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Development of acetate threshold tracking control algorithms for Fed-Batch control of recombinant E.coli.

Johnston, Wayne (2002) Development of acetate threshold tracking control algorithms for Fed-Batch control of recombinant E.coli. PhD thesis, Murdoch University.

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In this thesis, algorithms for fed-batch control of recombinant E.coli were developed to avoid acetate formation while maximising biomass and total productivity. In general, previous algorithms have addressed the former goal only. Acetate threshold tracking nutrient controllers were developed, which provide the maximum nutrient feed rate without overflow metabolism to detrimental acetic acid production. These were particularly designed for performance in complex media. The primary problem in the development of such controllers is the non-availability of on-line sensors which can directly detect the acetate threshold. Two approaches were undertaken to provide acetate threshold detection and control using standard sensors.

The first approach used a gam scheduled Proportional-Integral-Derivative (PID) algorithm, formulated to compensate for process non-linearity via a gain schedule based on biomass estimation. The sensor availability problem was overcome by using standard measured variables to provide an on-line signal (inverse ofrespiratory quotient, l/RQ) proportional to the difference between the actual growth rate and the threshold growth rate. This allowed control of 1/RQ in a transition zone that represented growth rates just below the acetate threshold (0.2h-1). Growth to 38 g dcw/l without acetate accumulation was achieved. However, the relationship between 1/RQ and the growth rate threshold, demonstrated at low biomass concentrations of 10 - 15 g dew/I via controlled threshold traverse, changed over the course of cultivation to 38 g dew/I. This caused a significant degradation in controller threshold tracking. Additionally, the relationship between 1/RQ and growth rate was demonstrated only in a complex media system (i.e. complex base plus glucose feed supplemented with complex media components), limiting the controller's industrial applicability.

The second control approach used culture probing, in which new controlled variables related to the acetate threshold, based on the dissolved oxygen tension (DO), were generated from disturbances in the glucose feed rate. Although preliminary work used a starvation based DO-transient controller (using feed cessations), subsequent work focussed on a feed-up DO-transient controller (using feed pulses). To minimise m the detrimental effects of operation above the acetate threshold, the feed-up DO-transient control algorithm was formulated and tuned conservatively to minimise unnecessary threshold penetration while tracking. This proved particularly critical in complex media, which does not tend to balance acetate accumulation just above the threshold with acetate depletion just below. Using the conservatively tuned feed-up DO-transient controller, it was possible to track the acetate threshold over a range of 10 to 30 g dew/I in non-induced fermentation runs. Thereafter, using a glucose feed highly supplemented in complex media components (yeast extract, tryptone) to provide some culture anabolic requirement, threshold tracking to high cell density (58 g dew/I) was achieved. However, acetate accumulation was high(> lOOmM).

High cell density operation was found not to increase total productivity in a case study of the production of a viral coat protein (Jembrana disease virus sub-unit, JDV SU), in which the feed-up DO-transient controller underwent induction at 20 g dcw/l and 60 g dcw/l respectively. Total productivity was decreased from 0.27 g/l.h to 0.12 g/1.h, despite final biomass levels of 32 g dcw/l and 67 g dcw/1 respectively. This was due to reduced induction success in the high cell density run, caused by acetate accumulation. However, medium cell density induction of the feed-up DO-transient controller allowed superior total productivity to the commonly used high-limit pH-stat nutrient controller (0.27 g/l.h versus 0.24 g/l.h), due to decreased process time by the former.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Engineering
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor: Cooney, Michael
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