Material Balances

One ofthe simplest concepts in process engineering is the material or mass balance. Because mass in biological systems is conserved at al! times, the law of conservation of rnasspro vides the theoretical framework for material balances. In steady-state material balances, masses entering a process are summed up and compared with the total mass leaving the system; the term balance implies that masses entering and leaving should be e!ual. "ssentially, material balances are accountingprocedur#s$ total mass entering must be accounted for at the end ofthe process, euen ifit undergoes beating, mi%ing, drying, fermentation or any other operation &e%cept nuclear reaction' within the system. (sually it is not feasible to measure the masses and compositions of all streams entering and leaving a system; unknown !uantities can be calculated using mass-balance principies. )ass-balance problems have a constant ibeme$ giuen the masses ofsome input and output streams, calculate the masses of others.

Mass balances provide a very powerful tool in engineering analysis. Many complex situations are simplified by looking at the movement of mass and equating what comes out to what goes in. Questions such as: what is the concentration of carbon dioxide in the fermenter off-gas? what fraction of rhe substrate consumed is not converted into products? how much reactant is needed to produce % grams of product? how much oxygen must be provided for this fermentation to proceed? can be answered using mass balances. his chapter explains how the law of conservation of mass is applied to atorns! molecular species and total mass! and sets up formal techniques for solv ing material-balance problems with and without reaction. "spects of metabolic stoichiometry are also discussed for cal culation of nutrient and oxygen requirements during fermentation processes.

real and tangible! such as the walls of a beaker or fermenter! or imaginary. #f rhe boundary does not allow mass to pass from system to surroundings and vice versa! the system is a closed system with consrant mass. $onversely! a system able to exchange mass with its surroundings is an open system. " process causes changes in the system or surroundings. %everal terms are commonly used to describe processes. (i) " batch processoperates in a closed system. "ll materials are added to the system at the start of the process& the system is then closed and products removed only when the process is complete. 'ii( " semi-batch processallows either input or output of mass! bur not both.

4.1

hermodynamic Preliminaries

Figure ).*

hermodynamic system.

*hermodynamics is a fundamental branch of science dealing with the properties of matter. hermodynamic principies are useful in setting up material balances& sorne terms borrowed from thermodynamics are defined below.

Surrounding

----System

4.1.1 System and Process

#n therrnodynamics! a system consists of any matter identified for investigation. "s indicated in +igure ).*! rhe system is set apart from the surroundings, which are the remainder of the uniuerse, by a system boundary. he system boundary may be

System boundary

) Material -alances

'iii( " ftd-batch process allows input of material to rhe system

but not output. 'iv( " continuous process allows matter to fiow in and out of rhe systern. #f rates of mass input and output are equal! continuous processes can be operared indefinitely.

change in both rhe system and the universe can occur! urge scale equilibrium does not ofcen occur in engineering systems: steady srares are more common.

4.2 Law of

onser!ation of Mass

).*.. Steady State and Equilibrium

#f all properries of a sysrern! such as rernperature! pressure! concenrration! volume. mass! etc. do not vary with time! rhe process is said to be ar steady state. hus! if we monitor any variable of a sready-srare sysrem. its value wil# be unchanging with time. "ccording to this definirion of steady state! batch! fed batch and semi-batch processes cannot operate under sready-state conditions! Mass of the systern is either increasing or decreasing wirh time during fed-batch and semi-batch pro cesses& in batch processes! even though rhe total mass is consrant! changes occurring inside the system cause che systern properties to vary wirh rime. %uch processes are called transient or unsteady-state processes. /n rhe other hand! conrinuous processes may be eirher sready srare or transient. #r is usual ro run continuous processes as close ro steady stare as possible& however! unsready-srare conditions will exist during starr-up

Mass is conserved in ordinary chemical and physical processes. $onsider the systern of +igure ).. operating as a conrinuous process wirh input and output streams conraining glucose. he mass flow rate of 0lucoseinto rhe :ystem is,Mi kg h - *& the mass fiow rate out is ) o kg h -1. #f Mi and )o are differenr! rhere are four possible explanations: 'i(
'ii(

rneasuremenrs of Mi and M o are wrong& the system has a leak allowing glucose ro enter or escape undetected: 'iii( glucose is consumed or generated by chemical reacrion wirhin che system& or 'iv( glucose accumulates within che systern.

#f we assume thar rhe measurements are correct and there are no leaks! rhe difference berween Mi and )o must be due ro consumprion or generation by reacrion! and1or accumularion. " mass balance for rhe system can be written in a general way ro account for rhese possibiliries: through 4 rhrough + gen.er,tcd 4 on!su,cd accu!mulated

ing analysis. 3owever! it is ofren confused wirh another and for sorne rime afrer any change in operaring condirions! %teady state is an important and useful concept in engineer rherrnodynamic terrn! e!uilibrium. " sysrem at equilibrium is one in which all opposing forces are exacdy counrer-balanced so rhat the properties of the system do not change with time. +rom experience we know rhar systems tend ro approach an equilibrium condirion when rhey are isolated from their sur roundings. "t equilibrium there *6 no net change in either rhe systern or the universe. 7quilibrium implies rhar there is no net driving force for change& rhe energy of the systern is at a mini mum and! in rough terrns! the system is 8static8! 8unrnoving8 or 8inerr8! +or exarnple! when liquid and vapour are in equilib rium in a c#osed vessel! although rhere may be constant exchange of molecules between rhe phases! rhere is no ner change in either the system or the surroundings. o converr raw materials inro useful products there must be an overall change in che universe. -ecause systems ar equi librium produce no net change! equilibrium is of little value in processing operations. he best strategy is to avoid equilib rium by continuously disturbing che system in such a way chat raw material wi3 always be undergoing transformation into

2 l 2 l 2 l 2 l
5
mass in mass out mass

mass

mass

the desired product. #n continuous processes at steady state! mass is constantly exchanged with the surroundings& chis dis turban ce drives the system away from equilibrium so thac a net

) sysrcrn Material -alances s98lem

boundaries boundaries

wirhin sysrem

wirhin systern

wlrhlIl'

sysrem

').*( he accumulation term in che aboye equation can be either positive or negarive: negarive accumulation represenrs deple rion of pre-exisring reserves. 7q. ').*( is known as che general mass-balance e!uation. he mass referred ro in :he equation can be total mass! mass of a particular molecular or arornic spe cies! or biomass. ;se of 7q. ').*( is illustrared in 7xample ).*.

+igure )..

+low sheet for a mass balance on glucose!

M;

( g ho l glu!ose)

%ystem

...... M"

( g hol glu!ose)

... -

7xample ).* <eneral mass-balance equation

" continuous process is ser up for treatment of wastewater! 7ach day! *=6 g cellulose and *=> kg bacteria enter in rhe feed stream! while *=) kg cellulose and *.6 ? *=) g bacteria leave in the effluent. he rate of cellulose digesti@n by the bacteria is A x *=) kg d -*. he rate of bacterial growth is . x *=) kg d - *& the rate of cell death by lysis is 6 x *=. kg d - l. B rite balances for cellulose and bacteria in the systern. +olution$ $ellulose is nor generated by the process! onlyconsumed. ;singa basis of* day! the cellulose balance in kg from 7q. ').*( is: 'l=6 - *=) + / - A ? *=)( " accumulation. herefore! . ? *=) g ccllulose accurnulates in the systern each day. Cerforming the same balance for bacteria:

herefore! 6.6 # *=$ kg bacterial cells accumulate in rhe systern each day.
---4.
...

4---------

)...*

ypes of Material -alance

he general mass-balance equation ').*( can be applied with equal case to rwo different types of mass-balance problem! depending on the data provided. +or conrinuous processes ir is usual to collect information about rhe process referring to a particular instant in rime. "mounts of mass entering and leav ing the system are specified using flow rares! c.g. molasses enters the system ar arate of 6= lb h -1; at rhe same instant in time! fermentation broth lcaves at arate of .= lb h - *. hese two quantities can be used directly in 7q. ').*( as the input and output terms. " mass balance based on rares is called a

continuous process is at steady state! the accumulation term on rhe righr-hand side must be Dero. his follows from rhe def inition of sready state: beca use all properties of rhe systern! including irs mass! rnust be unchanging wirh time! a sysrern at steady state cannot accumulate mass. ;nder these condirions! 7q. ').*( becomes: mass in + mass generared " mass out

+ mass consumed.
')..(

diffirential balance.
%n alternative approach is rcquired for batch and semi batch processes. #nformation about these systems is usually collected over a period of time rather than at a particular instant. +or example: *== kg subsrrate is added to the reactor& after > days8 incubarion! )6 kg product is recovered. 7ach terrn of rhe rnass-balance equation in this case is a quantity of mass! not arate. his type ofbalance is called an integral balance. #n this chapter! we will be using differential balances for continuous sysrerns operating at sready state! and integral bal ances for batch and semi-batch systems between initial and final states! $alculation procedures for rhe rwo types of material balance are very similar.

).... %implification of the <eneral

Mass--alance 7quation
7q. ').*( can be simplified in certain situations. #f a

7q. ')..( is called rhe general steady-state mass-balance e!ua tion. 7q. ')..( also applies over the entire duration of batch and fed-batch processes& 8rnass out8 in this case is the total mass harvested from the system so rhar at the end of the process rhere is no accumulation. #f reaction does not occur in the system! or if rhe mass bal ance is applied to a substance rhat is neither a reacrant nor product of reaction! the generarion and consumption terrns in 7qs ').*( and ')..( are Dero. -ecause total mass can be neither created nor destroyed excepr in nuclear reaction! generation and consumption terrns must also be Dero in balances applied to total mass. %imilarly! generation and consumption of atom ic species such as $! E! &' etc. cannot occur in normal chemical reaction. herefore at sready state! for balances =** total mass or atomic species or when reaction does not occur! 7q. ')..( can be further simplified ro: mass In " mass out.

(4.3)

able ).* summarises the rypes of material balance for which direct application of7q. ').>( is valido -ecause total number of moles does not balance in systems with reaction! we will carry out all material balances using mass. able).* "pplicarion of the simplified mass balance 7q. ').>(

out- )aterial
otal mass otal numberofmoles

,t steady sta te, does mass in " mass .ithout reaction .ith reaction
yes yes yes yes yes no no no

Mass of a molecular species E umber of moles of a molecular species Mass of an atomic species E umber of moles of an atornic species

yes yes

yes yes

4." Procedure For Material#Balance alculations

he Frsr step in material-balance calcularions is ro undersrand rhe problem. $ertain informarion is available abour a process& the task is ro calculare unknown quantities. -ecause ir is sorne rimes difficulr ro son rhrough all rhe derails provided! ir is besr ro use standard procedures ro rranslate process informarion into a form rhar can be used in calculations. Material balances should be carried )*t in an organised manner& this makes the solution easy ro follow! check! or use by orhers. #n chis chaprer! a forrnalised series of steps is fol lowed for each mass-balance problem. +or easier problems these procedures may seem long-winded and unnecessary& however a standard merhod is helpful when you are first learn ing mass-balance rechniques! he same procedures are used in rhe next chaprer as a basis for energy balances. hese poinrs are essential! 'i(

/rato a clearprocess0low diagram showing al! releuant infor matian. " simple box diagram showing all streams
entering or leaving rhe systern allows inforrnarion about a process ro be organised and summarised in a convenienr way. "3 given quanritative inforrnation should be shown on the diagram. Eote that the variables ofinteresr in marerial bal ances are masses! mass flow rates and mass compositions& if informarion about particular streams is given using volume

or molar quantities! mass flow rates and compositions should be calculated before labelling the flow sheet. 'ii( +elect a set 12 un3ts and state it clearly. $alcularions are

easier when all quanriries are expressed using consistent units. ;nits must also be indicared for a*lvariables shown on process diagrams. 'iii( +elect a basis for the calculation and state it clearly. #n approaching rnass-balance problems ir is helpful () focus on a specific quanrity of material entering or leaving the system. +or conrinuous processes at steady srare we usual #y base the calcularion on rhe amount of material entering or leaving the system within a specified period of rime. +or batch or semi-batch processes! ir is convenient to use eirher the rotal amount of material fed ro the systern or rhe amounr withdrawn at the end. %election of a basis for calculation makes ir easier :/ visualise rhe problem& the way rhis works wi*l become apparent in the worked examples of the next secciono 'iv( +ta te al! assumptions applied to tbc problem. o solve prob lems in this and the following chaprers! you will need () apply sorne 8engineering8 Gudgernenr. Heal-life siruarions are complexo and rhere will be rimes when one or more assumprions are required before you can proceed wirh calculations. o give you expericnce wirh rhis! problems posed in this texr may nor give you all rhe necessary inforrnarion. he derails ornirted can be assurned! pro vided your assumptions are reasonable. 7ngineers make assumprions all rhe time& knowing when an assumption is permissible and what constitutes a reasonable assump tion is one of rhe marks of a skilled engineer. Bhen you make assumprions about a problem ir is virally irnporrant rhar you stare thern exactly. /ther scientists looking through your calcularions need ro know rhe conditions under which your results are applicable& they wil# also want ro decide wherher your assumprions are acceptable or wherher rhey can be irnproved. #n this chapter! differential mass balances on continuous processes are performed with the understanding that the system is at steady state& we can assume thar mass flow rares and composirions do not change wirh rime and the accu rnularion term of7q. ').*( is Dero. #f steady srate does nor prevail in conrinuous processes! information abour che rate of accumulation would be required for solution of rnass balance problems. his is discussed furrher in $hapter I. "nother assumption we must make in mass-balance problems is that the sysrem under investigation does not leak. #n tota*ling up a3 the rnasses entering and leaving rhe systern! we must be sure that a*lstreams are taken into accounr. Bhen analysJng real systems it Js always a good idea ro check for leaks before carrying out mass balances. 'v( Identify which components olthe system, ifany, are in

volved in reaction. his is necessary for determining which mass

balance equation ')..( or ').>(! is appropriate.

he

) Material -alances

66

7xample ).. %etting up a flow sheet

3umid air enriched with oxygen is prepared for a gluconic acid fermentation. he air is prepared in a special hurnidifying chamber. *.6 l h -1 liquid water enrers the chamber ar the same time as dry air and *6 gmol min -1 dry oxygen gas. "ll the water is evaporated. he outHowing gas is found ro contain *K 'w1w( water. Lraw and labeM the 4ow sheet for rhis process. +olution $ Net us choose units of g and min for this process& rhe information provided is first converred to mass 4ow rates in these units. he densiry of water is taken to be *=> g *-*& therefore: *.6*h
-*

* --*.h-

"*-.-6 l

'*-=-> g(

*-- .6 gmm .

8- *

I= mm

"s the molecular weight of /! is >.: *6 gmol mm

+ , 1

*6 gm . ol mm

. * -->. g * 5 )O= g min-I.

* gmol

+igure )7..*

+lowsheet for oxygen enrichment and humidifi,ation of air.

3umid.oxygen-rich
arr

3gminoM
I mass$k -')

.i/uid water .6 g minoM

3umidifier

Cure oxygen )O= g min

o

Lryair Lgmino

;nknown 4ow rates are represented with symbols. "s shown in +igure )7..*! the 4ow rate of dry air is denoted / g min -1 and che flow rate ofhumid! oxygen-rich air is H g min -1. he water content in the humid air is shown as * massK.

simpler 7q. ').>( can be applied to molecular species which are neither reactants nor products of reaction.

).) Material--alance Borked 7xamples

) Material -alances

66

Crocedures for setting out mass-balance calculations are out-

lined in this section. "lthough not the only way to attack rhese problerns! the method shown wi; assist your problem-solving efforrs by formalising the marhematical approach. Mass balance calculations are divided inro four steps: assemble, ana fyse, calculare and 0inafise. Lifferential and integral mass balances with and without reaction are illustrated below.

0 Material 1alan!es

E$am%le ).>

ontinuous filtration

" fermentation slurry containing +treptomyces kanamyceticus cells is filrered using a continuous rotary vacuum filter. *.= kg h 1 slurry is fed to the filter& * kg slurry conrains I= g cell solids. o improve filtration rates! particles of diatornaceous-earth filrer aid are added at arate of *= kg h -1. he concentration ofkanamycin in the slurry is =.=6K by weight. Niquid filtrate is collected at arate of **. kg h - ; rhe concentrarion ofkanamycin in the filtrare is =.=)6K 'w1w(. +ilter cake containing cells and filrer aid is continuously removed from rhe filter cloth. 'a( Bhat percentage liquid is the filter cake? 'b( #f the concenrrarion ofkanamycin in the filrer-cake liquid is the same as in che filtrare! how much kanamycJn is absorbed per kg filter aid? +olution $ 2.

,ssemble
(i) /raw tbeflowsheet showing alf data with units. his is shown in +igure )7>.*. +igure )7>.* +lowsheet for continuous filtration.

12& g h-I IK cells &&34)o1 kanamyein +crmentation slurry I

Pl
I I
+ilter

%y tem boundary

I
I
+iller cake

+iller aid 1& g h-I

- - - - - -

..

+iltrare 112 g h-I &.&035 kanamycin

'ii(

/efine the system boundary by drawing on tbeflowsheet.

he sysrem boundary is shown in +igure )7>.*.

5. ,nalyse 'i( +tate any assumptions.

'ii(

process is operaring ar steady stare system does #lot leak filtrare contains no solids cells do not absorb or release kanamycin during filtration filter aid is dry the liquid phase of the slurry! excluding kanamycin! can be considered water

6ollect and state any e%tra data needed.

0 Material 1alan!es

Eo extra data are required.

0 Material 1alan!es

&'

'iii( +elect and state a basis. he calculation is based on *.= kg slurry entering rhe filter! or * hour. 'iv( 7ist the compounds, ifany, whieh are invofved in reaction. Eo compounds are involved in reaction. 'v( .rite dotan the appropriate general mase-balance

e!uation.
he systern is ar steady srate and no reaetion occurs: rherefore 7q. ').>( is appropriate: mass in 5 mass out.

8. 6alculase (i) +et up a calculation table show3ng all components 12al! streams passing acrosssystem boundaries. +tate tbe units usedflr the
tableo"nter al! known !uantities.

%s shown in +igure )7>.*! four streams eross the sysrem boundaries: fermenrarion slurry! filter aid! filtrare and filter cake.
he components of these streams: cells! kanamycin! filter aid and water are represented in able )7>.*. he table is divided into rwo maGor sections: #n and /ut. Masses entering or leaving the system eaeh hour are shown in rhe table& rhe units used are kg. -eeause filtrate and filter cake flow out of the sysrern! there are no entries for these streams on the lefr hand-side of the tableo $onversely! there are no entries for ferrnenration slurry or filter aid on the /ut side of the tableo he total mass of each stream is given in the last column on eaeh side of the tableo he total amount of each component flowing in and out of the systern is shown in the last row. Bith all known quantities entered! several masses remain unknown& rhese quantities are indicated by quesrion marks.

able )7>.* +tream

Mass-balance table 'kg(

In
6ells 9anamycin =.=I / :ilter aid .ater / *= *otal *.= *= / =.=6 6ells 9anamycin

Out

:ilter aid .ater *otal

+ermentation slurry +ilrer aid +iltrare +iltercake otal

A.. /

**.

'ii( 6alculare unknown !uantities, apply the mass-balance e!uation. o complete able )7>.*! ler us consider eaeh row and column separately. #n the row representing ferrnenrarion slurry! the total mass of the stream is known as *.= kg and the masses of each component except water are known. he entry for water can therefore be deterrnined as the differenee berween *.= kgand rhe sum of rhe known componenrs: '*.= - A.. - =.=I - /( kg " **..A) kg. his mass for water has been enrered in able )7>... he row for filter aid is already complete in able )7>.*& no cells or kanamycin are presem in rhe diatomaceous earrh! which we assume is dry. Be can fil# in rhe final row of the #n side of the table& numbers in this row are obtained by adding the values in each vertical column. he total mass of cells input ro rhe system in all streams is A.. kg! rhe total kanamycin enrering is =.=I kg! etc. he total mass of alM components fed inro the system is rhe sum ofthe last column of the left-hand side: '*.= + *=( kg5 *>= kg. /n rhe /ut side! wecan complete the row for filtrare. Be have assumed there are no solids such as ce#ls or filter aid in the filtrare: rherefore the mass of water in the filtrate is '**. - =.=6( kg ***.Q6 kg. "s yer! the entire cornposition and mass of the filter cake remain unknown.

) Material

-alances

able)7>.. +tream

$ornplered mass-balance table 'kg(

...- ............4 .. ..
'

4.4-4 ..

.. ...-

. . ..

---- - -4 ...4-----4.4 ..4-4.4-

------ .4-------4.--

In 6ells 9anamycin :ilteraid .ater *otal

*.= *=

Out 6ell 9anamycin :ilter aid .ater s

/ A.. =.=6 =.=* =.=I / *= *= ***. Q6 =.AQ **..A)

*otal

+ermentation slurry +ilter aid +iltrate +ilter cake otal

A.. /

=.=I /

/ *=

**..A) /

**. *O *>=

A..

=.=I

*=

**..A)

*>=

A..

o complete che rable! we must consider rhe mass-balance equation relevam ro this problern! 7q. ').>(. #n the absence of reaction! this equation can be applied to total mass and to the masses of each component of the system.

*otal balance

mass

*>= kg total mass in 5 total mass out . .-. otal mass out 5 *>= kg.

6el! balance
A.. kg cells in 5 cells out . .8. $e lis out 5 A.. kg.

9anamycin balance
/./, kg kanamycin in 5 kanamycin out. .8. Ranamycin out 5 =.=I kg.

:ilter-aid balance
*= kg filter aid in 5 filter aid out . .-. +ilter aid out 5 1&

kg. .ater balance

**..A) kg water in 5 water out. :. Baterout5 **..A) kg. hese results are entered in the last row of the /ut side of able )7>..& in rhe absence of reaction this row is always identi cal to the final row of #n side. he component masses for filter cake can now be filled in as the difference between numbers in the final row and rhe masses of each component in the filtrate. ake time to loo k over able )7>..& you should under stand how all rhe numbers were obtained. 'iii( 6heck tbat your results are reasonable and make sense. Mass-balance calculations must be checked. Make sute that all columns and rows of able )7>.. add up ro the totals shown. 4.

:inalise (i) ,nswer the specific !uestions problem.

asked in tbe

) Material

-alances

he percentage liquid in the filter cake can be calculated from the results in able )7>... Lividing the mass of water in the filrer cake by rhe total mass of this stream! the percentage liquid is: =.AQ kg *O kg
#

*== 5 ).>QK.

0 Material 1alan!es

59

#f the concentration ofkanamycin ro:

in this liquid is only =.=)6K! the mass ofkanamycin

is very close

=.=)6 -x =.AQ kg " >.I x *=-0 kg. *== 3owever! we know from able )7>.. that a total of =.=* kg kanamycin is conrained in rhe filter cake& therefore '=.=* - >.I x *=-0) kg " =.==QI kg kanamycin is unaccounred for. +ollowing our assumption rhat kanamycin is not adsorbed by the cells! =.==QI kg kanamycin must be retained by rhe filrer aid. *= kg filter aid is present& therefore the kanamycJn absorbed per kg filter aid is: /.==QIkg *=kg 'ii( " Q.Ixl/-)kgkg-l.

+tate the ansu;ers clearlyand unambiguously, figures.

'a( 'b(

checking significant

he liquid content of the filter cake is ).)K. he amount ofkanamycin absorbed by che filrer aid is Q.I x *=-0 kg kg -l.

Eote in 7xample ).> that the complete composition of rhe fer rnenration slurry was not provided. $ell and kanamycin concenrrations were given& however rhe slurry rnost probably contained a variery of other components such as residual car bohydrare! rninerals! vitamins! amino acids and addirional fermentarion products. hese components were ignored in the mass balance& the liquid phase of the slurry was considered ro be water only. his assumption is reasonable as rhe concen tration of dissolved substances in fermenrarion broths is usually very small& water in spent broth usually accounts for more than Q=K of the liquid phase. Eote also in this problem that rhe masses of sorne of rhe components were different by several orders of magnitude! e.g. rhe mass of kanamycin in the filtrare was of the order *=-2

whereas the rotal mass of this strearn was of the order *=.S $alculation of the mass of water by difference rherefore involved subtracting a very small number from a large one and carrying more significant figures than warranted. his is an unavoidable feature of most mass balances for biological pro cesses! which are characrerised by dilute solutions! low produce concentrations and large amounts of water. 3owever! although excess significant figures were carried in rhe rnass balance table! rhe final answers were reported with due regard ro data accuracy. he aboye example illustrates mass-balance procedures for a simple sready-state process wirhouc reaction. %n integral mass balance for a batch system without reaction is illustrated in 7xample ).).

E$am%le 4.4 Batc( mi$ing

$orn-steep liquor contains ..6K invert sugars and 6=K water& the rest can be considered solids! -eer molasses containing 6=K sucrose! *K invert sugars! *OK water and rhe remainder solids! is mixed with corn-steep liquor in a mixing tank. Bater is added to produce a diluted sugar mixture comaining .K 'w1w( invert sugars. *.6 kg corn-steep liquor and )6 kg molasses are fed into the tank. 'a( 3ow much water is required? 'b( Bhar is the concentration of sucrose in the final mixture?

+olution$ l. ,ssemble 'i( :low sheet.

he flow sheet for this batch process is shown in +igure )7).*. ;nlike in +igure )7>.* where the streams represented continuous #y-flowing inpurs and outputs! the streams in +igure )7).* represent masses added and removed at the beginning and end of the mixing process! respectively.

Figure 4E4.1

+lowsheet for batch mixing process.

*.6 kg 2.5'i invert sugars

SOCk water <=.+ek solids

$orn-stcep *iquor

' '
'

!-------

.:
'

%ystern boundary

... l6
Mixing tan) I

'

Molasses )6kg * k inverl sugarx *BMf water ."11 r?, solids

+O 3! sueroso

'-

.. .

.9
:
Bater Bkg

-- -

7rodu!t mi8ture

'

> kg .K invcrt sugars

'ii( +ystem boundary. he system boundary is indicated in +igure )7).*. 5. ,nalyse

'i(

,ssumptions.
-noleaks - no inversion of sucrose ro reducing sugars! or any other reaction

'ii( "%tra data. Eo extra data are required. 'iii( Basis. *.6 kg corn-steep liquor. 'iv( 6ompounds inuolued in reaction. Eo compounds are involved in reaction. 'v( )ase-balance e!uation. he appropriate rnass-balance equation is 7q. ').>(: mass in
"

mass out.

8.

6alculate
'i( 6alculation tableo able )7).* shows all given quantities in kg. Hows and columns on each side of the table have been complered as much as possible from the information provided. wo unknown quantities are given symbols& rhe mass of water added is denoted ., the total mass of product mixture is denoted P.

able 0;0.1 Mass-balance table ( g) -------------------------4.4---------4.4------4.4----------------

+tream
Inuert

In +ucrose
)

Out *otal I..6 O.* . A=.I

+

sugars
$orn-steep liquor Molasses Bater Croduct mixture otal

+olids 6Q.>A6 *>.Q6

/

Inuert

+ucrose

+olids

*otal

sugars ...6
)

>.*.6 =.)6
)

*.6 )6 .
O.02P

> >

>.6A6

...6

A>.>.6

*A=
+

0.02P

'ii( )ase-balance calculations.

*otal mass balance '*A= + W) kg total mass in 5 7 g total mass out. $.2=1@ .A P.
'*(

Inuert sugars balance >.6A6 kg invert sugars in 5 '=.=. P) kg invert sugars out. :.>.6A6 5 =.=. > >A *AO.A6 kg.
;sing this result in '*(:

.AB.=C g.
(2)

+ucrose balance ...6 kg sucrose in 5 sucrose out . .8. %ucrose out " ...6 kg. +olids balance A>.>.6 g solids in " solids out. .8. %olids out " A>.>.6 kg.

'A=.I + W) kg in " 3.& out. ;sing the result from '.(:

AQ.>6 kg 3.= in 5 3.= out. .'.-.=out5AQ.>6 kg. hese results allow rhe mass-balance table to be cornplered! as shown in able )7)... able)7).. $ompleted rnass-balance table

'kg( In
Out H 50
I..6 O.* O.A6

+tream
Inuert +ucrose

+olids
6Q.>A6 *>.Q6 /

*otal
*.6 )6 O.A6

lnuert

+ucrose

+olids

H 50

*otal

sugars
$orn-steep liquor Molasses Bater Croduct mixture otal >.*.6 =.)6 / ...6

sugars

/
>.6A6

/
...6

>.6A6 A>.>.6 AQ.>6 *AO.A6 >.6A6

...6 ...6

A>.>.6 A>.>.6

AQ.>6 AQ.>6

*AO.A6 *AO.A6

'iii( 6heck the results. "l# columns and rows of able )7).. add up correcdy.

<.

:inalise 'i( *he specific !uenions.

he water required is O.A6
---

kg. he sucrose concentration in che product mixture is:

...6

*AO.A6

? *== 5 *..IK

'ii( ,nswm. 'a( O.A6 kg water is required. 'b( he product mixture contains *>K sucrose.

Material balances on reactive systems are slightly more corn plicated rhan 7xamp#es ).> and ).). o solve problems wirh reaction! sroichiomerric relarionships must be used in con-

Gunction wirh mass-balance equations. illusrrated in 7xamples ).6 and ).I.

hese procedures are

7xample ).6 $ontinuous acetic acid fermentation

,cetobacter aceti bacteria convert ethanol to acetic acid under aerobic conditions! " continuous fermenration process for vinegar
production is proposed using non-viable A. aceti ce#ls immobilised on rhe surface of gelarin beads. he production rarget is . kg h -1 acetic acid: however the maximum acetic acid concentratJon tolerated by rhe cells is *.K. "ir is purnped into the fer menter at arate of.== gmol h -l. 'a( Bhat minimum arnount of ethanol is required? 'b( Bhat minimum amount ofwater must be used to dilute the ethanol to avoid acid inhibition? 'e( Bhat is the eomposition of the fermenter off-gas?

) Material -alances

+olution$ l. ,ssemble (i) :low sheet.

he flow sheet for this process is shown in +igure )76.*. +igure )76.* +low sheet for continuous acetic acid ferrnentation.

)ff-gas < g h'l

+eed siream E = g g h'l h'l !thanol water

ir84I I I I

-Gi
I
I
,.>

%ystern
boundary

+ennenter

8-- - 9

I I

.-.

7rodu!t > g h'l 2 g h'l or 125 a!eti! a!id

#nlet air
3.?@A g hB 1.$00 g h'I=. 0.020 g h'l
.

'Gi( +ystem boundary. he system boundary is shown in +igure )76!l. 'iii( .rite down tbe reaction e!uation. #n the absence of cell growth! maintenance or other metabolism of substrate! the reaetion equation is:

$23s/3 + =. , $3$$//3 + 32=.

'ethanol( 2. 'acetie acid(

,nalyse 'i( ,ssumptions.

-steady state -noleaks -inlet air is dry -gas volumeK 5 moleK -no evaporation of ethanol! 32= or acetie acid -complete conversJon of ethanol -ethanol is used by the eells for synthesis of acetic aeid only& no side-reactions oecur

-oxygen transfer is sufficiendy rapid to meet the demands of rhe cells -concentration of acetic acid in the product stream is *.K 'ii( "%tra data. Molecular weights: erhanol " )I acetic acid " I=

T.5>.
$ornposition of air: .8 'iii( Basis. he calculation is based on . kg aceric acid leaving the system! or * hour. 'iv( 6ompounds in volved in reaction, he compounds involved in reaction are ethanol! acetic acid! =. and 3.=. E 2 is not involved in reaction. 'v( )ass-balance e!uations. +or erhanol! aceric acid! =. and 3.=! the appropriate mass-balance equation is 7q. ')..(: mass in + mass generated " mass out C25.O -2&" *O .* K =.8 AQK E

+ mass consumed.

+or total mass and E.! the appropriate mass-balance equarion is 7q. ').>(: mass in 5 rnass our.

8.

6alculare
'i( 6alculation table. he mass-balance table with data provided is shown as able )76.*& the units are kg. 7t/3 denotes ethanol: 3"c is acetic acid. #f . kg acetic acid represents *. massK of the produce strearn! rhe total mass of rhe producr stream must be .1=.*. " *I.IA kg. #f we assume complete conversion of erhanol! the only components of the product stream are acetic acid and water& therefore water must account for OO massK of the product stream " *).IA kg. #n order to represent what is known about the inlet air! sorne preliminary calculations are needed. =. content " '=..*( '.== gmol. ->- . g * " *>)) g " *.>)) kg * gmol E2 content " '=.AQ( '.== gmol(. -. - O g gmol
*

" )).) g " ).).) kg.

herefore! rhe total mass of air in " 6.AIO kg. he masses of /& and E. can now be entered in the rable! as shown. able)76.* --4.4---- . .'+tream
......

Mass-balance table 'kg( In

-4.4-4.4----4.4!4 ..',.,-, ..... ..4-4.-.4--4.4-------4.. ...4--4.

.B'

..

----------4 ....

Out

+eed strearn #nlet air Croduct stream /ff-gas otal

"tOD " /

D,c / /

D51 . /

/ *.>))

/ ).).)

*otal "@. 6.AIO

"tOD

D,c

D51

2
/

*otal

/ / " / . *.>)) ).).) 6.AIO /

. / .

*).IA / / *).IA

*I.IA G *I.IA

+
"@.

+
G

" and . denote the unknown quantities of ethanol and water in 'he feed stream! respectively& E represents rhe total mass of off-gas. he question marks in the rabie show which orher quantities must be calcula red.
'ii( )ass-balance and stoichiometry calculations. "s E . is a tie component! its mass balance is straighrforward.

F5balance
).).) kg C. in 5 C. out. :. E. out5).).) kg. o deduce the other unknowns! we rnust use sroichiornetric analysis as well as mass balances.

D,cbalance

o kg 3"c in + 3"c generated 5 . kg 3"c out U / kg 3"c consumed.

:. 3"c generated 5 . kg. . kg 5 . kg. * kgmoll I= kg 5 >.>>>
?

*=-. kgmol.
?

+rom reaction sroichiometry! we know that generation of>.>>> x *=-. kgmol 3"c requires >.>>> 7t/3 and =.8 and is accompanied by generarJon of>.>>> ? *=-. kgmol 3.=: :.>.>>> x *=-2 kgmol. )I kg # * kgmol 5 *.6>> kg 7t/3 is consumed
*

*=-. kgmol each of

>.>>>

>. kg x *=-2 kgmol. * kgmol ::& *.=IA kg =. is consumed

*

*O kg * >.>>> ? *=-. kgmol . * kgmol 5 =.I== kg 3.& is generated.

*

Be can use this inrormation to complete 'he mass balances for 7t/3! =. and 3.=.

"tOD balance
7r/3 in U / kg 7r/3 generated 5 / kg 7t/3 out U *.6>> kg 7t/3 consumed. :. 7t/3 Jn5 *.6>> kg5 " ). balance

:. .

* >)) kg =. in U /kg =. generated 5 =. out U *.=IA kg =. consumed. ./ut 5 =..AA kg.

herefore! summing the =. and E 2 componenrs of rhe off-gas:

G! '=..AA U ).).)( kg5 ).A=* kg. D51balance

Bkg 32= in U=.I== kg 32= generared5 *).IA kg 3.= outU /kg 32= consumed. $. .A *).=A kg.

) Material -alances

66

hese resulrs allow us ro complete the mass-balance rable! as shown in able )76... able)76.. $ornplered mass-balance rabie 'kg(
B-B9'

..+ ..
'

9---4.,.4---4.4!-..4 Out

+tream "tOD
+eed stream #nlet air Croducr srream /ff-gas otal *.6>> /

In D,c
/ /

D51
*).=A /

2 /
*.>)) *.>))

N_2
/ ).).)

*otal
*6.I=> 6.AIO

"tOD

D,c

D51

F5

*otal

/ / *.6>> / *).=A ).).) .*.>A* /

. / .

*).IA / / =..AA ).).) /

*I.IA ).A=*

*).IA =..AA ).).) .*.>A*

'iii( 6heck the results. "ll rows and columns of able )76.. add up correctly!

<.

:inalise
'i( *he specific !uestions. he ethanol required is *.6>> kg. he water required is *).=A kg. he off-gas eonrains =..AA kg =. and ).).) kg ED. %ince gas compositions are norrnally expressed using volume or moleK! we must convert rhese values ro moles:

=. eontenr 5 =..AA kg.

E. contenr 5).).) kg.

* kgmoll >. kg 5 O.I6I x *=-> kgmol * kgmoll .Okg 5=.*6O=kgmol.

herefore! rhe rotal molar quanriry of off-gas is =.*IIA kgmol. he off-gas cornposition is:
-----:-----8..!!---

O.I6I x *=-> kgmol =.*IIA kgmol

? *== 5 6.*QK

--:-:----:-8:....4....!.# x *== 5 Q).OK E 2'

=.*6O= kgmol =.*IIA kgmo

'ii( ,nswers. QuanrJties are expressed in kg h -1 rather than kg ro reflecr the continuous nature of the process and the basis used for ealculation. 'a( *.6 kg h -1 ethanol is required. 'b( *).* kg h -1water must be used to dilute the ethanol in rhe feed stream. 'e( he composition of the fermenter off-gas is 6..K =. and Q).OK E..

here are several points ro note about the problem and cal culation of7xample ).6. +irsr! cell growrh and its requirement for substrate were not eonsidered beeause the eells used in this process were non-viable. +or fermentation with live eells!

growth and other metabolic actlVlty must be taken into aeeount in rhe mass balance. his requires knowledge of growth sroichiornetry! which is eonsidered in 7xample ).I and discussed in more detail in %ection ).I. ;se of non-growing

0 Material 1alan!es

immobilised cells in 7xample ).6 meant that the celis were not components of any stream flowing in or out of the process! nor were they generated in reaction. herefore! cell mass did not have to be included in the calculation. 7xample ).6 i##ustrates the importance of phase separations. ;nreacted oxygen and nitrogen were assumed ro leave the system as off-gas rather than as components of the liquid prod uct stream. his assumption is reasonable due to the very poor solubility of oxygen and nitrogen in aqueous liquids& although the product stream most likely contains sorne dissolved gas! the quantities are relatively small. his assumption may need to be reviewed for gases with higher solubiliry! e.g. ammonia. #n the aboye problem! nitrogen did not react! nor were there more than one stream in and one stream out carrying nitrogen. " material8which goes directly from one stream to another is called a tie component; the mass balance for a tie component is relarively simple. ie components are useful because they can provide partial solurions ro mass-balance

problems making subsequent calculations easier. More than one tie component may be present in a particular process. /ne of the listed assumptions in 7xample ).6 is rapid oxy gen transfer. -ecause cells use oxygen in dissolved form! oxygen must be transferred into rhe liquid phase from gas bub bles supplied to the fermenter. he speed of this process depends on the culture condirions and operation of the fer menter as described in more detail in $hapter Q. #n mass-balance problems we assume that alM oxygen required by the stoichiometric equation is irnrnediately available ro the celMs. %ometimes it is not possible to solve for unknown quan tities in mass balances until near rhe end of the calculation. #n such cases! symbols for various components rather than numerical values must be used in the balance equations! his is ilMustrated in rhe integral mass-balance of 7xample ).I which analyses batch culture of growing cells for production of xanthan gum.

E$am%le 4.* %roduction

+ant(an

gum

?anthan gum is produced using Ganthomonas campestris in batch culture. Naborarory experiments have shown that for each gram of glucose utilised by the bacteria! =..> g oxygen and =.=* g ammonia are consumed! while =.A6 g gum! =.=Q g cells! =..A g gaseous $/. and =.*> g 3.= are formed. /ther components of the system such as phosphate can be neglected. Medium con taining glucose and ammonia dissolved in .= === litres water is pumped into a stirred fermemer and inoculated with " campestris. "ir is sparged into the fermenter& rhe total amount of off-gas recovered during the entire batch culture is *.6= kg. -ecause of rhe high viscosity and difficulty in handling xanthan-gum solutions! the final gum concentration should nor be allowed to exceed >.6 wtK. 'a( 3ow much glucose and ammonia are required? 'b( Bhat percentage excess air is provided?
+olution $

l. ,ssemble
'i( :low sheet.

he -ow sheet for this process is shown in +igure )7I.*.

'ii( +ystem boundary.

he system boundary is shown in +igure )7I.*. 'iii( 4eaction e!uation. * gglucoseU=..> g =. U=.=* g E3> , =.A6 ggumU =.=Q gcellsU =..A g$=. U=.*> g 3.=.

0 Material 1alan!es

*,

+igure )7I.*

Clowsheet for xanthan gum fermentation.

/ff-gas
123& g

I
I I
+ccd
2& (#I) g water 1_

4 4 4

4 4 4

%ystem boundary

1*
I I
I Croduct > g

Eermenter

. .
I I I
W.4

I I I
,.>

8.CHkxanthan gum

4 4 4 4

4 4 4

%ir % g

.>.> massK =! AI.A massK E&

5.

,nalyse 'i( ,ssumptions. -noleaks -inlet air and off-gas are dry -conversion of glucose and E3> is *==K complete -$=. leaves in the off-gas 'ii( "%tra data. Molecular weights: 'iii( Basis. *.6= kg off-gas. 'iv( 6ompounds in volved in reaction. he compounds involved in reaction are glucose! =.8 E3>8 gum! cells! $/. and 3.=. E. is not involved in reaction. 'v( )ass-balance e!uations. Cor glucose! &2' E3$' gum! cells! $/2 and 32&' the appropriate mass-balance equation is 7q. ')..(: mass in + mass generated " mass out

+ mass consumed.

+or total mass and E.! the appropriate mass-balance equation is 7q. ').>(:

0 Material 1alan!es

*,

mass in " mass out.

) Material -alances

8.

6alculate
'i(

6alculation

tableo
%orne preliminary calculations are required to start the rnass-balance table. +irst! using * kg *-* as the densiry of water! .= === litres water is equivalent to .= === kg. Ner , be the unknown mass of air added. "r low pressure! air is cornposed of .* molK =. and AQ molK E.& we need ro determine the composition of air as mass fractions. #n *== gmol air: > . g =. content 5 .* gmol. - - - * * gmo . Og E . content 5 AQ gmol. --- * * gmo

I 5 IA. g.
+ IA.( 5 .OO) g! the composition of air

I 5 ..*. g.

#f the total mass of air in *== gmol is '..*. is: IA.g --)x *==5.>.> massK =.X .OO g ..*. g --)x *== 5 AI.A massK E .X .OO g

herefore! rhe mass of=. in rhe inlet air is 1.588,; the mass of E! is =.AIA ,. Net +denote the total mass offeed medium added& let > denote the total mass of product. Be will perform the calculation to produce the maximum-allowable gum concentration: therefore! rhe mass of gum in rhe product is 1.18C>. Bith rhe assumption of *==K conversion of glucose and E3>! these cornpounds are not present in the product. Quantiries known at the beginning of the problem are shown in able )7I.*. able)7I.*
+tream Elucose +eed "ir /ff-gas Croduct otal
/

Mass-balance table 'kg(

.4. -4.= .. 4.. .... 4. .. 4 S....

In

Out

O2
/

F5
/

6O5 Eum
/ / /

cu.
/ /

FD8
/

D5 1

---4 ..4-4----!.

*otal

.==== :
/ ,

4 ..

Elucos e

O2

F5

6O5 Eum

6ells FD8 D51

*otal

1.588, 1.=I=, )

) )

) ) / /

1.18C> 1.18C>

/ ) /

) )

*.6=
>

1.588, 1.=I=, /

.==== :@,

*.6=
@>

'ii(

)ass-balance and stoichiometry calculations.

*otal mass balance

&:@ ,' g total mass in 5 '*.6= $. :@,A 25C1@>.

+ >' g total mass out.

(1)

Eumbalance

) Material -alances

o kg gum in + gum generated

:. <umgenerated5

5 &1.18C>' kg gum out &1.18C>' kg.

+ /kg gum consumed.

0 Material 1alan!es

'-

+rom reaction stoichiornetry! synthesis of #0.0$5P) kg gum requires: -=.A6 -=.A6

0.0$5P

(1 kg( 5 '=.=)IA P) kgglucose

0.0$5P

'=..> kg( 5 '=.=*=A P) kg =.

0.0$5P

-=.A6

'=.=* kg( 5 '=.===)A P) kg E3$ .

and produces: -=.A6

0.0$5P

'=.=Q kg( 5 #0.0042P) kg cells

0.0$5P

=.A6
0.0$5P

'=..A kg( 5 #0.012%P) kg $/2

=.A6

'=.*> kg( " '=.==I=A P) kg 3p.

15 balance

:. .out5
F2ba;ance

#0.2$$A) kg =. in + / kg =. generated 5 =. out #0.2$$A & =.=*=A P) kg.

+ '=.=*=A

P) kg =. consumed. (2)

E. is a tie component.
#0.'%'A) kg E. in 5 E. out. :. E. out 5 '=.AIA A) kg.

(3)
$/2 balance
/kg $/. in + '=.= 12%P) kg $/. generated 5 $/. out :. $/. out 5 #0.012%P) kg.

+ /kg $/. consumed. (4)

he total mass of gas out is *.6= kg. herefore! adding the amounts of=.! E. and $/. out from '.(! '>( and ')(: *.6=
:.A!

" #0.2$$A & =.=*=A P) !A(0.001)P *.6=-=.==*QC.

+ '=.AIA A) + '=.= 12%P)

(5)
Elucose balance
glucose in + /kg glucose generated 5 /kg glucose out + '=.=)IA P) kg glucose consumed. :. <lucose in 5 '=.=)IA P) kg.

(6)

) Material-alances

'1

FD8balance
E3> in + / kg E3> generated 5 / kg E3> out consumed. :. E3> in " '=.===)A P) kg.

+ '=.===)A

P) kg E3>

(7)

Be can now calculare the total mass of the feed! *! glucose in + E3> in + water in. +rom 'I( and 'A(:

:$

*! '=.=)IA P) kg + '=.===)A P) kg + .= === kg 5 '.==== + =.=)A*A P) kg. Be can now use (A) and '6( in '*(: '.==== + =.=)A*A P) + '*.6= - 0.001)P) " '*.6= .= === " =.Q6)A> > :. P! .= Q)O.> kg. :. <um out " A>>.. kg. %ubstituting this result in '6( and 'O(:

(A)

+ *)

,A *.*=.. kg
*! .= QOO.* kg. +rom able )7I.*:

=. in .O..= kg C. in " Q.O.. kg.

;sing the results for P+ A and +in '.(! '>(! ')(! 'I( and 'A(:

=. out " 6A.O kg E. out " Q.O.. kg $/2 out .I>.Q kg <lucose in 5 QAO.> kg E3> in5Q.O kg.

6efl balance

o kg cells in + #0.0042P)

kg cells generated " cells out :. $ells out 5 #0.0042P) kg $ells out " OO.= kg.

+ / kg cells consumed.

D51balance
.= === kg 3.= in + '=.==I=A P) kg 3.= generated 5 3.= out :. 32& out5 .==== + '=.==I=A P) kg. 3.& out 5 .= *.A.. kg. hese entries are induded in able )7I...

+ /kg 3.= consumed.

0 Material 1alan!es

.able4E*.2
+tream

$ompleted mass-balance table 'kg(

In
Out

Elucose

+eed
"ir /ff-gas Croduct otal

QAO.>
)

F5 )

6O5 )

Eum ) )

e&,.
) )

FD;

D51

*otal

Elucose

Q.O
)

.====
)

.O..= Q.O.. )

.=QOO.* *.*=..
) )

2
)

F5

6O5

Eum

6ells FD8D5O

*otal

6A.O Q.O.. .I>.Q

) )

A>>.. OO.= )

) *.6= .=*.A.. .=Q)O.>

QAO.>

.O..=

Q.O.. )

Q.O

.====

..*QO.>

6A.O Q.O.. .I>.Q A>>.. OO.= )

.=*.A.. ..*QO.>

'iii( 6heck the results. "ll the columns and rows of able )7I.. add up correctly to within round-off error. 4. :inalise 'i( *he specific !uestions. +rom rhe cornplered mass-balance rable! QAO.> kg glucose and Q.O kg E3> are required. $alcular ion of percentage excessair is based on oxygen as oxygen is the reacting componem of air. Cercemage excesscan be calculared using 7q. '..>6( in units ofkg: kg =. present - kg =. required ro react ( ' cornpletely with rhe limiting substrate kg =. required ro react cornplerely ( ' with the limiting substrate

K excess air 5

x *==.

#n this problem! both glucose and ammonia are limiting substrates. +rom stoichiometry and the rnass-balance table! the mass of oxygen required *= reacr cornplerely with QAO.> kg glucose and Q.O kg E3> is: QAO.> kg k '=..> kg( 5 ..6.= kg =.8 1 g he mass provided is .O..= kg& therefore: Kexcessair5 .O..= - ..6.= ..6.= *==5.6.>K.

'ii( ,nswers. 'a( QO= kg glucose and Q.O kg E3> are required. 'b( .6K excess air is provided.

4.& Material Balances /it( Hecycle! By# Pass and Purge Streams
%o far! we have performed mass balances on simple single-unir processes. 3owever! steady-state systerns incorporating re cycle! by-pass and purge streams are common in bioprocess industries& flow sheets i3ustrating these modes of operation are shown in +igure ).>. Material-balance calculations for such systems can be somewhat more involved than those in

7xamples ).> to ).I& several balances are required before all mass flows can be determined. "s an example! consider the system of +igure ).). -ecause ce3s are rhe catalysts in fermentation processes! it is often advantageous *= recyele biomass from spent fermentation broth. $e3 recyele requires a separation device! such as a cen trifuge or gravity settling tank! to provide a concentrared recyele stream under aseptic conditions. he flow sheet for cell recyele is shown in +igure ).6& as indicated! at least four

0 Material 1alan!es

A>

+igure ).>

+low sheet for processes with 'a( recyele! 'b( by-pass and 'e( purge streams. (a)

'b(
-y-pass stream

+eed
#,

.. -

Crocess

Croduct

+eed

Crocess

.-.

Crod;$#

Hccycle

tream

'e(

+ecd
j~

.. -

Crocess 9
Hccycle stream

...

Croduct

Curge strcam

+igure ).)

+ermenter with cell recycle.

+resh feed

Eermeruarion broth +ermenter

E""9----l9
%ettling tank

Croduct stream

$ell concentrate

Hecycle stream

different system boundaries can be defined. %ystem # repre sents the overall recycle process& only the fresh feed and final product streams cross this system boundary. #n addition! separ ate material balances can be performed over each process unir: the mixer! the fermenter and the settler. /ther systern bound aries could also be defined: for example! we could group the mixer and fermenrer! or sertler and fermenter! together. Material balances with recycle involve carrying out individual mass-balance calculations for each designated system.

Lepending on which quantities are known and what informa don is sought! analysis of more than one system may be required before the flow rates and compositions of all streams are known. Mass balances with recycle! by-pass or purge usually involve longer calculations than for simple processes! but are not more difhcult conceptually. "ccordingly! we will not treat these types of process further! 7xamples of rnass-balance pro cedures for multi-unit processes can be found in standard chemical-engineering texts! e.g. :*->Y.

) Material

-alances

+igure ).6

%ysrem boundaries for cell-recycle system. (1)

--------,
I
I

I
BG

'**(

'###(

I
+resh

feed

I I I I

,
I

'-

FW

I
.*.

r:
I

- -

'#V(

I I
I
Croduct

I
Mixer

+ennenter
I

W...4-

l-

-F,)

I
,)
I

%ettler 9
t--

8---

'F,:

I I
I I
I

stream

--

.
).I %toichiometry of <rowth and Croduct +ormation

Hecycle stream

3i(! we can wrire rhe following equation for aerobic cell growrh: $;!3!/yE! UaZ.U bD,p?,FiH c6Da&'pFB@d615@eD51. (0.0)

%o far in chis chaprer! the law of conservation of mass has been used to determine unknown quantiries entering or leaving bioprocesses. +or mass balances with reaction such as 7xamples ).6 and ).I! the sroichiomerry of conversion musr be known before the mass balance can be solved. Bhen cell growrh occurs! cells are a product of reaction and must be represented in rhe reaction equarion. #n rhis section we wilP discuss how reaction equations for growrh and product syn thesis are formulated. Merabolic stoichiometry has many applicarions in bioprocessing: as well as in mass and energy balances! it can be used to compare theoretical and actual product yields! check the consisrency of experimental ferrnen tation data! and formulare nutrient medium.

).I.* <rowth %toichiometry and 7lemental -alances

Lespire its complexiry and rhe rhousands ofintracellular reac tions involved! cell growth obeys the law of conservation of matrer. "3 atorns of carbon! hydrogen! oxygen! nitrogen and orher elements consumed during growth are incorporated into new cells or excreted as producrs. $onfining our artention ro those compounds taken up or produced in significant quan riry! if rhe only extracellular producrs formed are $/2 and

#n 7q. (0.0)' 6wD%OyFJis rhe chemical formula for the sub mate 'e.g. forglucose wAI, %A *.!y5Iand JAO', 3 1hF? is rhe chemical formula for the nitrogen source! and $3':/pEO is the chemical 8formula8 for dry biomass. a, b, e, d and e are stoichiometric coefficients. 7q. (0.0) is written on the basis of one mole of substrate& therefore - moles =. are consumed and d moles $/2 are formed per mole substrate reacted! etc. "s illusrrated in +igure ).I! the equation represents a macro scopic view of metabolism& ir ignores the detailed structure of the system and considers only those components which have net interchange with the environment. Lespite irs sirnplicity! the macroscopic approach provides a powerful tool for ther modynamic analysis. 7q. (0.0) does not indude a multitude of compounds such as " C and E"L3 which are integral ro metabolism and undergo exchange cycles in cells! but are not subGect to net exchange with the environment. $ompounds such as vitamins and minerals taken up during metabolism could be included: however! since these growth factors are gene rally consumed in small quanriry we assume here that their conrriburion ro the sroichiornerry and energerics of

0 Material 1alan!es

A6

+igure ).I

eonversion of substrate! oxygen and nitrogen for cell growth.

%ubstrate $F.3F/FE: aOJ b E,/hEi Eitrogen source

-iomass
.CH-0+/

$7NN

a co,

reaction can be neglected. /ther substrates and products can easily be added if appropriate. #n 7q. ').)(! biomass is represented by the formula eH-Op + here is no fundamental obGection to having a molecular formula for cells! even if it is not widely applied in biology. he formula is a refiection of the biomass cornposi tion. "s shown in able )..! microorganisms such as "scherichia coli contain a wide range of elernenrs: however Q=-Q6K of biomass can be accounted for by four maGor ele ments: e! 3! and E. $ompositions of several species in terms of these four e*ements are listed in able ).>. he for mulae in able ).> refer to dry biomass and are based on one e atom& the total amount ofbiomass formed during growth can be accounted for by the stoichiometric coefficient e in 7q.

').)(. -acteria tend to have slightly higher nitrogen contents '**-*)K( than fungi 'I.>-Q.=K( :)Y. +or a particular species!
cell cornposition depends also on culture conditions and sub strate utilised! hence the different entries in able ).> for rhe same organismo 3owever! the results are remarkably similar for different cells and conditions& e3l.%//!sEo.. can be used as a general formula when composition analysis is not avail able. he average 8molecular weight8 of biomass based on H' 3! and E content is therefore .).I! although 6-*=K resid ual ash is often added to account for those e*ements not included in the formula. 7q. ').)( is not complete unless the stoichiometric coeffi cients a, b, e, d and e are known. /nce a formula for biomass is obtained! these coefficients can be evaluated using normal pro cedures for balancing equations! i.e. elemental balances and solution of sirnultaneous equations.

able ).. bacteria

7lemental cornposirion

of "scherichia coli

e balance: 3 balance:

wAc@d

&:rom 4. y. +tanier, ".,. ,delberg and 0. Ingraham, *Q*I! he Microbial Borld! <th edn, >rentice-Dall, Few Kersey' "lement e L dry weight

').6(
%@bgAca@5e

').I(
balance: y@5a@bhAcM8@5d@e

D
E 3

6= .= *)
O

').A( J@b iAc B.

').O(

E balance:

C %
I Ea ea Mg el +e "3 others

> * =.6 =.6 =.6 =.. =.>

Eotice that we have five unknown coefficients &a, b, e; d and e' but only four balance equations. his means rhat additional information is required before the equations can be solved. ;sually this informarion is obtained from experiments. " use fu# measurable parameter is the respiratory!uotient &4N'$ moles eo produced 1 l . d " mo =. ume es cons ').Q(

respiratory quotient! 4N "

0 Material 1alan!es

.able ).>

7lemental composition and degree of reduction for selecred organisms

&>rom;,. 4oels, 2OB1, ,pplication ofmacroscopicprincipies to microbial metabolism, -iotechnol. -ioeng. ..! .)6A-.6*)( Organism "scherichia coli 9lebsiellaaerogenes 9l. aerogenes 9l. aerogenes 9l. aerogenes >seudomonas612B ,erobacter aerogenes >aracoccusdenitrificans >. denitrificans +accharomycescereuisiae %. cerevisiae %. cerevisiae 6andida utilis H. uti;is H. utilis H. uti;is
"verage

"lemental formula
$3#.AA==.)QEo..) $3*.A%==.)>E/... $32.A>==.)>E/..) $3#.A%==.)AE/.*A $3 *.A>==.)>E/..) $3..====.%.Eo..> $3 *.O>==.%%E/..6 $3*.O*==.6*Eo..= $3*.6*==.)IEo.*Q $3 *.I)==.6.E/.PI $3*.O>==.%IE/.*A $3#.O*==.%#Eo.*A $3*.O>==.6)E/.*= $3#.OA==.6IEo..= $3*.O>==.)IE/.*Q $3 *.OA==.%IE/..= $3 *.AQ==.6=E/..=

/egree
).8=A )..> ).*6 ).>= ).*6 )..A >.QO ).*Q >.QI ).*. )..= )..O ).)6 ).*6 ).>) ).*6 ).*Q

&relatiue to FD8'

12reduction r

'standard deviation " >K( Bhen an experimental value of 4N is available! 7qs ').6( to ').Q( can be solved to determine the stoichiometric coeffi cients. he results! however! are sensitive ro small errors in 4N which must be measured very accurately. Bhen 7q. ').)( is cornpleted! the quantiries of substrate! nitrogen and oxygen required for production ofbiomass can be determined direcrly.

E$am%le ).A Stoic(iometric coefficients for cell growt(

Croduction of single-cell protein from hexadecane is described by rhe fo#lowing reaction equation:

where $3 *.II==..AE/..=represents rhe biomass. #f 4NA =.)>! determine the stoichiometric coefficienrs. +olution$ $ balance: 3 balance:

*I " c@ d >) + > b " *.II e + . (1) '.( '>( ')( '6(

balance:

5aA1.5= c@5d@e bA1.51 e

=.)> " dla.

E balance:

4N$

0 Material 1alan!es

77

Be muse solve this ser of simultaneous equations. %olution can be aehieved in many different ways& usually ir is a good idea to express each variable as a function of only one orher variable. bis already written simply as a function of cin ')(& let us try express ing the othervariables solely in terrns of c. +rom '*(:

dA2I-c. 'I(
+rom '6(: -

1 5 -)). $

" ..>.I

1.

(7)

$ombining 'I( and 'A( gives an expression for - in terms of conly:

a " ..>.I '*I - c' a " >A... - ..>.I c.

%ubstituting ')( into '.( gives:

(A)

>) + > '=..= c'5 *.II c@ . e >) 5 *.=I c@ . e eA2=-1.C8c.

%ubstituting 'O(! 'I( and 'Q( into '>( gives:

(9)

. '>A... - ..>.I c' " =..A c@ . '*I- c' + '*A - =.6> c' .6.)) 5 ..>Q e cA 21.I<.
;sing this result for cin 'O(! ')(! 'I( and 'Q( gJves:

a " *..)O bA5.28 dAC.8= eA 22.8I.

$heck rhat these coefficient values satisfy 7qs (1)-(3). he complete reaction equation is:

"lthough elemental balances are useful! rhe presence of water in 7q. ').)( causes sorne problems in practicaM application. -ecause water is usuaM#y present in great excess and changes in water concentration are inconvenient ro measure or

experimentally verify! 3 and / balances can present difficulr ies. #nstead! a useful principie is conservation of reducing power or available elecrrons! which can be applied ro deter mine quanritative relationships berween substrates and

) Material

-alances

products. %n electron balance shows how available electrons from the substrate are distributed in reaction.

).I.. Electron Balances

,uailable electrons refers ro the number of electrons available for transfer ro oxygen on combustion of a substance ro $/2' 32& and nitrogen-containing compounds. he number of available electrons found in organic material is calculated from the valence of the various elements: ) for H' * for 3! -. for =! 6 for C! and I for %. he number of available electrons for E depends on the reference state: -> if ammonia is rhe reference! / for molecular nitrogen E2' and 6 for nitrare. he reference state for cell growth is usually chosen ro be the same as rhe nitrogen source in rhe medium. #n the following discussion ir will be assumed for convenience that ammonia is used as nitro gen source& rhis can easily be changed if other nitrogen sources are employed '6(. /egree of reduction, y! is defined as rhe number of equiva lents of available electrons in that quanriry of material containing * g atorn carbono herefore! for substrate 602%OyFJ, the number of available electrons is #42 + 3 & 24 & $5). he degree of reduction for the subsrrate! r6i+s therefore #42 + 3 & 24 & $5)72. Legrees of reduction relative to E3> and E2 for several biological compounds are given in able -.. in "ppendix -. Legree of reduction for 6Ol 32& and E3> isDero. "lectrons auailable flr trans0er to o%ygen are conserued during metabolismo #n a balanced growth equation! number of avail able electrons is conserved by virtue of the fact that the amounts of each chemical element are conserved. "pplying this principie ro 7q. ').)( wirh ammonia as nitrogen source! the available-electron balance is:

of stoichiornetric coefficients. 3owever! one electro n balance! two elemental balances and one measured quantiry are still inadequate information for solution of five unknown coeffi cients& another experimental quantiry is required. "s cells grow there is! as a general approximation! a linear relationship between the amount of biomass produced and the amount of substrate consumed. his relationship is expressed quantira rively using rhe biomassyie;d, P%s$

P%s !

g cells produced g subsrrare consumed . ').**(

" large number of facrors influences biomass yield! including medium composition! nature of the carbon and nitrogen sources! p3 and temperature. -iomass yield is greater in aero bic than in anaerobic cultures& choice of e*ectron acceptor! e.g. =.8 nitrare or sulphate! can also have a significant effect :6! I(. Bhen P%s is constant throughout growrh! irs experirnen tally-determined value can be used ro determine rhe stoichiometric coefficient e in 7q. ').)(. 7q. ').**( expressed in terms of rhe sroichiometric 7q. ').)( is:

P%s !

c'MB cells( 'MB subsrrare( ').*.(

7qs ').6( and ').O( and a measured value of 89:or evaluation

wPs - <aA .r/

').*=( where Ps and 9- are the degrees of reduction of substrate and biomass! respectively. Eote rhat the available-elecrron balance is not independent of the complete set of elemental balances& if the sroichiometric equation is balanced in terms of each ele ment including 3 and /! the e*ectron balance is implicitly satisfied.

).I.> Biomass 0ield

ypically! 7q. ').*=( is used with carbon and nitrogen balances

) Material

-alances

where MB is molecular weight and 8MB cells8 means the biomass formula-weight plus any residual ash. 3owever! before applying measured values of P%s and 7q. ').*.( ro evaluate e, we must be sure that rhe experimental culture sys tem is well represented by the stoichiometric equation. +or example! we must be sure that substrate is not used to synrhe sise extracellular products other than 6Ol and 3.&. /ne complication with real cultures is that sorne fraction of sub strate consumed is always used for maintenance actiuities such as maintenance of membrane potential and inrernal p3! turnover of cellular components and cell motiliry. hese metabolic funcrions require substrate but do not necessarily produce cell biomass! $/. and 3.& in the way described by 7q. ').)(. Ir is important to account for maintenance when experimental information is used to complete stoichiometric equations& maintenance requirements and the difference between observed and true yields are discussed further in $hapter **. +or rhe time being! we will assume that available values for biomass yield reflect substrate consumption for growrh only.

0 Material 1alan!es

AQ

).I.) Croduct %toichiometry

$onsider formation of an extracellular product

6Q,DkO,Fm during growth. 7q. (0.0) can be extended to

indude product synthesis as follows:

6wD%O FJ@a12@bD;OhF? H e6lMOpF.,@d615@ e3.=U f60DkO,Fm

(0.1$) where fis rhe stoichiometric coefficienr for producto Croducr synthesis introduces one extra unknown stoichiornetric coeffi cient to the equation& thus! an additional relationship between coefficients is required. his is usually provided as another experimentally-deterrnined yield coefficient! the produet yield from substrate, Pp<: g product formed f&). product( 9 Js 5 -gFF:s:F&uA-b-st-ra-t-e-c-o 5-ns-'uM-mB-esdu-b:strate( . (0.10)

7q. ').*I( is a very useful equation. #r means that if we know which organism '9- (! substrate #2 and Ps' and product #0 and 9p( are involved in cell culture! and the yields of biomass #e) and product &f', we can quickly calculare the oxygen demando /f course we could also determine a by solving for all the stoi chiometric coefficients of 7q. ').*>( as described in %ection ).I.*. 7q. ').*I( allows more rapid evaluation and does not require that the quanrities ofE3>! $/. and 3.= involved in rhe reaction be known.

).I.I Maximum Cossible9ield

+rom 7q. ').*6( the fractional allocation of available electrons in the subsrrate can be written as: 1

5 ,

e=/

+ f0yp.
wPs
').*A(

wPs

wys

%s menrioned aboye wirh regard ro biomass yields! we must be sure that the experimental system used ro measure 9Js con
forms to 7q. ').*>(. 7q. ').*>( does not hold if product formation is not directly #inked with growth: accordingly it cannot be applied for secondary-merabolire production such as penicJllin fermentation! or for biotransformations such as steroid hydroxylation which involve only a small number of enDymes in cells. #n rhese cases! independenr reaction equa tions must be used ro describe growth and product synthesis!

#n 7q. ').*A(! the first term on the righr-hand side is rhe frac rion of available electrons transferred from substrate to oxygen! the second term is the fraction of available electrons transferred to biornass! and the third term is che fraction of available electrons transferred to product. his relationship can be used to obtain upper bounds for the yields of biomass and product from substrate. Net us define8 1 as the fraction of avaJlable elecrrons in che substrate rransferred to biomass:

).I.6

heoretical /xygen Lemand

').*O( #n rhe absence of produce formation! if all available electrons were used for biomass synrhesis! would equal uniry. ;nder these conditions! the maximum value of the stoichiometric coefficient cis$

/xygen demand is an irnportant parameter in bioprocessing as oxygen is often the limiting substrate in aerobic ferrnentations. /xygen demand is represented by the stoichiometric coeffi cient ain 7qs ').)( and ').*>(. /xygen requirement is related direcdy to rhe electrons available for transfer to oxygen& the oxygen demand can therefore be derived from an appropriate electro n balance. Bhen product synthesis occurs as repre sented by 7q. ').*>(! the electro n balance is:

8-

wPs - <a "

er1 + f0yp
').*6( of the product.

').*Q( emax can be converted to a biomass yield with mass units using 7q. ').*.(. herefore! even if we do not know the sroichiorn etry of growth! we can quickly calculate an upper limit for biomass yield from the molecular formulae for substrate and product. #f the composition of the cells is unknown! 9- can be

where 9p is the degree of reduction Hearranging gives:

').*I(

) Material -alances

80 80

taken as 4.2 corresponding ro the average biomass formula $3#.%/o.sEo..8 Maximum biomass yields for several sub strates are listed in able ).)& maximum biomass yield can be expressed in terms of mass '*8:('[ !max (! or as number of $ atoms in the biomass per substrate $ atom consumed '$max1w)' hese quantities are sometirnes known as thermodynamic ma%imum biomass yields. able ).) shows that substrates with high energy content! indicated by high 9svalues! give high maxi mum biomass yields. Nikewise! che maximum possible producr yield in rhe able 0.0 hermodynamic maximum biomass yields

absence of biomass synthesis can be determined from 7q.

(4.17):

(4.20)
7q. (4.20) allows 8us to quickly calculate an upper limie for product yield from the molecular formulae for substrate and product.

&,dapted from 7.". "rickson, >. E. )inkevich and R 9 "roshin, *QAO!,pplication 12mass and energy balance regularities in fermen tation, -iotechnol. -ioeng. .=!*6Q6-*I.*(
+ubstrate

:ormula

9s

*hermodynamic ma%imum yield correspondingto ; ;l " *

6arbon yield
'max1 w (

)ass yield
9?%!max

"lkanes Methane 3exane #?) 3exadecane #?) "lcohols Methanol

$3) $I32) $2I-$3)= $.3'!/ $.3I=. $>3s=> $3.= $I32.=I $2.3..=22 '$I3l//%(x $3.=2 $.34=. $>3I=. $>3I=> $434=) $.3.=)

8.0 6.3 6.1 6.0 6.0 5.0 4.7 4.0 4.0 4.0 4.0 2.0 4.0 4.7 4.0 3.0 1.0

1.9 1.5 1.5 1.4 1.4 1.2 1.1 0.95 0.95 0.95 0.95 0.5 0.95 1.1 0.95 0.7 0.24

2.9 2.6 2.5

1.1

7thanol
7thylene glycol <lycerol $arbohydrates +ormaldehyde <lucose %ucrose %tarch /rganic acids +ormicacid "ceticacid Cropionic acid Nacticacid +umaric acid /xalicacid

1.5 0.9 0.9 0.8 0.8 0.8 0.9 0.3 0.8

*.*

0.8 0.6 0.1

E$am%le ).O Product yield and o$ygen demand

he chemical reaction equation for respiration of glucose is:

6andida utilis cells convert glucose to $/. and 3.= during growth. he celM composition is $3#.O)==.6%E/.. plus 6K ash. 9ield ofbiomass from substrate is =.6 g g -*. "mmonia is used as nJtrogen source.
'a( Bhat is the oxygen demand with growth compared ro that withouti 'b( H. utilisis also able to grow with ethanol as substrate! producing cells of the same cornposition as aboye. /n a mass basis! how does rhe maximum possible biomass yield from ethanol compare with the maximum possible yield from glucose?

+olution$
Molecular weJghts: glucose 5 *O= ethanol5)I

MB biomass is '.6.)) + ash(& since ash accounts for 6K of the total weighr! Q6K of the total 5 .6.)). herefore! MB biomass 5 .6.))1=.Q6 5 .I.AO. +rom able -..! 9s for glucose is ).==& 9s for ethanol is I.==. 9- 5 ') ? * + * ? *.O) - . ? =.66 - > ? =..( ).*). +or glucose w I& for ethanol w 5 ..

'a( P%s 5=.6 g g-#. $onverting this mass yield to a molar yield:

P%s
* !

=.6 g biomass g glucose

**O= g glucose *** gmol biomass * . * gmol glucose . .I.AO g biomass

x s5

>>

I gmol biomass . gmol

5 c.

glucose /xygen demand is given by 7q. ').*I(. #n rhe absence of product formation:
-

5 *1):I ').==( - >.>I ').*)(Y 5 ..6..

herefore! :he oxygen demand for glucose respiration with growth is ..6 gmol =. per gmol glucose consumed. -y comparison with the chemical reaction equation for respiration! rhis is only about ).K that required in che absence of growrh. 'b( Maximum possible biomass yield is given by 7q. ').*Q(. ;sing 'he data aboye! for glucose:
cmax 5 ,

I').==(

5 6.O=.

$onverting this to a mass basis:

9?%!max

6.O= g biomass ** gmol glucose **.I.AO g biomass * gmol glucose . *O= g glucose . * gmol biomass

9?%!max " =.OI

FFFFB,-F-lgg ucose

gbiomass

+or ethanol: .'I.==(

8:max. 5 ,

5 ..Q=

and

) Material

-alances

9?%!max

.!Q= gmol biomass gmol erhanol gbiomass g erhanol

* gmol echanol **.I!AO g biomass * . * )I g erhanol . * gmol biornass

y:

?%!max

" .I Q

herefore! on a mass basis! rhe maximum possible amount ofbiomass produced per gram erhanol consumed is roughly twice that per gram glucose consumed. his result is consistenr wirh rhe data listed in able ).).

7xample ).O illustrares two irnportanr poinrs. +irsr! the chem ical reacrion equation for conversion of subsrrare wirhour growrh is a poor approximation of overall sroichiornerry when cell growrh occurs. Bhen estimating yields and oxygen requirements for any process involving cell growth! rhe full stoichiornetric equation including biomass should be used. %econd! rhe chemical nature or oxidarion state of the substrato has a maGor influence on producr and biomass yield through rhe number of available elecrrons.

rhe membrane into the buffer. $ells in rhe brorh are too large ro pass rhrough the membrane and pass out of rhe tubes as a concentrare. +igure )C *.* cells. 3ollow-fibre membrane for concentration of

Eerm!ntation broth

4.' Summary of (a%ter 4

"r rhe end of $haprer ) you should: undersrand rhe terms: system, surroundings, boundaryand processin thermodynamics& 'ii( be able to idenrify open and closedsystems, and batch, 'i(
1uffer solution m!mhran!

semi

batch, ftd-batch and continuous processes; 'iii( understand rhe difference between steady state and e!ui librium$
'iv( be able ro write appropriare equations for conservation of mass for processes wirh and wirhour reaction: 'v( be able ro solve simple rnass-balance problems with and without reacrion& and 'vi( be able ro apply sroichiornerric principies for macroscop ic analysis of cell growth and product formarion.

he aim of che membrane syscem is to produce a cell suspen sion contaJning IK biomass. 'a( Bhar is rhe -ow rare from rhe annular space? 'b( Bhat is :he flow rate of cell suspension from the mern brane rubes? "ssume that the cells are not active! i.e! they do not grow. "ssume further that the membrane does not allow any mol ecules orher than water ro pass from annulus ro inner cylinder! or vice versa.

Problems
4.1 ell concentration using membranes

).. Membrane reactor

" battery of cylindrical membranes similar ro that shown in +igure )C *.* is used for an extractive bioconversion. 7xtractive bioconversion means thar ferrnentation and extrae tion of produce occur ae rhe same time. 9east cells are imrnobilised within the membrane walls. " *=K glucose in water solurion is passed through the annular space at arate of)= kg h -l. %n organic solvent! such as .ethyl*!>-hexanedJol! enters rhe inner tube at arate of )= kg h -l.

" battery of cylindrical hollow-fibre membranes is operared at seeady stare ro concentrare a bacteriaY suspension from a fer menter. >6= kg min -1 fermenter brorh is pumped through a srack of hollow-fibre membranes as shown in +igure )C*.*. he broth comains *K bacteria& the rest may be considered water. -uffer solution enters the annular space around che membrane tubes at a -ow rate of O= kg min - *& because broth in rhe membrane rubes is under pressure! water is forced across

) Material -alances

-ecause the membrane is constructed of a polymer which repels organic solvents! rhe hexanediol cannot penetrate the membrane and rhe yeast is relatively unaffected by its toxicity! /n the other hand! because glucose and water are virtually insoluble in .-ethyl-* !>-hexanediol! these compounds do not enter rhe inner tube to an appreciable exrent. /nce imrnobil ised in the mernbrane! the yeasr cannot reproduce but convert glucose to ethanol according to rhe equarion:

dehydrated egg product leaving the enDyme reactor is =..K. Letermine: 'a( 'b( 'e( 'd( which is the limiting substrate: the percentage excess substrate& the composirion of rhe reactor off-gas& and rhe composition of the final egg produce

).6 "Deotropic distillation

7thanol is soluble in .-ethyl-*!>-hexanediol& ir diffuses into che inner tube and is carried out of the sysrern. $/2 gas exits from the membrane unit through an escape valve. #n the aque ous stream leaving the annular space! che concentration of unconverted glucose is =..K and the concenrration of ethanol is =.6K. #f rhe system operares at steady state: 'a( Bhat is the concentration of ethanol in the hexanediol stream leaving che reactor? (b) Bhat is the mass flow rare of$=.? "bsolute or *==K ethanol is produced from a mixture ofQ6K ethanol and 6K water using rhe Reyes distillation process. " third component! benDene! is added to lower the volarility of the alcohol. ;nder these conditions! rhe overhead produce is a constant-boiling mixture of *O.6K ethanol! A.)K 3.= and A).* K benDene. he process is outlined in +igure )C6.*. +igure )C6.* process. +lowsheet for Reyes distillation

..----*:.....
).> 7thanol distillation
Niquid from a brewery fermenter can be considercd ro contain *=K erhanol and Q=K water. 6= === kg h - 1 of this fermenta rion product are pumped ro a distillation column on the factory site! ;nder eurrent operaring condirions a disrillate of )6K erhanol and 66K water is produced from the top of the eolumn at arate one-tenth that of the feed. 'a( Bhat is rhe composition of rhe waste 8botrorns8 from the
OC i& ethanoI 35 water
1enKene

?0.15 benKene 1A.35 ethanoI

water
?.05

.... ..-G

Listillation tower

srill?
'b( Bhat is the rate of alcoholloss in the borroms? in the

).) Hemoval of glucose from dried egg

he enDyme! glucose oxidase! is used commereially to remove glucose from dehydrared egg to improve colour! flavour and shelf-life. he reaetion is: $I3*.=I 'glucose(

+ =. + 3.=

-?

$I3*.=A + 3.=.8 'gluconic acid(

" continuous-3ow reactor is ser up using immobilised-enDyrne beads which are retained inside the vessel. Lehydrared egg slurry containing .K glucose! .=K water and the remainder unreactive egg solids! is available at arate of > === kg h -1. "ir is pumped through the reactor contems so that *O kg oxygen are delivered per hour. he desired glucose level

*8-----*.....

1&&5 ethanoI

;se rhe following data to calculare the volume of benDene which should be fed ro rhe still in order ro produce .6= litres absolute ethanol: p '*==K alcohol( 5 =.AO6 g cm ->& p 'ben Dene( " =.OA. g cm
->.

).I $ulture of plant roots

Clant roots produce valuable chemicals in vitro. " batch cul ture of ,tropa belladonna roots at .6T$ is esrablished in an air-driven reactor as shown in +igure )CI.*. -ecause roots cannot be removed during operarion of rhe reactor! ir is proposed to monitor growth using mass balances.

0 Material 1alan!es

+igure )CI.*

Heactor for culture of plant roots!

/ff-gas

).O Croduct yield in anaerobic digestion

"naerobic digestion of volatile acids by methane bacteria is represented by the equation: $3$$//3 'acetic acid(

medium

Eutrient -----!

+ E3$

biomass + $/2 'methane(

+ 3.= + $30+

Ho'(ts

%ir-driMen

he cornposirion of methane bacteria is approximated by the empirical formula $3P.)==.)=E/..=8 +or each kg acetic acid consumed! =.IA kg $/. is evolved. 3ow does the yield of methane under these conditions compare with rhe maximum possible yield?

reactor

).Q %toichiometry of single-cell protein synthesis

'a( 6ellulomonas bacteria used as single-cell protein for human or animal food are produced from glucose under anaerobic conditions. "3 carbon in the subsrrare is converted inro biomass& ammonia is used as nitrogen source. he molecular formula for rhe biomass is $3P.6I==.8i)E/.PI& rhe ce#ls also contain 6K ash. 3ow does rhe yield ofbiomass from substrate in mass and molar terms compare with the maximum possible biomass yield? 'b( "nother system for manufacture of single-cell protein is )ethylophilus methylotrophus. his organism is produced aerobically from rnerhanol with ammonia as nitrogen source. he molecular formula for rhe biomass is $3 *.I%==.>IE/...r&hese cells contain IK ash. 'i( 3ow does rhe maximum yield of biomass compare with 'a( aboye? Bhat is the main reason for rhe differ ence'ii( #f the actual yield of bJomass from methanol is ).K the thermodynamic maximum! whar is rhe oxygen demand?

Lrain!d li/uid

*).6 g nutrient medium contammg >K glucose and *.A6K E3> is fed into rhe reactor& rhe remainder of the medi um can be considered water. "ir ar .6F$ and * arm pressure is sparged into the fermenter at arate of.. !mN min - P. Luring a l /-day culture period! )A litres =. and *6 litres $/. are col lected in rhe off-gas. "fter *= days! ### ) g liquid containing =.=I>K glucose and *.AK dissolved E3> is drained from rhe vessel. he ratio of fresh weighr to dry weight for roots is known to be *): *. 'a( Bhat dry mass of roots is produced in *= days? 'b( Brite the reacrion equation for growth! indicaring :he approximate chemical formula for the roots! $3a=!aEo8 'e( Bhat is the limiting substrate? 'd( Bhat is the yield of roots from glucose?

).*= 7thanol production by yeast and bacteria

and Symomonas mobilis bacteria produce ethanol from glucose under anaerobic condi tions without externaY electron acceptors. he biomass yield from glucose is =.** g g- for yeasr and =.=6 g gB P for @ mobilis. #n borh cases the nirrogen source is E3>F -oth cell cornpositions are represented by the formula $3P.%//.6E/..8 -orh 'a( Bhat is the yJeld of ethanol from glucose in both cases? 'b( 3ow do the yields calculated in 'a( compare with the thermodynamic maximum?

+accbaromyces cereuisiae yeast

).A /xygen requirement for growth on glycerol

9lebsiella aerogenes is produced from glyceroM in aerobic cul ture with ammonia as nitrogen source. he biomass contains OK ash! =.)= g biomass is produced for each g glyceroM con surned! and no maGor metabolic products are formed. Bhat is the oxygen requirement for this culture in mass terms?

4.11 1etecting un)nown %roducts

9east growing in continuous culture produce =.>A g biomass per g glucose consumed& about =.OO g =. is consumed per g cells formed. he nitrogen source is ammonia! and the bio mass composition is $3*.AQ==.6IEo.# "re orher products also synthesised?

3eferences
*. +elder! H.M. and H.B. Housseau '*QAO( "lementary >rincipies 12 6hemicaf >rocesses,$hapter 6! Pohn Biley! Eew9ork. .. 3irnmelblau! L.M. '*QA)( Basic >rincipies and 6alculations in 6hemical "ngineer3ng, >rd edn! $haprer .! Crenrice-3all! Few Persey. >. Bhitwell! P.e. and H.R. oner '*QIQ( 6onseruation 12 )ass and "nergy, $hapter )! -laisdell! Baltham! Massachuserrs! ). $ordier!P.-N.! -.M. -utsch! -. -irou and ;. von %toekar '*QOA( he relationship between elemental eomposition and heat of combustion of mierobial biomass. Appl. )icrobio7 Biotecbnol. .6! >=6->*.. 6. Hoels! P .". '*QO>( "nergetics and 9inetics in

4.12 Medium formulation

>seudomonas 6)=* is to be used for production of single-cell protein for animal feed. he subsrrate is fueM oiM. he composi tionof >seudomonasC<12 is$3*.O>==.66E/..68 #fthe final cell concentration is .6 g *-*! what minimum concentration of 'E3)(.%=) must be provided in the medium if 'E3)(.%= ) is rhe sole nitrogen source?

Biotecbnoiogy,

4.1" 2$ygen demand for %roduction of recombinant %rotein

Croduction of recombinant protein by a genetically engineered strain of "scherichia coli is proportional ro cell growth. "mmonia is used as nitrogen source for aerobic respi rarion of glucose. he recombinant protein has an overall formula $3 *.66==.>*E/..6X he yield ofbiomass from glucose is measured at =.)O g g-l& rhe yield of recombinant protein from glucose is about .=K rhat for cells. 'a( 3ow much ammonia is required? 'b( Bhat is rhe oxygen demand? 'e( #f rhe biomass yield remains ar =.)O g g-2! how much dif ferent are the ammonia and oxygen requirements for wild-type E. coli unable to symhesise recombinant protein?

$hapter >! 7lsevier -iomedical Cress! "msterdam. I. "tkinson! -. and +. Mavituna '*QQ*( Biochemical "ngineering and Biotechnology Dandbook, .nd edn! $hapter )! Macmillan! -asingstoke.

Suggestions for Furt(er 3eading Process Mass Balances 'see also refs *->(
3ougen! /.".! R.M. Batson and H.". HagatD '*Q6)(

6hemical >rocess >rincipies$ )aterial and "nergy Balances,

.ndedn! $hapterA!Pohn Biley! Eew9ork. %haheen! 7P. '*QA6( Basic >ractice 12 6hemical "ngineering; $hapter )! 3oughron Miffiin! -oston.

4.14 Effect of growt( on o$ygen demand

he chemical reaction equation for conversion of ethanol '$\3I=( to acetic acid '$.3)=.( is: $.3I= 3.=. "ceric aeid is produeed from ethanol during growth of ,cetobacter aceti, whieh has the composition $3#.O==.%E/..8 -iomass yield from substrate #s/.*) g g-l& productyieMdfrom substrate #%=.Q. g g -1. "mmonia is used as nitrogen source. 3ow does growrh in this culture affect oxygen demand for acetic acid production?

Metabolic Stoic(iometry 'see also ref 6(

7rickson! N.7.! #.<. Minkevich and V.R. 7roshin '*QAO( "pplication of mass and energy balance regularities in fer mentation. Biotechnol. Bioeng. .=!*6Q6-*I.*. 3eiGnen! P.P. and P.". HoeMs '*QO*( " macroscopic model describing yield and maintenance relationships in aerobic fermentation processes. Biotecbnol. Bioeng. .>! A>Q-AI>. Eagai! %. '*QAQ( Mass and energy balances for microbial growth kineties. ,dv. Biocbem. "ng. **!)Q-O>. Hoels! P.". '*QO=( "pplication of maeroseopic principies ro microbial metabolism. Biotecbnol. Bioeng. 55, .)6A-.6*).

+ =. , $.3)=. +