flo$ bet$een inlet +subscript i, and outlet +subscript o,. =L, volume of li)uid; !g, volumetric gas flo$ rate; A, gas phase concentration of ?2 *ecause gas phase concentrations are usually measured as partial pressures, ideal gas

&.

la$ can be incorporated:


J = Fg p Fg p & ++ ,i + ,o , $V L # #

$here p is the partial pressure at the inlet and outlet. he difference bet$een pi and po is usually small thus pi and po need to be measured very accurately +e.g. by mass spectrometry,. !or (no$n p, !g, =L, 3 and , N is obtained, and (La can be calculated from
J = k L a +C O C , $hen AO and A are measured.

2.

1ynamic method: +based on unsteady state measurement, At time t0, the broth is de-o%ygenated by sparging 02 or by stopping the air flo$ if the culture is consuming ?2 A drops Air is then pumped into the broth at a constant flo$ rate A increases and reaches a s.s. value, C . A& and A2 are t$o o%ygen concentrations measured at t & and t2. 1uring the re-o%ygenation step +unsteady state,
dC = k L a+C O C , dt
o

At s.s., k L a+C O C , =
dC = k L a +C C , dt o

x is substituted into the above e)uation to yield

Assume (La is a constant, integrating the above e)uation from t& to t2 yields

ln+ , C C2 kLa = t 2 t&

C C&

&#

*as-)i+uid mass transfer in real s%stems (,- transfer) *ubble size is critical in determining o%ygen transfer, smaller bubbles leads to: higher a slo$er bubble rise velocity higher gas hold-up +volumetric fraction of gas in the li)uid,.
VG V L+VG

VG: volume of gas bubbles in the reactor, VL: volume of li)uid

$o e%treme cases: Aoalescing + , li)uid +a li)uid $hich greatly stimulates bubble coalescence,: mass transfer is poorest 0on-coalescence fluid: highest mass transfer.

%tirred tank reactor& /parged gas is usually rapidly collected in the gas cavities behind the rotating impeller blades. bubbles. he cavities flo$ in a highly turbulent vorte% and the gas is dispersed into smaller hese follo$ the li)uid flo$, but $ill also rise to the surface. hey $ill coalesce

in areas that are relatively calm and re-disperse in places $here the shear stress is high. A part of the bubbles is re-circulated into the cavities and the rest escapes at the surface. Impirical values for gas hold-up
= 0.&. + P ' V , 0... +' g p 0 ' p , 0.4:

+for coalescing fluid,

$here +;'=,Fpo$er input per unit volume, pFpressure in the system Impirical e)uations of (La: +coarse e)uations $ith accuracy.0E, valid for ;'=F0.5&0 (<'m.,.
k L a = 0.024 + P ' V , 0.# +' g p 0 ' p, 0.5

for coalescing fluid +e.g. clean air-$ater system,

k L a = 0.002 + P ' V , 0.: +' g p 0 ' p , 0.2 for non-coalescing fluid +e.g. fermentation broth,

'g, superficial gas velocity ; po, reference pressure of & bar. (ubble colu)n& <hen air flo$ rate is high enough,
= 0.4+' g p o ' p , 0.:
k L a = 0..2 +' g p o ' p , 0.:

&5

2n non-coalescing li)uids +e.g. some fermentation broths,, the bubble rises and does not mi% $ith other bubbles, provided that the size is smaller than the e)uilibrium average diameter 4 mm.

2n a large bubble column +850 m.,, the bubbles $ill significantly e%pand as they rise because of the decreasing hydrostatic pressure, $hich influence the mass transfer. (L: usually .-#&0-# m's for bubbles82-. mm diameter, could be do$n to &&0-# m's for smaller bubbles, depending on bubble rigidity

*ir+li,t reactor& Although the riser resembles a bubble column, the gas-hold up is lo$er than predicted by the above e)uation due to the interaction $ith the li)uid flo$. Aorrespondingly, (La $ill be lo$er, up to a third of the bubble column. A precise )uantification, ho$ever, cannot be easily made.

&4

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