A multivariate analysis of aerobic growth conditions for E.
coli strain AFP184 and the effects on anaerobic succinic acid
productivity

Abstract: If fossil fuels are to be removed as the main raw material for production of
fuels and chemicals an economically competitive process based on renewable
resources is required. Succinic acid, produced by microbial fermentation of
renewable feed stocks, can substitute chemicals based on benzene and other
intermediate petrochemicals for production of polyester and solvents. Other
chemicals that can be produced from succinic acid are food ingredients, fuel
additives and plant growth stimulants. The micro organism used for succinic
acid production in this project is the bacterium Escherichia coli strain
AFP184. This strain has been metabolically modified to increase the succinic
acid production. The process in this project is a dual-phase fermentation
which constitute of an aerobic growth phase and an anaerobic succinic acid
production phase. After transition to anaerobic conditions the cells starts
to produce succinic acid in large quantities using glucose as the main
carbon source. Formation of succinic acid requires CO2 input and hence the
process does not contribute to any atmospheric CO2 accumulation. The media
used for all the fermentations is a low cost media based on corn steep
liquor. Corn steep liquor is an industrial by-product from wet milling of
maize and it contains nutrients needed for the cells. The use of E. coli
strain AFP184 for fermentative succinic acid production can provide a stable
process where the productivity of succinic acid is high and the productivity
of other mixed acid fermentation products are low or non. The use of a low
cost media makes the strain AFP184 a suitable candidate for large scale
succinic acid production.

In order to optimize the growth phase of AFP184 and to get knowledge if a
fluctuation within certain domains of four controlled variables will affect
biomass production and further succinic acid production, a designed
experiment was used. Using design of experiment (DOE) data was collected
from the growth phase and analyzed with multivariate data analysis. The
controlled variables that were varied in chosen domains were stirring (400
rpm to 1200 rpm), airflow (2 vvm to 10 vvm), temperature (32 °C to 42 °C)
and pH (5.5 to 7.5). The responses in the designed experiment were eight
different organic acids, dry cell weight, specific growth rate, viable cells
and glucose consumption. The results from the analysis were validated by
additional experiments, where also the effect on the anaerobic succinic acid
production phase was evaluated.

From the analysis of the designed experiment it was shown by additional
experiments that the growth rate and cell viability could be increased and
the acetic acid formation decreased. The controlled variables temperature
and pH could be varied in the chosen domain to produce biomass with moderate
acetic acid concentration at the chosen time of transition from aerobic to
anaerobic conditions. The additional experiments also showed during the
anaerobic succinic acid production phase that productivity and yield of
succinic acid decreased compared with the three centre point runs from the
designed experiment and that could depend on the physiological state of the
cells at the time of transition.