Fed-Batch Bioproduction of Spectinomycin
17
sample, process the sample and measure the antibiotic concentration. Microbiological techniques employ Escherichia coli for assaying the chloride and sulphate
salts respectively. The turbidimetric methods developed for spectinomycin require
a substantial amount of time and are subject to interference from a variety of
factors. Moreover, the assay reflects the potency of the sample and does not
provide information about the degradation products and intermediates.
4 Fed-Batch Cultivation
Spectinomycin bioproduction proceeds in three steps. First, a spore suspension
is thawed for inoculating the seed culture. Alternatively, the slants are incubated
and plated on petri dishes to generate spores to be used in seed culture
preparation. Petri dishes incubated at 32~ require 4 days to produce spores.
Next, the seed culture is grown at 32~ for 33-36 h to obtain a rich and heavy
inoculum in exponential growth phase. Finally, the production bioreactor is
inoculated with 10%o of the final medium volume of seed culture for growth and
production. The age of the seed culture is critical in determining the quality of
growth and production, and should not cross 36 h. In the case of inoculum
preparation, some degree of variability is expected. Although small variations in
the inoculum size will not significantly affect the bioproduction, a reasonable
degree of control is necessary.
For the fed-batch cultivation, the bioproduction medium is prepared according to a standardized composition and procedure as described in Sect. 3.1
and 3.2. The bioreactor operates with an agitation of 200 rpm, at 28~ and
pH adjusted to 6.4 before inoculation, dissolved oxygen is preset to some defined
initial value. The pH, temperature, dissolved oxygen, air flow rate and agitation
speed were monitored on-line with a computer using a software developed
for data acquisition and control. The dissolved oxygen was controlled by
manipulating only the air flow rate, and the pH was controlled by adding acid or
base. Whereas temperature and agitation were controlled with B-Braun control
modules, glucose was measured as described in Sect. 3.3, and spectinomycin was
assayed by a turbidimetric method using Escherichia coli.
The fed-batch production was conducted for glucose feed concentrations of
100, 125, 150, 175, 200 and 400 g l-1. The dissolved oxygen in the medium was
controlled at 50% of the saturation value for all bioproduction runs. In this
manner the glucose feed concentration for the highest yield and the correlation
between glucose and oxygen demand were determined.
4.1 Glucose Feed Optimization
This optimization accounts only for the carbon and oxygen resource requirements for production in a 15 1 bioreactor. Since these two variables are critical
17
sample, process the sample and measure the antibiotic concentration. Microbiological techniques employ Escherichia coli for assaying the chloride and sulphate
salts respectively. The turbidimetric methods developed for spectinomycin require
a substantial amount of time and are subject to interference from a variety of
factors. Moreover, the assay reflects the potency of the sample and does not
provide information about the degradation products and intermediates.
4 Fed-Batch Cultivation
Spectinomycin bioproduction proceeds in three steps. First, a spore suspension
is thawed for inoculating the seed culture. Alternatively, the slants are incubated
and plated on petri dishes to generate spores to be used in seed culture
preparation. Petri dishes incubated at 32~ require 4 days to produce spores.
Next, the seed culture is grown at 32~ for 33-36 h to obtain a rich and heavy
inoculum in exponential growth phase. Finally, the production bioreactor is
inoculated with 10%o of the final medium volume of seed culture for growth and
production. The age of the seed culture is critical in determining the quality of
growth and production, and should not cross 36 h. In the case of inoculum
preparation, some degree of variability is expected. Although small variations in
the inoculum size will not significantly affect the bioproduction, a reasonable
degree of control is necessary.
For the fed-batch cultivation, the bioproduction medium is prepared according to a standardized composition and procedure as described in Sect. 3.1
and 3.2. The bioreactor operates with an agitation of 200 rpm, at 28~ and
pH adjusted to 6.4 before inoculation, dissolved oxygen is preset to some defined
initial value. The pH, temperature, dissolved oxygen, air flow rate and agitation
speed were monitored on-line with a computer using a software developed
for data acquisition and control. The dissolved oxygen was controlled by
manipulating only the air flow rate, and the pH was controlled by adding acid or
base. Whereas temperature and agitation were controlled with B-Braun control
modules, glucose was measured as described in Sect. 3.3, and spectinomycin was
assayed by a turbidimetric method using Escherichia coli.
The fed-batch production was conducted for glucose feed concentrations of
100, 125, 150, 175, 200 and 400 g l-1. The dissolved oxygen in the medium was
controlled at 50% of the saturation value for all bioproduction runs. In this
manner the glucose feed concentration for the highest yield and the correlation
between glucose and oxygen demand were determined.
4.1 Glucose Feed Optimization
This optimization accounts only for the carbon and oxygen resource requirements for production in a 15 1 bioreactor. Since these two variables are critical