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cell immobilization to produce butanol. In addition to being simple, it has
the potential to be used in large-scale reactors. In the present study, clay
brick was chosen as the cell adsorption support. It is available at a low cost
and is easy to dispose of after use.
The previous cited studies were performed using P2 medium, which
is composed of expensive chemicals such as biotin, thiamin, and yeast
extract. The use of these chemicals at the commercial level would not be
commercially viable. Hence, it is suggested that the use of cheaper and
simpler nutrient sources be investigated. The use of corn steep liquor (CSL)
has been reported in pilot plant trials for butanol batch production employ-
ing C. beijerinckii BA101 (6). CSL is a byproduct of the corn wet-milling
process and contains nutrients leached out of corn during the soaking pro-
cess. The reader is advised that there are few studies on the use of CSL in
continuous immobilized cell biofilm reactors. Similarly, the use of exog-
enous sodium butyrate and sodium acetate has not been reported in con-
tinuous immobilized cell biofilm reactors. Hence, these studies areconsidered novel. The successful application of CSL, sodium butyrate, and
sodium acetate is expected to benefit the economics of butanol production
by fermentation.
One objective of the present study was to examine the effect of CSL on
the performance of high-productivity biofilm reactors. Another objective
was to study the effect of supplementing acetate and butyrate into the feed
of the biofilm reactor. Supplementation of acetate to the feed medium of
batch reactors (not continuous biofilm reactors) has been shown to be ben-
eficial to this fermentation. It is also anticipated that butyrate added to the
feed would be converted to butanol.
Materials and Methods
Organism and Culture Maintenance
For fermentation studies, the microorganism used was the C. beijerinckii
BA101 hyperbutanol-producing strain (7). Laboratory stocks of C. beijerinckii
BA101 were routinely maintained as spore suspensions in sterile double-
distilled water at 4°C. C. beijerinckii BA101 spores (200 µL) were heat shocked
in 50 mL of cooked meat medium (CMM) for 10 min at 80°C followed by
cooling in ice-cold water. Pyrex bottles (100-mL volume) containing heat-
shocked spores were incubated anaerobically for 15 to 16 h at 36 ± 1°C before
inoculating the reactor.
Fermentation Media
The feed medium contained glucose (60 g/L), yeast extract (Difco,
Detroit, MI) (1 g/L), and P2 medium ingredients (buffer, minerals, and
vitamins) (8) unless stated otherwise. Glucose and yeast extract solutions
were sterilized separately at 121°C for 15 min followed by cooling to room
temperature by sweeping oxygen-free N2 gas across the surface of the
medium. Stock solutions of buffer, mineral, and vitamins were added
aseptically to the cooled glucose-yeast extract solution. The stock solu-
tions were filter sterilized through a 0.2-µm filter. The medium was kept
anaerobic by continuously sweeping oxygen-free N2 across the surface.
When needed, sodium butyrate or sodium acetate solutions were added
to the P2 feed medium. The pH of the feed medium was adjusted to 6.8
prior to autoclaving.
Thirty-two grams of CSL (500 g/L of solids slurry) was added to each
liter of CSL medium. The composition of CSL has been given elsewhere (9).
The CSL was dissolved in approx 300 mL of water and boiled for 30 min.
After cooling to room temperature, the mixture was centrifuged to remove
solids. To the supernatant, 60 g of glucose was added and the volume
adjusted to 950 mL with water. The pH of the feed solution was adjusted to
6.8 prior to autoclaving at 121°C for 15 min. The autoclaved medium or