12
J. Gomes and A.S. Menawat
Since ATP synthesis depends directly on the respiratory action, a bioenergetic consideration indicates that both glucose and oxygen requirements require
proper balancing for high yields in spectinomycin bioproduction. Further, on
examining the mechanism of the two possible rate limiting steps, and considering that the microorganism possesses an enhanced anaploretic pathway for
assimilating amino acids for growth, we find that methylation controls the rate
of antibiotic synthesis. This implies that spectinomycin biosynthesis begins after
the growth phase. However, since the organism requires nutrients for maintenance and survival even during production, an optimum glucose concentration
exists for maximal antibiotic production. These aspects should be considered in
formulating a scheme for spectinomycin bioproduction.
3 Bioproduction of Spectinomycin
3.1 Preparation of Inoculum
The inoculum for spectinomycin bioproduction is started from a spore suspension. The spore suspension obtained either from 20% glycerol suspensions or
sporulating petri dishes, is transferred aseptically into the seed culture vessel.
For laboratory scale bioproduction, the inoculation medium contains dextrose,
cotton seed flour, brewers yeast, potassium phosphate and antifoam agent. In
larger scale productions the dextrose may be replaced with a more economical
source of carbon such as cerelose. Cotton seed flour and brewers yeast are
excellent sources of amino acids and essential minerals for healthy growth of the
microorganism. The initial pH is normally about 5.7 and is not adjusted before
inoculation. The seed culture vessel is normally incubated at 32~ for about
33-36 h to obtain a heavy inoculum. This duration of incubation is sufficient to
ensure that the seed culture is in the mid-exponential phase of growth. The
amount of dextrose and potassifim phosphate is critical in determining the
quality of the seed culture in terms of spectinomycin productivity.
The seed culture vessel may be simply an Erlenmeyer flask for laboratory
scale or a small bioreactor for pilot scale bioproduction. In the case of large scale
industrial bioproduction the seed culture vessel is a well equipped reactor with
options for in-situ sterilization and compressed air transfer facility to the
main production bioreactor. In both cases the inoculum is grown in the batch
mode.
Alternatively, two or three petri dishes may be plated from a healthy slant
and incubated at 32~ The dishes sporulate in about 34 days. This essential
extra time requires planning and may not be acceptable for certain production
facilities. However, for laboratory scale operations this technique is often followed to ensure that the microorganism is still good for antibiotic production.
The spores may then be dry-scraped and suspended with sterile water and used
J. Gomes and A.S. Menawat
Since ATP synthesis depends directly on the respiratory action, a bioenergetic consideration indicates that both glucose and oxygen requirements require
proper balancing for high yields in spectinomycin bioproduction. Further, on
examining the mechanism of the two possible rate limiting steps, and considering that the microorganism possesses an enhanced anaploretic pathway for
assimilating amino acids for growth, we find that methylation controls the rate
of antibiotic synthesis. This implies that spectinomycin biosynthesis begins after
the growth phase. However, since the organism requires nutrients for maintenance and survival even during production, an optimum glucose concentration
exists for maximal antibiotic production. These aspects should be considered in
formulating a scheme for spectinomycin bioproduction.
3 Bioproduction of Spectinomycin
3.1 Preparation of Inoculum
The inoculum for spectinomycin bioproduction is started from a spore suspension. The spore suspension obtained either from 20% glycerol suspensions or
sporulating petri dishes, is transferred aseptically into the seed culture vessel.
For laboratory scale bioproduction, the inoculation medium contains dextrose,
cotton seed flour, brewers yeast, potassium phosphate and antifoam agent. In
larger scale productions the dextrose may be replaced with a more economical
source of carbon such as cerelose. Cotton seed flour and brewers yeast are
excellent sources of amino acids and essential minerals for healthy growth of the
microorganism. The initial pH is normally about 5.7 and is not adjusted before
inoculation. The seed culture vessel is normally incubated at 32~ for about
33-36 h to obtain a heavy inoculum. This duration of incubation is sufficient to
ensure that the seed culture is in the mid-exponential phase of growth. The
amount of dextrose and potassifim phosphate is critical in determining the
quality of the seed culture in terms of spectinomycin productivity.
The seed culture vessel may be simply an Erlenmeyer flask for laboratory
scale or a small bioreactor for pilot scale bioproduction. In the case of large scale
industrial bioproduction the seed culture vessel is a well equipped reactor with
options for in-situ sterilization and compressed air transfer facility to the
main production bioreactor. In both cases the inoculum is grown in the batch
mode.
Alternatively, two or three petri dishes may be plated from a healthy slant
and incubated at 32~ The dishes sporulate in about 34 days. This essential
extra time requires planning and may not be acceptable for certain production
facilities. However, for laboratory scale operations this technique is often followed to ensure that the microorganism is still good for antibiotic production.
The spores may then be dry-scraped and suspended with sterile water and used