Journal of Scientific & Industrial Research

Vol. 65, July 2006, pp. 599-602

Process strategies for cephalosporin C fermentation

Pradeep Srivastava*, Punita Mishra and Subir Kundu

School Of Biochemical Engineering, Institute of Technology, Banaras Hindu University, Varanasi 221 005

Received 19 December 2005; accepted 04 April 2006

Cephalosporin C (CPC), a β-lactam antibiotic, is starting molecule for industrial production of semi synthetic

cephalosporins. CPC fermentation has been carried out in Air Lift Reactor (indigenously made up of borosilicate glass) in

different fermentation modes i.e., batch and fed batch modes using a mold Acremonium chrysogenum (synn.

Cephalosporium acremonium). A definite correlation exists between the antibiotic productivity and flow rate of

supplementary feed. Fed batch process was shown to be more efficient than the batch one, and the process in which the

lowest supplementary feed flow rate was used resulted in significantly higher antibiotic production.

Keywords: Acremonium chrysogenum, Cephalosporin C, Batch fermentation, Fed batch fermentation, Flow rates

IPC Code: C12P17/00

Introduction Cephalosporin-C (CPC), a β-lactam antibiotic,

which like all secondary metabolites, is synthesized in

bioprocesses involving microorganisms. Industrial

production of this antibiotic is still carried out in

conventional batch fermentation in aerated stirred

tank bioreactors by fermentation of submerged

cultures of wild and mutant strain of Acremonium

chrysogenum1. Regulatory mechanisms for secondary

metabolism in this process include repression and

inhibition of the β-lactam synthetases by glucose and

other carbon and energy sources easily metabolized

by the microorganism. These conditions are essential

for cell growth, but drastically suppress the

production of many antibiotics2. The development of

an improved strategy for CPC fermentation requires

proper control of specific growth rates of cells using

certain key nutrients. Various strategies have been

evaluated which include batch, fed batch, semi

continuous and continuous modes. Another strategy

uses, repeated batch reactors3 in tower reactors for

CPC production. Some earlier studies4 suggest the

requirement of a certain level of specific growth rate

of the organism for its maximum biosynthetic

activity.

In batch process, CPC production by

A. chrysogenum in a synthetic medium containing

glucose and sucrose, diauxic phenomenon is

observed. It is characterized by the rapid

consumption of sucrose to form biomass

(trophophase) at the beginning of the process. After

depletion of this carbohydrate by the slow

consumption of sucrose, the more difficult

carbohydrate to assimilate, during which the

majority of the CPC is produced (idiophase) and

cell growth is insignificant. Even when added

slowly, sucrose tends to accumulate in the reaction

medium in virtue of the low activity of the enzyme

that hydrolyzes this carbohydrate, thereby limiting

its consumption rate5. The use of sucrose is,

therefore, a good strategy to obtain high antibiotic

concentration in the conventional batch process, but

is unsuitable for the fed batch process. A viable

alternative is to use hydrolyzed sucrose in the

supplementary medium, which together with the

low cost of this sugar in relation to other

carbohydrates such as glucose and fructose may

make the fed batch process even more

advantageous.

Control on biomass and hence specific growth

rate in fermentation with sufficient supplies of

dissolved oxygen enables maximization of

antibiotic production. A direct correlation has been

developed between morphological variation and

oxygen availability in the broth6. Present study

describes fermentation of CPC in batch and fed

batch and an attempt is made to compare the

various kinetic analyses of the processes. The feed

strategy employed was effective in avoiding oxygen

limitation during the fermentation and helped in

process improvement.

_____________

*Author for correspondence

E-mail: drpradeep19@sify.com

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600

Materials and Methods Organism

A. chrysogenum, obtained from M/s J K

Pharmaceuticals Ltd, Cuddalore, Pondichery, was

maintained on Potato-Dextrose-Agar after incubation

for 5-8 days at 28°C. The surface growth from a 7-day

old slant was suspended in sterilized distilled water

(10% inoculum) and used for inoculation of seed

media.

Culture Medium and Conditions

Germination carried and inoculum prepared7. The

medium for main fermentation of the batch process

contained sucrose, DL methionine and the remaining

components in the same amounts as those in

preculture8. Composition of the initial batch medium

of the fed batch process was the same as that used in

the conventional batch process (Run # 1). The

supplementary medium utilized in Run # 2, #3 and # 4

contained the same components as the initial batch

and previously hydrolyzed sucrose with different flow

rates.

The sucrose used in the supplementary medium

was hydrolyzed at 40°C in a buffer solution (pH 4.5)

of 10-2

M sodium acetate, employing invertase enzyme

in soluble powder form. The fermentation Runs

(Table 1) were carried out in borosilicate fabricated

Air Lift reactor (ALR; Internal loop, 2l) equipped

with instruments for temperature and dissolved

oxygen monitor, and a pH meter. The optimum

temperature is maintained by flowing water through

the jacket and DO by adjustment of air supply. Basic

design of the lab scale ALR was determined9. All

Runs were carried out at 28°C and dissolved oxygen

concentration was maintained around 40% or above

with appropriate airflow. Silicone oil was added to

control foaming whenever necessary.

The batch fermentation Run #1 contained 1.8 l of

main fermentation medium. The feed profile was

designed to control the growth at a constant rate

through the addition of growth limiting carbon source.

The high product formation rate, required at slow

growth rate, depends on having a high viable cell

concentration. Initially, sub optimal substrate

concentration (S0= 20gl-1

) of sucrose was used during

the early growth and after trophophase, substrate feed

was made at constant rate. Fed batch fermentations,

Runs #2, #3 and #4, were started with 1.2 l of medium

and after the completion of trophophase i.e.,

maximum growth attained (37 h), supplementary

medium was fed at established flow rates until 1.8,

2.0 and 1.6 l, respectively were attained at the end of

100 h of feed supply. Purpose of the first fed batch

experiment, Run #2, was to reproduce the sucrose

consumption rate during the idiophase of the

conventional 1.8 l batch Run (Run #1).

Fermentation Runs were performed in triplicate.

During each Runs, samples were collected and

analyzed for substrate, cell mass and CPC. Substrate

consumption rate was determined by DNS method10

.

The weight of growing cell mass was estimated by

dry cell weight method9. CPC was estimated by

Hydroxylamine method11

and confirmed by HPLC.

Dissolved oxygen measurements were carried using a

pre calibrated DO probe (Bioengineering AG,

Sweden).

Results and Discussion

Under batch mode of CPC fermentation Run #1 in

ALR (internal mode), variation of growth and product

profiles (Fig. 1) is in accordance with earlier studies6.

Maximum CPC titer (3.1 gl-1

) was obtained after

120 h of fermentation while cell growth was highest

at 47 h of fermentation. In batch culture, some process

leading to production of antibiotics are sequential i.e.

they exhibit a distinct growth phase (tropophase)

followed by a production phase (idiophase).

Maximum antibiotic yield is obtained by achieving

high cell mass concentration, which leads to high rate

Table 1 Operational condition of the fed batch Runs

Fermentation

Run

Final

volume, l

Flow rates

ml/h

CPC production

g/l

Growth

rate µ h-1

#1 1.8 Batch Run 3.1 0.048

#2 1.8 6 3.1 0.040

#3 2 8 2.7 0.046

#4 1.6 3.8 3.6 0.037

Fig. 1 Biomass, substrate and cephalosporin C fermentation in

Air Lift Reactor (batch fermentation), Run # 1

SRIVASTAVA et al: PROCESS STRATEGIES FOR CEPHALOSPORIN C FERMENTATION

601

of CPC production and maintaining these values as

long as possible12

. Cell growth (maximum, 13.5 g/l)

took approx 47 h. Synthesis of CPC increased up to

3.1 g/l then decreasing together with cell

concentration possibly due to substrate depletion in

total processing time.

Cell concentration results show that maximum

values are reproducible and are close to those for the

biomass in conventional batch process (about 13.5 g

cell/l). Due to addition of the medium, cell

concentration of the all fed batch fermentation process

remained practically constant and was near its

maximum value during the entire phase; however, the

substrate was used for its maintenance. Depletion of

carbon source and the subsequent start of the

idiophase in the fed batch Runs were anticipated in

relation to the simple batch fermentation. Addition of

supplementary medium was started after about 37 h

fermentation at all flow rates.

In Run # 2 (Fig. 2), with the operational conditions

equivalent to the batch fermentation (Run #1), the

maximum CPC concentration was on the order of that

obtained in the batch fermentation (3.1 g CPC/ l), the

difference being that this concentration was attained

after 75 h of fermentation and remained practically

constant during the entire feeding stage. The growth

rate observed was quite low compared to growth rate

during batch fermentation. Run # 3 (Fig. 3) was

performed at a flow rate 33% higher than that in Run

#2 and the maximum CPC concentration obtained

(2.1 g CPC /l) was 33% less. The results suggest the

occurrence of significant catabolic repression due to

the higher mass of sugars supplied in relation to the

former condition. Run # 4 (Fig. 4) reproduced the

production phase of the conventional batch process

for 1.6 l, supplying feed at a flow rate 33% lower than

that in Run #2. High production rates can be observed

after 70 h of fermentation. After this, production can

be seen to be increasing although at lower rates,

attaining 3.9 g /l at 120 h and 3.6g /l. at the end of the

process without any signs of apparent degradation of

the antibiotic was approx 30% higher.

Results showed high rates of CPC production as

Fig. 2 Concentration profile of biomass, substrate and

cephalosporin C fermentation during fed batch fermentation in Air

Lift Reactor, Run # 2 (flow rate, 6 ml/h)

Fig. 3 Concentration profile of biomass, substrate and

cephalosporin C fermentation during fed batch fermentation in Air

Lift Reactor, Run # 3 (flow rate, 8 ml/h)

Fig. 4 Concentration profile of biomass, substrate and

cephalosporin C fermentation during fed batch fermentation in Air

Lift Reactor, Run # 4 (flow rate, 4 ml/h)

J SCI IND RES VOL 65 JULY 2006

602

well as upkeep of maximum cell concentration during

the entire idiophase under all feeding conditions.

Several conditions studied resulted in a significantly

higher productivity in the lowest flow rate at a lower

specific growth rate (0.0441 h-1

), certainly due to

lower catabolic repression. A lower flow rate may still

be investigated, taking care to maintain the integrity

of the cell population by supplying a minimum of the

remaining nutrients.

Conclusions The increase in production of CPC is achieved as

the flow rate of supplementary medium is reduced in

the fed batch fermentation process. Lower growth

rates are observed during CPC production in fed batch

mode. In general, slow addition of supplementary

feed containing hydrolyzed sucrose resulted in

reduction in catabolic repression and thus it can be

more advantageous strategy than the batch process in

ALR. The fed batch fermentation favored

maintenance of higher antibiotic production rates

during the process in relation to those obtained in the

batch process, resulting in a higher specific antibiotic

production.

Acknowledgement

Authors thank UGC, India, for the financial

assistance.

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