Fed-Batch Bioproduction of Spectinomycin
15
are capable of giving high precision measurements of bioreactor off-gas. This
enables the application of elemental balancing or related techniques to obtain
information about biological rates directly. A comparison of off-line measurements with on-line estimation of state variables (poly-fl-hydroxybutyric (PHB)
acid, residual biomass, nitrogen source and glucose) using an elemental balancing technique [38] shows excellent reconstruction.
The only disadvantage with mass spectrometry is its high cost. The design
and cost of the mass spectrometer varies considerably with the species to be
analyzed and the resolution desired. Consequently, research has continued in
the development of new instruments for measuring off-gas. Christensen et al.
[39] have developed an acoustic off-gas analyzer with performance comparable
to that of a mass spectrometer. Carbon dioxide and oxygen can be analyzed
continuously with this instrument with a relative accuracy of 0.014).33% in
0.2 s. Sipior et al. [40] have designed a phase fluorometric optical carbon
dioxide gas sensor that can be made at a cost less than US$1000. The sensor is
based upon the change in lifetime of a donor (sulforhodamine 101) fluorophore
induced by fluorescence resonance energy transfer to a pH sensitive non fluorescent acceptor, m-cresol purple. On-line carbon dioxide measurements every
6 s with this sensor compared well with off-line measurements by GC requiring
20 min analysis time. The authors indicate that this sensor has potential in vivo
applications.
In industrial scale spectinomycin production, temperature, power input,
dissolved oxygen and pH are measured on-line. Off-gas analysis is normally
performed using paramagnetic oxygen analyzers and infrared carbon dioxide
analyzers. Glucose and spectinomycin are however measured off-line. Cell mass
concentration is not reported since it cannot be measured accurately in complex
medium, especially because Streptomyces exhibits filamentous growth. Sometimes viscosity measurements are correlated to the physiological state of the
bioreactor and used to determine the start of spectinomycin production.
For the data presented here, temperature, dissolved oxygen and pH were
measured on-line. The residual glucose concentration was measured off-line by
an enzymatic method. Samples were taken from the bioreactor and first centrifuged in 50 ml sterile tubes at 3000 rcf for 15 rain. Then 10 ml of the resulting
supernatant was spun down at 14 000 g in a microcentrifuge to remove the finer
suspended particles. Finally, the resulting clear supernatant was used for
measuring glucose in a YSI model 27 industrial analyzer. Spectinomycin was
assayed using a turbidimetric method. Problems associated with its measurements are discussed in the following section.
3.4 Spectinomycin Estimation
The absence of UV absorbing chromophores and the inherently weak chemical
structure of spectinomycin makes it a difficult molecule to assay. Consequently,
many different types of assay methods have been tried [23,41-45]. The
15
are capable of giving high precision measurements of bioreactor off-gas. This
enables the application of elemental balancing or related techniques to obtain
information about biological rates directly. A comparison of off-line measurements with on-line estimation of state variables (poly-fl-hydroxybutyric (PHB)
acid, residual biomass, nitrogen source and glucose) using an elemental balancing technique [38] shows excellent reconstruction.
The only disadvantage with mass spectrometry is its high cost. The design
and cost of the mass spectrometer varies considerably with the species to be
analyzed and the resolution desired. Consequently, research has continued in
the development of new instruments for measuring off-gas. Christensen et al.
[39] have developed an acoustic off-gas analyzer with performance comparable
to that of a mass spectrometer. Carbon dioxide and oxygen can be analyzed
continuously with this instrument with a relative accuracy of 0.014).33% in
0.2 s. Sipior et al. [40] have designed a phase fluorometric optical carbon
dioxide gas sensor that can be made at a cost less than US$1000. The sensor is
based upon the change in lifetime of a donor (sulforhodamine 101) fluorophore
induced by fluorescence resonance energy transfer to a pH sensitive non fluorescent acceptor, m-cresol purple. On-line carbon dioxide measurements every
6 s with this sensor compared well with off-line measurements by GC requiring
20 min analysis time. The authors indicate that this sensor has potential in vivo
applications.
In industrial scale spectinomycin production, temperature, power input,
dissolved oxygen and pH are measured on-line. Off-gas analysis is normally
performed using paramagnetic oxygen analyzers and infrared carbon dioxide
analyzers. Glucose and spectinomycin are however measured off-line. Cell mass
concentration is not reported since it cannot be measured accurately in complex
medium, especially because Streptomyces exhibits filamentous growth. Sometimes viscosity measurements are correlated to the physiological state of the
bioreactor and used to determine the start of spectinomycin production.
For the data presented here, temperature, dissolved oxygen and pH were
measured on-line. The residual glucose concentration was measured off-line by
an enzymatic method. Samples were taken from the bioreactor and first centrifuged in 50 ml sterile tubes at 3000 rcf for 15 rain. Then 10 ml of the resulting
supernatant was spun down at 14 000 g in a microcentrifuge to remove the finer
suspended particles. Finally, the resulting clear supernatant was used for
measuring glucose in a YSI model 27 industrial analyzer. Spectinomycin was
assayed using a turbidimetric method. Problems associated with its measurements are discussed in the following section.
3.4 Spectinomycin Estimation
The absence of UV absorbing chromophores and the inherently weak chemical
structure of spectinomycin makes it a difficult molecule to assay. Consequently,
many different types of assay methods have been tried [23,41-45]. The