PROCESS FOR THE FERMENTATIVE PRODUCTION OF CEPHALOSPORIN C USING ACREMONIUM CHRYSOGENUM

METHODE DE PRODUCTION DE CEPHALOSPORINE C PAR FERMENTATION A L'AIDE D'ACREMONIUM CHRYSOGENUM

English Abstract

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Abstract of the Disclosure
Process for the fermentative production of cephalo-
sporin C using Acremonium chrysogenum
There is described a process for the fermentative produc-
tion of cephalosporin C in which the fermentation solu-
tion is filtered during the fermentation through a cross-
flow filtration system. The amount of filtrate which has
been drawn off from the fermenter can be replaced.

Claims

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Patent claims:
1. A process for the fermentative production of cephalo-
sporin C using Acremonium chrysgenum, which comprises
filtering the fermentation solution during the
fermentation through a cross-flow filtration system and
the possibility of replacing the amount of filtrate in
the fermenter.
2. The process as claimed in claim 1, wherein a polymer
filter or ceramic filter is used as the cross-flow
filtration system.
3. The process as claimed in claim 1 or 2, wherein the
filter has a pore size of between 4 and 200 nm.
4. The process as claimed in one or more of claims 1
to 3, wherein the flow rate over the filter surface is 1
to 10 m/s.
5. The process as claimed in one or more of claims 1
to 4, wherein the filtrate solution in the fermenter can
be replaced by water.
6. The process as claimed in claim 5, wherein the filtr-
ate solution is replaced by a nutrient solution.
7. The process as claimed in claim 6, wherein the filtr-
ate solution is recycled to the fermenter after the
valuable product has been removed.

Description

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De~criptlon
Process for the f~rmontatlve production o~
cephalosporin C u~ing Acromonium chry~ogenum
Antibiotic~-containing culturo liquid~ are genorally
worked up a~ter the maxlmum contont ha~ b~on roachod. In
most ca~e~, the first step i~ a separation of cells and
solids by filtratlon or by contrifugation. Thi~ may be
followed by concsntrating tho valuable con~tituents by
extraction or by adsorption. As a rule, tho purifiod
product is thon crystallized and can be proceosod to give
~emisynthotic antibiotics.
A disadvantage in thls step-by-~tep method ie the insta-
bility of a large number oi antibiotic molecules such as,
for example, cephalosporin C (CPC). CPC ie already
degraded during the fermentation. This can be effected
either purely chemically, by water attacking the ~-lactam
ring, or onzymatically, for example by ostorasos ~Ronocny
et al., 1973, J. of Antlb., 26, 3, 135-141). A series of
aecondary products such a~ deacotoxycephaloeporin C
(DoCPC) and deacetylcephalosporin C (DCPC), which must bo
removed from the CPC during the purification step, is
also formed cluring the fermentation. Thl~ ro~ult~ ln
considerable yleld losses. Cross-flow flltratlon ~ystoms
using polymer or ceramics membranes have already been
used for the worklng-up of culture ligulds which contain
antibiotics (Harris et al., J. Chem. Techn. Biotechnol.,
1988, 42, 19-30). A reduced decomposltlon of
cephalosporin C ln thoso proco~sea has not boen
demonstrated as yet. It 18 known to lsolate cephalosporln
C after the fermentatlon u~lng cross-flow flltration (M.
Ralyanpur et al., Contrlbuted Paper, Chapter 32, pagos
455-470).
It is an ob~ect of the pro~ont invontion to flnd a
proces~ in which the dogradation of cophalo~porin C and
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HOE 91/E~' 262-Dr.TI~/~ch
the formation of ~econdary productl~ can be roduced and
the yield relative to the amount of ~ub~trate e~ployed
can be increa~ed.-
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Surprisingly, it has now been found th~t th- u~ of a
cross-flow filtration module during the f~rm-ntation of
Acremonium chry~ogonum allowed the CPC ylold to be
increasod, the formation of DCPC to bo roducod and the
production time to be extendod. Tho filtration and the
fact that small amounts of DCPC aro formod furthermoro
simplify th0 working-up of cephalo~por~n C ~ubetantlally.
The invention therefors relatos to a procoss ~or the
preparation of CPC wh~ch comprl~es fllterlng tho ferment-
ation solut~on durlng the formont-tlon through a cross-
flow filtratlon systam and the poselblllty of replaclng
the withdrawn amount of flltrate ln the formenter.
Acremonium chry~ogenum (Cephalosporlum acremonlum) as
well as mutants and eeloctant~, as long as thoy produco
CPC derivatlves, aro suitable for u~o in tho procoss
according to the lnventlon.
The nutrient ~olutlon contalns carbon sources auch as
~ucrose, corn 13tarch, doxtrose or molasses, and nitrogen
sources 13uch a6 ~oyboan meal, groundnut meal, malt
extract or ammonium acetate.
The nutrient modlum all30 contalns lnorganlc salts such as
~odium hydrogsn phosphate, sodium chloride, calcium
chloride, calclum ~ulfato, calcium carbonate, magnesium
sulfate or potassium hydrogen phosphate. Fat such as
methyl oleate or soybean oil can also be added to the
nutrient medium. Trace elements such a~ salts of iron,
manganese, copper, zinc, cobalt or othor metals are also
added.
, Acremonium chrysogenum ~Cephalosporium acremonium),
i preferably DSM 6473, 18 cultured at temporaturee betwoon
20C and 30C, proforably ~t 25C, and at a p~ of betwoon
and 8, preferably pH 7. It le flr~t cultured
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aerobically in a ~haker fla~k and th~n in ~ fermont~r
with s~irring and 4eratlon with air or pur~ oxygen. The
microorgani~ms are culturod in the formontorn ovQr A
period of from 120 to 2~0 hour~, preferably b~tw~qn 130
and 170 hours.
The cross-flow filtration ~yst~m~ uoed can be plate
module~, tube modules, cap~llary modules, wound moduleA
or hollow-fiber membrano module~ made of polymers, carbon
or Gsramic~ and having ~eparation bordoro from the
ultrafiltration to tho ~terile-filtration rango. Filtra-
tion modules which are preferably employod are tho~e
which have a pore size of 0.2 ~m or 4 nm. The membrane
materials which can bo omployed are poly~ulfones, poly-
amide~, cellulose acetate, aluminum oxide or zlrconium
oxide. The filtration can be affected continuously or
batchwise. It ~tarts approximately 2 - 3 day3 after
inoculation of the fermenter and can be continued until
the fermentation ha~ ended. The flow rate of the ferment-
er ~olution over the filtration ~urface is between 0.5
and 20 m/~, preferably 1 to 10 m/s.
The filtrate which is drawn off from the fermenter during
the fermantation can be replaced by a corresponding
amount of li~lid or, alternatively, the filtrate can be
recycled to the fermenter after the valuablo product has
been ~eparated, for oxample by absorption. To this end,
water, enriched with suitablo salts or other nutrient
medium components, can be pumped in.
The liquid volume which has not permeated the membrane is
recycled to the fermenter.
The fermenter and the cross-flow filtration ~ystem are
connected to ~uitable pipes or tubes and sterilized
before the fermentation. Approximatoly 0.2 m' of filtra-
tion surface are required for a 100 1 fermenter. It is
also possible to use greater and smaller filtration
~urfaces.
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Example 1:
Fermentation o$ Acr~monium chry~ogenum DgM 6473
The fermentation was cArried out u~lng th~ followlng
nutrient aolution:
5 Preculture medium g/l
Corn~teep 11.75
Ammonium acetate 4.5
Sucro~e 20.0
CaSO~.2H2O
10 MgSO~7.H2O o 5
p~ 7.0 (ad~usted using 15 % by wt. NaOH)
Fermentation medium g/l
Fat-free groundnut meal 100.0
Ammonium acetate 6.0
15 Glucoee monohydrate 5.0
Methyl oleate 5.0
D,L-Methionino 3.0
CaSO~-2H2O
MgSO47-~2O
20 CaCO3 5.0
A~tifoam o.5
Fed-batch solution:
Gluco~e monohydrate 500.0
D,L-Methionine 24.75
Agar slant~ are used for inoculating 100 ml of preculture
medium (500 ml shakor flas~s eguipped with 4 baffles).
The flaeke are incubated for 48 hours at 150 rotations
per minute (rpm) at 25 to 28C. These culturos are used
for inoculating a furthor preculture (1000 ml of medium,
5000 ml flasks, 25 to 281:, 120 rpm, 58 to 60 hour~). The
eecond preculture i~ ueed for inoculating 60 1
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fermentation medium in a ~tlrred formontor. The
fermentation is aarried out at 25C. The gas flow i~
controlled in such a way that ~he pO, in th- ~erm-ntatlon
nutrient ~olution is above 20 %.
After 74 hours f~rm~ntation, tho filtration 18 ~t-rtod
usin~ a croes-flow coramics modulo (manufacturod by
Membraflow, a-Al~O3) having a filtoring ~urface of 0.2 m'
and a pore sizo of 0.2 ~m. Tho following procoss para-
meters were adhered to:
10 Flow rate: 2 m/s
Pumping rate: 1500 l/h
Filtration capacity: 2 l/h
Filtration tlme: 68 hours.
The table which follows shows the test rosults after15 142 hours of a fermentation without (A) and with (B)
cross-flow filtration:
Table l:
CPC DCPC/ Productivity CPC Productivity
CPC DCPC
t%] l%~ max. t%] after 6 after 6
days [%l days [~]
IA 100 I 100 100 100 100
25 B ~ 44 200 400 53
Example 2
The fermentation is as in Exumple 1. Table 2 shows the
results after 167 hours of a fermentation with cross-flow
~0 filtration (B) comparod with a parallol formontation
without filtration, which was stopped aftor 142 hours:
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Table 2:
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CPC DCPC/CPC
- I ~%) (%)
1~ (142 hour~)100 100
B (167 hour~)129 69
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