Note: Descriptions are shown in the official language in which they were submitted.
`~ Title ef the inve~tio~ zSr~
Proce~ for the high cell-den~ity far~entation of
E~cherlchia coli in an agita~ed tank ferment2r
Field of thQ inventio~
S The invantion relate~ to a process for the high
cell--den~ity fermentation of E. coli ~train~, specific
ally tho~e Yuitable a~ ho ts for vectors with recombinant
expression systems. The field of the invention lie~ in
those branches of indu~try in a national economy which
hsve a biotechnological ~ector and employ in the la~tar
B. coli strainY as producer organisms.
Prior art
E~ coli i~ often used a~ cellular host for the
production of recombinant DNA product~. Beside-~ a high
intracellular concentration of the de3ired product, a
crucial preregui~ite for a high overall yield is ~he
achievement of high concentrations of cells in the
fer~enter.
The problems which ari~e in the cul~ivation at
high cell den~ities in a conventional agitated ~ank
fermenter are those of growth inhibition owing to the
initial ~ub~trate concentxations being too high, the
con3umption of a~sential con~tituent~ of the nutrient
medium during the cour~e of cultivation, the formation of
metabolic by-product~ which have inhibitory effect~, and
the limited capacity of oxygen introduction.
Variou~ ~trategies c.~n b~ used to attempt to meet
the increasing oxygen dema~d o~ a growiny B. coli
~ulture, nam~lys increa~ing the ~tirrer ~peed and the ga~
introduction rate, introducing air enriched in o~ygen
(Jung et al. 1988; Pa~3 et al. 1989; RrUger 1989;
~ppstein et al. 1989), introducing pure oxygen ~a3 (Bauer
and Sh~loach 1974; Shiloach and Bauer 1975; Bauer and
White 1976; ~auer and Ziv 1976; ~ori et al. 1979; Gleiser
and ~au~r 1981; ~itzutani et al. 1986; Bailey et al.
1987 ; ~an et al . 1987; ~r~ger 1989), cultivation at low
temperature~ (Shiloa~h and Bauer 1975; ~auer and Whi~e
1976; Bauer and Ziv 1976) and under elevated pre~ure
(T~ai et al. 1987).
3 7~
E. coli ha~ been fermented in a glucose/mineral
~alt medium with exce~s oxygen up to 16 . 5 g of dry
biomass of X/l (Reiling et al. 1985). R~Uger (1989) and
Eppstein et al . ( 1989 ) achieved 19 g of X/l . Growth at
higher ~ell densitie~ required a fed-batch technique in
order, on the one hand, to eliminat~ inhibikion and
limitation of ~ub~tancss and, on the other hand, to
prevent the formation of inhibitory metabolic by-
product~. It was necessary in all casas to metex in other
nutrient~ as well besides the C source and ammonia as N
qour~e and pH regulator. U~ing a glucose/mineral salt
medium, Eppstein et al . ( 1989 ) achieved 39 g of a~/l by
additional subsequent feeding with salts. Fas~ et al.
(lgB9) achiered 45 g of XJl by metering~ glut:ose ~olution,
which al~o contained magnesium 3ulphate, into a
glucoYe/mineral salt ~edium which had been initially
introduced. Additional ~ubsequent feeding with salts by
Pan et al. (1987) resulted in ~5 g of X/l.
Other fed-batch fermentations were carried out in
gluco~s/mineral ~alt media whic~, beforQ inoculation
thereof, additionally contained yea~t extract or auto-
lysad yeast powder. 38 g of ~Jl were obtained without
additional ~ub~equent feeding during cell growth apar~
from glucose and ammonia (Mori et al. lg79~. Additional
matering in of ~alt-~ produced higher biomas~ concentra-
tions with 47 g of ~/l (Bausr and White 1976), 55 g of
~/1 (Shiloach and Bauer 1975) and 68 g o~ X~l (B~uer and
ZiY 1976). Additional metering in of ~alts, trace ele-
ments and vitamins re ulted in 70 g of X/l (Fie~chko and
3n Rit~ch 1986). It wa~ possible to obtain very high call
densitie~ of 110 g o~ X/l ~Cutayar and Psillon 1989) and
125 g o~ X/l (~ori et al. 1979) by co~plex ub~equant
feeding with ~alt~, yea~t extract and trace el~ment~ in
addition to the customary me~ering in of gluco~e and
ammonia. Further ~uppl~mentation of a glucose/mineral
~alt medium which already contained yea~t extrac~ before
inocula~ion with Bacto tryp~one or pep~one, and the
metering in of thase complax substrat2~ ~uring the
fermontation re~ulted in no further increase in th
_ 3 ~
bioma3s concentration (~ailey et al. 1987; Tsai et al.
1987). Very high biomass concentrations o~ 80 to 105 g of
X/l (~ppstein et al. 198~) were achievad by combina~ion
of th~ ~ed-batch technique de~crib~d above (me~ering in
of glucose and salts) by introduction of air enriched in
oxygen. Kr~ger (1989) achieved 112 g of ~/1 by introd~c-
ing pure oxygen gas.
All the proc~s~es described above are associated
with the di~ad~an~age tha~ thay represent very co~plex
subsequent feeding ~y~tem~. ~he addition of all the
additional n~trients apart fro~ glucose, or gluco~e mixcd
with magne~ium sulphate, a~d æmmonia requires specific
metering in of nutrient sub~trates, which co~pri~e
either o~ly intermittent addition~ or time-controllQd
addition phas3~. This also applie~ to the inpst of the
ga~eous oxygen ~ub~trata. The aim is a process for
achieving high cell den~ities u~ing a gluco~e/minsral
salt medium without the nece~sity for fur~her nu~rient~,
apart from tha usual gluco~e ~olution mixed with mag-
ne~ium sulphate, and ammonia, with the u~u~l introductiono~ air, and where po~sible without the nec2s~ity for
enrichment with pure oxygan.
E. coli cells which act as ho~t~ for vectors and
thu~ are used for the production of recombinant DNA
product3 must b~ fermented appropriately for the ex-
pression y~tem to be employed for ~ha recombinant
product. ThiY mean~ that E. coli ho~t-~ with expre~ion
systæms which c~n b~ ~witched on are inltially fermentQd
at high eell d~nsitis~ and then the ~witching on of the
recombinant D~A e~pre~ion i~ carried outc Al~o known for
. coli ar~ a n~mber o~ homologou~ and h2terologous
expre~ion ~y3tem~ which are con~titutive or ~or which
expr~ssion i5 higher at low~r SpQCi~iC growth rate~ than
at the ~aximu~ ~pecific growth rate pos~ible, ~, in the
3S particular nut~ient medium. ~he ~pecific growth rate i~
defined as follow~:
~ . d~/dt.
~ 4 -
In .~ome ca~e a ~rowth of E. coli with ~
has bèen achieved. Thus, Zabriskie and Arcuri (1986)
achieved growth of the culture with ~ by feeding
the carbon source into the fermentation solution at a
constant rate which was lower than that which would have
resulted in ~. However, the di ad~antage of this
procedure i8 that ~ change continuously. If tha metering
rate were kept con~tant throughout the fed-batch proces~,
~ would f~ll continuously.
Allen and Luli ~1985) developed a ~trategy for
the intermittent read~u~tment of the ~pecific growth rate
~ with increasing cell den~ity by readjusting the ~eeding
rate~ of the carbon source from time to tLme. The
implicit disadvantage of thi~ ~trat~gy i~ that ~ changes
lS co~tinuou~ly in a tolerance range which d~pend~ on the
frequency of the off-line measurement~ of gluco~e and
cell d3nsi~y, although growth is at ~ < ~. Changes in
the 3pecific growth rate entail fluctuation3 in mata-
boli~m and may ha~e a de~tabili~ing effect, especially
when there i~ production of m~tabolic by-product~ with
inhibitor~ effect~. Lee and Mohler (1989) prs~anted a
controlled growth-rate fermentation in order to achieve
grow~h at constan~ ~. The core of the procedure is
continuou~ mea~urem~nt of ths carbon dioxide released by
the cell~ during growth, the instan~a~eous growth ra~e
calcula~ed from this by c~mputer, and the imm~diate
ad~ust~nt, which i3 nece~ary to main~ain the required
gxowth rate, o~ th~ metering rate of th~ carbon souxco
with the coupled feeding o~ the C ~ource. ~ee and Mohler
~0 (1989) were able in ~hi~ way to cultiva~a ~. coli a~
approximately con~tant ~ < ~ the range of cell
d~nsities ~om 0.3 t~ 60 g o~ ~/1 (EP 031594~ ~2). T~
3trategy of maintaining ~ constant via msa3urement of ~he
variable C02 is, however, as~ociated in principle with the
di~ad~antage that CO2 is ~ot in every case a growth-corre-
lated ~ignal. ~his particularly applies when change~ inmetaboli~m take place or a~aplerotic bio ynthetic path-
way~ are u~ed by the cell~, which may ocour par~iculaxly
~ith very high cell den~ities and carbon deficiency.
-- 5 --
Another disadvantage of the hee and Mohler procedure is
that growth rapidly Rtop~ after exhau6tion of all ~he
po3sibilitie~ for increa~ing the oxygen input in the
particular ferment0r 3y8tem U ed, because the di~olvsd
oxygen concentration decrea~Qs immediately. Thu~, after
the growth phase at ~ - const ~ , no detectable
prolongation o~ growth to increase the cell density
further at ~ c ~3~ iq posYibla when ~ i3 continuou31y
decreasing. Furtharmore, the di~solved carbon dioxide
concentration depends ve~y greatly on ~he pH in the
ad~usted p~ range; small pH change~ have adverse effect~
on the measured signal and may result in falsification of
the ~alculation of ~
It is therefore al~o the intantion that this
inYention de~criba a fermentation proces~ ab12 ~pecifi-
cally to re~ult in ve~y high cell densitie~ of E. coli
with ~table metabolism.
Ai~ o~ th~ i ven~io~
Tha aim of the invention compri~e~ de~cribing a
proceoR for achi~ving high cell densitie~ of E. coli in
an agitated tank fermanter, where srowth at the maximum
specific growth rate is followed by forcing growth at
~ubmaximum ~pQCi~iC growth rate, and the dissolved oxygen
concentration in the fer~entation msdium is maintained at
or above a defined value.
Su~ary of thQ iN~e~ion
~ he invention ha3 the ob~ect of describing, with
avoidance of the di3advantages of th~ prior art, a
proc~ with which B. coli can be fermented in a gluco~e/
~i~eral ~alt m~dium in ~n agitated tank fermenter to high
cQll dQnsities without further metering in of other
nutrient 3ub~trate~, apart fro~ tha subsequent feeding
with gluco~e ~upplemented with magnesium ~ulphate,
ammonia and an~ifoam agent, being nece~ary and without
oxygen-enrichment of ~he air which i3 introduced bein~
necessary. The intention in thi~ connection wa~ that
formentation be carried out in ~uch a way that the growth
~- 6 - 2~ 7~
of the E. coli cells takes place initially at the ma~imu~
specific growth rat~ and then ater an appropriate
interval at ~ubmaximum ~pecific growth rate.
The object on which the invention i~ bas~d i~
S achieved by a proce~ for the high call-density fexmenta-
tion of Eschorichia coli in an agitated tank fermenter,
which i~ characteri~ed in that fsrmhntation is carried
out in a medium containing a nitrogen source, organic
carbon source and mineral salt~, and ~he f~rmentation is
carxied out in ections by providing
- fir~tly a batch section with maxi~um specific growth
ra~e and
- then a fed-ba~ch section with ~ubmaximum specific
growth rata, the latter being co~trolled and/or regulated
15via the o~ygen i~put~ using a ~lucose solution supple-
mented with ma~ne~ium ulphate.
j. According to a spacific embodimen~, a time
I profile with an approximat~ly con3tant ~ubmaximum
~pecific growth rate can be provided in the fed-batch
20~ction. It is furthermora pos~ible for the fed-batch
s2ction to be comple~Qd, after the maximum oxygen input
predete~mined by the agitated tank fermenter ha~ bee~
r~ached, by a sub~ection in which the ~pecific growth
rate decre sa~.
25To carry out the proGess according to the in~e~-
tion it i~ pos~ible to fermant E. coli R12 or a deriva-
tive thareof and, preferably, the derivatiYe ~. coli TGl.
It i po~sible to carry out ~he ferment~tion in
the pr~e~ce of ~upplin~ a~d thereby to compensate
30au~o~rophi~s. In the c~e of a thia~in~ auxotrop~y, the
~. coli ~rain can b~ ~e~m~nted in ~he ~re~once of
thiamine. Fo~ exam~le, ~. coli ~Gl can be fermen~ed in
the prRsen~e o a thihmine concentration s 4.5 m~/l.
It is pos~ible to introduce the gaseous o~yqen
35only with the aid of air throughout the ferm~ntation.
~owover, it i~ al~o pos~ible for the sa~eou~ oxygen ~irRt
to be in~roduced with ~he aid of air and then to use air
onriched with oxy~en, or oxygen.
~ono~accharides, di~accharide~ and/or polyols
- 7 ~ J~J~ 7~
can be used a~ organic carbon source. ~xæmple8 are
glucos~, ~ucrosa, lactosa and glycerol.
According to a qpe~if ic embodiment, a glucose
~olution which i~ ~upplemented with magnesiu~ ~ulphato
S and contain~ an antifoam agent can be u~ed in tho fed-
- batch section.
According to another ~pecific embodiment o~ the
p~oce~s according to ~ha i~vention, gluco~e ~olution (for
example of a concentration s 700 g/l) supplemented with
0 magnesium ulphate of a concen~ration 19.2 g of
~gS0~.7H20/l can be used in th~ fed-~atch section.
According to another ~pecific e~bodiment, the
nutrient mediu~ ~mployed in the process according to the
in~ention ean already contain all the nutrient 5ub~rate8
for the entire ferme~tation with the exceptions tha~
- aquaou3 ammonia solution (for example of a concentra-
tion < 25%) i9 metered in f or pH ad~ust~ent,
- gluco~e 301ution ( for example of a concentration
~ 700 g/l) ~upplemented with ma~ne~ium ~ulphate (for
example of a conc~ntration < 19.2 g of MgSO~.7H20/l) i~
mete~ed in during the fed-~a~ch ~ection and
- where appropriats an~ifoam age~t, for example Ucolub
N115, iQ metered in.
The nutrient medium employ~d i~ the proce~s
according to the inv~ntion can qualitati~ely compri~ tha
following compono~t~, for example:
gluco~e, pota~sium dihydrogen pho~phate, diammonium
hydrog~n phosphate, magnesiu~ ~ulphate, iro~ ~itrat~,
cobalt chloride, mansane~e chloride, copper chloride;
borlc acid,-~odium ~olybdate, zinc ac~tate, Titriplex III
and/or citric acid and, wher3 appropriate, antifoa~
agent~ and, whare appropriata, ~uppline~ to compensate
auxo~rophies.
In particul~r, the nut~i3nt ~edlum employed can
compri~, for exa~ple, ~he following component~: gluco~e
(< 50 g/l~, RH2PO, (s 13.3 g/l), (~H4)2HPb (S 4 g/1),
NgSO,.7HzO (s 1.2 g/l), iron Gi~rate (5 60 mg/l),
CoCl2.6H~O ( 2.5 mg/l), MnCl2.4H~O (s 15 mg/l), CuCl2.2~2O
(s 1.5 mg/l), H3BO3 (s 3 mg/l), Na~0O~.2H2O Is ~.5 mg/l),
2~a~9 7
~ 8 ~
Zn(CH3C00)z.2H~O (s 8 mg/l), Titriplex III (s 8.4 mg/l)
and/or citric acid ~ 1.7 g/l) and, where apprspriato,
antifoan agent Ucolub ~115 (s 0.1 m~
To carry out the proce3s according to the inve~-
tlon, the constituent~ o~ the nutrisnt medium can beintroduced into th~ fermenter in the following _equence:
- first potassium dihydrogen phosphate, dia~onium
hydrogen phospha~o a~d/or citric acid;
- then a aolution, which i3 made up ~rom ~tock solution~
where appxopriate, of cobalt chloride, manganase chlor
ids, COppGr chloride, boric acid, sodium molybdate9 zinc
acetate andJor Titriplex III,
- then iron citra~e,
- then, wh~re appropriate, an antifoam agent,
after which ~terili~ation i~ carried ou~.
Titriple~ III can be introduced fir~t for the
~olution mada up rom stock ~olution3.
After th& solution which ha~ been introduced
initially into ths fermenter ha~ been 3terili~ed it is
po~sible to intxoduce a ~terili~ed ~olu~ion o~ glucosa
and/or magne3ium sulphatQ into the f~rmenter.
A lag pha3~ may occur after the inoculation o~
the nutrient medium and before ths batch section.
Farmentation in the batch ~ection can be carried
out, ~or exampl~, at a p~ s 7 . 5, in particular in tha
range fro~ 6.6 to 6.9 and preferably at ~bout 6.8. ~he
pEI can b~ ad~u~ted u~ing aqlleous ammonia 8dUtiOIl ( of a
concentration of, for exa~pl~, s 25~).
A~cording to a specific e~bod~men~ of ~he proceRs
a~cording to the inven~ion, a P02 2 1%, praferably in the
range ~rom 5 to 20% and, in p~rticular, about 10%, can be
ad~u~ted in ~he ba~ch section by gradually increa~ng the
sti~rar speed.
The transition to the ~ubmaximum ~peci~ic grow~h
~5 rate in th~ fed-batch sec~ion after the b~tch section i~
complete ca~ be effected by
- ei~her ~educing the s~irrer 3peed, for example in the
range fro~ 5 to 60 min,
- or keeping th~ ~tirrer qpeed con~ant, for example in
-- 9 --
~he ransa from 0.5 to 10 h.
A P02 2 1%, preferably in the range from 5 ~o 20~
and, in par~icular, of abou~ 10%, can be ad~usted in ~he
fed-batch section by means of ~he glueose metQring.
It i~ also possible to ad~ust the requirad sub-
- maximum ~pecific growth rata in the fed~batch section by
inc~easing the o~ygen input. It i~ po-~ible for this
purposa to increase the ~tirrer speed, the gas introduc-
tion rate, the pres~ure and/or the oxygen content o~ the
air, or to introduce oxygen gas.
Whon the ~ubmaximum sp~cific growth rate in the
fed-batch ection i~ monitored, it is po~sible with its
aid to ad~ust the oxygen inpu~ appropriately. ThQ 3ub
maximu~ ~pacific growth rate can be monitored, for
1~ e~ample, by m~ans of the oxyge~ con~ent of the air
l~aaving tho f~ nter.
~ccording to a speciic embodimant of the proce~3
according to the invention, after the maximum technically
po~ible oxygen input has been reached in the fed-batch
section it is po~sible to ferment the cultura further ~y
meanY Of P02 control by me~ering in glucose while the
su~maximum ~pecific ~rowth rate falls.
It qhould also be mentioned, for the ~ake of
complet~ne~s, that th~ concentration of dry biomass can
be altered by alteration of
- the xatio of the duration o~ the ba~ch ~action to the
duration of the fed-batch section andJor
the level of the submaximum specific growth rate, which
; i3 to ba kept approx~ately constant, in the ~ed-batch
~ctionO Thu~, it is pos~i~le to ach~ve a~ least abou~
95 g of X/l, for examplo, on culti~ation of E. coli ~G1
at ~ = 0.107 l/h - con~ in thæ f~d-batch ec~ion.
After it ha~ cea3ed to be pos~ible to increa~e
further the oxyge~ inpu~ effi~iency or the particular
fermsnter ~y8~em, surprisingly ~he presen~ procas~
pe~lt8 further growt~, al~hough with ~alling ~ without
the occur~nce of oxygen d~ficie~cy ~o~ the cell~ in the
culture solution by means of the pre~e~ P02 control. A
dry bioma~3 concen~ration o~ at least 110 g o X/l ha~
- 10 ~ 2~ 3~',,J~
been obtained in the medium describad abov~ with ~. coli
~Gl in thi~ mannar, the final volu~e not e~cesding 76% of
tha fermantar ne~ volume.
The invention i~ explained in detail hereinafter
by a fi~ure and an exemplary embodimen~.
~xempl~ry ~bodiment
~he high cell-den~ity fermen~ation proces~ i9 to
be described in datail taking the example of ~he cultiva-
tion of E~ coli TGl in a 72 1 fermenter (B.Braun
Mel~ungen A~, type 8015.1.01).
The 37 l o~ nutrient 501ution to ba inoculated
cont~ined ~he following con~tituent~: glucoss (25 g/l),
R~2PO4 (13.3 g/l), (NH4)2HP04 (4 g/l), MgSO4-7H20 (1.2 g~l),
iron(III) citrate (60 mg/l), CoCl2 6H2O (2.5 mg~l),
~hC12 4HzO (15 mg/l), CuCl2-2H20 (1.5 mg~l), H3BO3 (3 mg/l),
Na2~oO42H20 (2.5 mg/l), Zn(CH3COO)2-2H20 (~ mg/l)/ thiamine
(4.s mg~l), Titriplex III (8.4 mg/l), citric acid (1.7
g/l) and Ucolub N115 (0.1 ml/l). ~his nutrlent ~olution
wa~ prepared in th~ following way: firstly, 148 g of
~0 (NH4)2HP04/ 492.1 g of KH2PO~ and 62.9 g of citric acid a~
dry ~ubstance were dis~ol~ed in about 30 1 of deioni~ed
watar i~ tha fermanter; this wa~ followed by ~ddition of
257.4 ml of trace mixture ~olution, addition o an
iron(III) citrate solution (2.22 g of iron(III) citrate
in 300 ml) and o 3.7 ml of ~colub NllS. The trace
mixtur~ solu~ion wa~ prepar~d from ~tocX olutions in the
following way: 62.2 ml o~ Titriplax III (5 g/l), 34O3 ml
o~ CoClz-2HzO (2.7 g/13, 34.7 ~1 of CuCl8-2~2O (1.6 g~l),
34.7 of ml M~Cl2-4H20 (16 g/l), 27.8 ml of H3BO3 (4 g~l),
30.8 ml o~ Na2MoO~2H20 (3 g/l) a~d 32~9 ml of
Zn(C~3COO)2-2~O (9 g/l). The ~olution in th~ fer~n~e~
wa~ terili~d in the u~ual way. Also added sub~eq~ently
for c~m~letion-w~re sepaxately ~terili~ed glucose solu-
` tion (1.0175 kg of glucose monohydrate in 2.5 1 of H20)/
sterili~ed magn~ium sulpha~e solu~ion (44.4 g of
NgSO~-7H2O in 1 1 of ~2O), thia~ine ~olution ~terilised by
filkration (166.5 mg in 100 ml of H2O), the pH was
ad~usted t~ 6.8 with aqueous ammo~ia Rolution (25%), and
finally the mixture wa~ made up to 36.8 l with ~eril~
H20. It wa subsequently inoculated with 200 ml of a
thawed glycerol pre~erved cultur~ (20% v/v3 of E. coli
TG1.
E. coli TG1 wa~ cultivated at a tempera~ure of
28C, a pre~sure of 1.5 bar, a pH of 6.8 and a gas
intxoduction rate of 85 l/min. The pH was kept constan~
throughout the fermentation b~ controlled ~e~ering in of
25% NHa. Fig. 1 ~hows the tine cour~e~ of the stirrer
speed, the concentration of dry biomas~ X, the glucose
concentration and the di3solved oxygen concentration.
In the ba~ch ~ection of the high cell-den~ity
farmentation, the culture grew, after the inoculation at
time t ~ O and after a ~hort lag pha~e o about 2 h, at
th~ maximum s~ecific growth rate (~ 0.456 1/h~. Ater
tha PO2 had reached 10% it was subsequently kept con~tant
by controlled increase of the ~irrer speed. The batch
section wa~ co~pletQd after con~umption of the gluco~e
întroduced at the start. The PO2 increased draxtically.
A~t2r thiC there was no further control of the PO2 via
the ~tirrer speed. To reduce tho specific growth rate
fro~ ~ = O.456 to ~ ~ 0.11 1/h, the ~tirrer ~psed wa~
reduced from 420 rpm to 250 rpm over a 30-minute period.
~he PO2 control was ubsequently operated via the meter-
ing in of glucose ~olution (700 g/l) ~upple~ented with
magnesium ulphate (lg.2 g of MgSO~-7~20/l). ~he main-
tena~ce of ~ constant in pha~e 1 of ~he fed-batch ~ection
wa~ effected by increa3ing the oxygen input. For thi~
purpo~e I the ~pead wa~ 9et via a proportional plu~
integral con~rollex ~i~h the control variable ~6. The
~peciic growth rate wa~ determined i~directly from the
o:scyçle~ balance~ of tha 3y~tem by th~ followi3lg fonnula:
a,,~(t)
t
K~ao2(~
where: Qo2 i~ the o~ygen con~ump~ion rate and
R i3 the quoti~nt of the dry bioma~
12 ~ 'al~
concentration at time to and ths yield cesf~ici0nt
for oxygell.
A dry bioma3~ concen~ration of 95 g/l ~a~ reached
at l:h~ ~d of the first pha~e of the fadbatch ~ection
S Becau~e it wa3 no lon~er po~i~le to increase the oxys~er
inpu~ into the culture ~olution ~fter the maaci;mum techni~
cally possi~le Rtlrrar ~peed had been ~ached, ths
culture ~hen g:re~ in phase 2 of the ~ed batch section at
a constantly falling ~. A dry bioma~s of 110 g of X/l wa~
obtained at the end of thi~ pha3e and thus at the comple-
tion o~ the ~srmentation.
