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Continuous measurement of water activity
T I B T E C H - A U G U S T 1987 [Vol. 5]
Modelling insufficient mixing W h e n scaling up or scaling d o w n aerobic culture processes, the variation in mixing requirements is the most likely root of performance differences between different scales of reactors of similar geometry. In a large reactor there are likely to be areas of oxygen gradient and oxygen free zones. These can be simulated on the small scale by the inclusion of an oxygen-free c o m p a r t m e n t in an other-
wise aerated reactor. In a recent study, the gradients of oxygen were simulated b y p u m p i n g culture ' m e d i a from a w e l l - m i x e d and aerated reactor along a length of silicon tubing. The tubing was m a i n t a i n e d at the temperature of the aerated reactor and effectively° acted as a plug-flow reactor (no mixing at all). Oxygen-flee areas were simulated by stripping the culture of
A promoter-probing vector for Brevibacterium Brevibacterium ]actofermentatum and related Gram-positive glntamic acid producing bacteria are important in the p r o d u c t i o n of amino acids. Genetic m a n i p u l a t i o n of these industrial organisms is being explored and one of the i m p o r t a n t tools for research on the expression of heterologous genes is a vector to probe the activity of heterologous promoters in Brevibacterium spp.
Such a vector, pEB003 has been constructed. It contains at least four genetic elements: a promoter-indicator gene (chloramphenicol acetyltransferase (CAT) w h i c h confers resistance to chloramphenicol); a selectable gene (kanamycin resistance) placed in the reverse direction to the indicator gene so that read through is prevented; a replication origin for Brevibacterium spp. and a
Low shear bioreactor for plant cell aggregates Plant cell cultures often form aggregates in s u s p e n s i o n culture w h i c h can make the provision of oxygen difficult: the high aeration or mechanica] agitation necessary can both cause shear damage to the cells. However, a reactor has been d e v e l o p e d w h i c h effectively creates separate cell growth and aeration zones. The reactor is
analogous to fluidized bed c o l u m n s except that the cells are i m m o b i l i z e d naturally (by aggregation) rather than artificially. Aeration is performed in a bubble column. Aggregates of Coffea arabica (which forms a high p r o p o r t i o n of large aggregates) demonstrated a grading effect in the reactor (small aggregates at the top
Stabilizing plasmids using cloned indispensible genes A n alternative to p l a s m i d stabilization by antibiotic selection is to clone an i n d i s p e n s i b l e gene, in w h i c h the host c h r o m o s o m e is deficient, onto the plasmid. This m e t h o d has been demonstrated using strains of E. coli w h i c h have a temperature sensitive valyl-tRNA synthestase: pSGS13, a p l a s m i d derived from pBR322 coding for valyl-tRNA (and also for n-tryptophan production) was transformed into the temperature sensitive E. coli strain. In fed batch cultures in the laboratory, pSGS13 gave a high t r y p t o p h a n p r o d u c t i o n and was comp l e t e l y genetically stable. Mortensen, U. and Nilsson, J. (1987) Proc. 4th European Congress on Biotechnology, Vo]. 3 (Neijssel, O. M., van der
Meer, R.R. and Luyben, K. Ch. A. M., eds), p. 303-304, Elsevier: AC Biotechnics AB, Box 946, S-220 09 Lund, Sweden.
Continuous measurement of water activity A sterilizable device has been d e v e l o p e d for the continuous m e a s u r e m e n t of the water activity in fermentation media. It consists of a capacitive e l e m e n t w h i c h measures the relative h u m i d i t y in a small chamber separated from the m e d i u m by a thin ethylenepolytetrafluor
oxygen using nitrogen a n d passing it through an anaerobic coil. In this way the relative volumes of aerobic, anaerobic and gradient zones could be systematically varied. Larsson, G. and Enfors, S-O. (1987) Proc. 4th European Congress on Biotechnology, Vo]. 1 (Neijssel, O. M., van der Meer, R.R. and Luyben, K. Ch.A.M., eds), p. 236-239, Elsevier: Department of Biochemistry and Biotechnology, Royal Institute of Technology, 100 44 Stockholm, Sweden.
replication origin for E. coil Using the vector a n u m b e r of promoters were inserted into the restriction sites w h i c h i m m e d i a t e l y precede the CAT gene. It was found that these also gave rise to expression in B. lactofermentatum. Moringa, Y., Tsuchiya, M., Miwa, K. and Sano, K. ( 1 9 8 7 ) J . Biotechnol. 5, 305-312: Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki-shi, 210 Japan.
and larger ones lower down), a feature with important engineering implications. Hegglin, M., Prenosil, J. E. and Bourne, J. R. (1987) Proc. 4th European Congress on Biotechnology, Vol. 2 (Neijssel, O. M., van der Meer, R.R. and Luyben, K. Ch. A . M . , eds), p. 400, Elsevier: Chemical Engineering Department (TLC), Swiss Federal Institute of Technology (ETH), CH-8092 Ztirich, Switzerland.
membrane. The h u m i d i t y in the chamber d e p e n d s on the water activity of the m e d i u m outside the membrane. For high water activity media, where the h u m i d ity inside the chamber is likely to be high, circulation of dried gas through the chamber prevents saturation of the sensor. The sensor has been u s e d to regulate the water activity during a fermentation over a p e r i o d of five days: its response time at 90% water activity was two minutes. Gervais, P. (1987) Proc. 4th European Congress on Biotechno]ogy, Vo]. 2 (Neijssel, O. M , van der Meer, R. R. and Luyben, K. Ch. A. M., eds), p. 89, Elsevier: D~partement de Biologie PhysicoChirniqne, ENS.BANA, C a m p u s Universitaire de Montmuzard, 21100 Dijon, France.
Modelling insufficient mixing W h e n scaling up or scaling d o w n aerobic culture processes, the variation in mixing requirements is the most likely root of performance differences between different scales of reactors of similar geometry. In a large reactor there are likely to be areas of oxygen gradient and oxygen free zones. These can be simulated on the small scale by the inclusion of an oxygen-free c o m p a r t m e n t in an other-
wise aerated reactor. In a recent study, the gradients of oxygen were simulated b y p u m p i n g culture ' m e d i a from a w e l l - m i x e d and aerated reactor along a length of silicon tubing. The tubing was m a i n t a i n e d at the temperature of the aerated reactor and effectively° acted as a plug-flow reactor (no mixing at all). Oxygen-flee areas were simulated by stripping the culture of
A promoter-probing vector for Brevibacterium Brevibacterium ]actofermentatum and related Gram-positive glntamic acid producing bacteria are important in the p r o d u c t i o n of amino acids. Genetic m a n i p u l a t i o n of these industrial organisms is being explored and one of the i m p o r t a n t tools for research on the expression of heterologous genes is a vector to probe the activity of heterologous promoters in Brevibacterium spp.
Such a vector, pEB003 has been constructed. It contains at least four genetic elements: a promoter-indicator gene (chloramphenicol acetyltransferase (CAT) w h i c h confers resistance to chloramphenicol); a selectable gene (kanamycin resistance) placed in the reverse direction to the indicator gene so that read through is prevented; a replication origin for Brevibacterium spp. and a
Low shear bioreactor for plant cell aggregates Plant cell cultures often form aggregates in s u s p e n s i o n culture w h i c h can make the provision of oxygen difficult: the high aeration or mechanica] agitation necessary can both cause shear damage to the cells. However, a reactor has been d e v e l o p e d w h i c h effectively creates separate cell growth and aeration zones. The reactor is
analogous to fluidized bed c o l u m n s except that the cells are i m m o b i l i z e d naturally (by aggregation) rather than artificially. Aeration is performed in a bubble column. Aggregates of Coffea arabica (which forms a high p r o p o r t i o n of large aggregates) demonstrated a grading effect in the reactor (small aggregates at the top
Stabilizing plasmids using cloned indispensible genes A n alternative to p l a s m i d stabilization by antibiotic selection is to clone an i n d i s p e n s i b l e gene, in w h i c h the host c h r o m o s o m e is deficient, onto the plasmid. This m e t h o d has been demonstrated using strains of E. coli w h i c h have a temperature sensitive valyl-tRNA synthestase: pSGS13, a p l a s m i d derived from pBR322 coding for valyl-tRNA (and also for n-tryptophan production) was transformed into the temperature sensitive E. coli strain. In fed batch cultures in the laboratory, pSGS13 gave a high t r y p t o p h a n p r o d u c t i o n and was comp l e t e l y genetically stable. Mortensen, U. and Nilsson, J. (1987) Proc. 4th European Congress on Biotechnology, Vo]. 3 (Neijssel, O. M., van der
Meer, R.R. and Luyben, K. Ch. A. M., eds), p. 303-304, Elsevier: AC Biotechnics AB, Box 946, S-220 09 Lund, Sweden.
Continuous measurement of water activity A sterilizable device has been d e v e l o p e d for the continuous m e a s u r e m e n t of the water activity in fermentation media. It consists of a capacitive e l e m e n t w h i c h measures the relative h u m i d i t y in a small chamber separated from the m e d i u m by a thin ethylenepolytetrafluor
oxygen using nitrogen a n d passing it through an anaerobic coil. In this way the relative volumes of aerobic, anaerobic and gradient zones could be systematically varied. Larsson, G. and Enfors, S-O. (1987) Proc. 4th European Congress on Biotechnology, Vo]. 1 (Neijssel, O. M., van der Meer, R.R. and Luyben, K. Ch.A.M., eds), p. 236-239, Elsevier: Department of Biochemistry and Biotechnology, Royal Institute of Technology, 100 44 Stockholm, Sweden.
replication origin for E. coil Using the vector a n u m b e r of promoters were inserted into the restriction sites w h i c h i m m e d i a t e l y precede the CAT gene. It was found that these also gave rise to expression in B. lactofermentatum. Moringa, Y., Tsuchiya, M., Miwa, K. and Sano, K. ( 1 9 8 7 ) J . Biotechnol. 5, 305-312: Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki-shi, 210 Japan.
and larger ones lower down), a feature with important engineering implications. Hegglin, M., Prenosil, J. E. and Bourne, J. R. (1987) Proc. 4th European Congress on Biotechnology, Vol. 2 (Neijssel, O. M., van der Meer, R.R. and Luyben, K. Ch. A . M . , eds), p. 400, Elsevier: Chemical Engineering Department (TLC), Swiss Federal Institute of Technology (ETH), CH-8092 Ztirich, Switzerland.
membrane. The h u m i d i t y in the chamber d e p e n d s on the water activity of the m e d i u m outside the membrane. For high water activity media, where the h u m i d ity inside the chamber is likely to be high, circulation of dried gas through the chamber prevents saturation of the sensor. The sensor has been u s e d to regulate the water activity during a fermentation over a p e r i o d of five days: its response time at 90% water activity was two minutes. Gervais, P. (1987) Proc. 4th European Congress on Biotechno]ogy, Vo]. 2 (Neijssel, O. M , van der Meer, R. R. and Luyben, K. Ch. A. M., eds), p. 89, Elsevier: D~partement de Biologie PhysicoChirniqne, ENS.BANA, C a m p u s Universitaire de Montmuzard, 21100 Dijon, France.