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solutions were cooled anaerobically by sweeping oxygen-free N2 gas across
the surface. One gram of cysteine hydrochloride was dissolved in 25 mL of
water and filter sterilized (0.2 µm) before aseptically adding to the cooledCSL medium. Twenty-five milliliters of FeSO4·7H2O (0.012 g) solution was
made in water followed by filter sterilization (0.2 µm) and adding to the
CSL medium. FeSO4·7H2O is an essential mineral for solventogenesis and
was included in both P2 and CSL media. The CSL medium was fed to the
reactor.
Bioreactor and Cell Immobilization
The reactor was composed of a 312-mL total volume jacketed poly-
acrylic (192 × 46 mm) vessel. The reactor was sterilized using 30% (v/v)
ethanol solution for 48–72 h, after which it was drained and washed thor-
oughly with sterilized deionized water. Brick pieces (4 to 5 mm, total weight
of 220 g) were washed with deionized water several times followed by
sterilization in an oven at 250°C for 2 h. The particles were then cooled in
an anaerobic chamber to room temperature. The void volume inside the
reactor was 186 mL. The reactor was packed aseptically with the sterilized
brick particles. After the reactor was packed, oxygen-free N2 was passed
through the column overnight. This was done to ensure that anaerobic
conditions were attained inside the column and inside the particles.
Forty milliliters of actively growing C. beijerinckii cells from a CMM
bottle were inoculated into the reactor, and the reactor was filled with fresh
P2 medium. The composition of P2 medium has been published elsewhere
(8). Cell growth was allowed inside the reactor for 4 h, after which the P2
medium was continuously fed (using a peristaltic pump [Cole-Parmer,
Vernon Hills, IL] and silicone tubing) at a flow rate of 92 mL/h (dilution
rate of 0.29 h–1
). Water (35°C) was circulated through the jacket of the col-
umn to control temperature using a circulating water bath (Polystat; Cole-
Parmer). Samples were taken after 3 d. The dilution rate was altered
whenever a steady state was reached in terms of average solvent produc-
tion and glucose utilization. Samples were collected over a period of five to
seven steady-state residence times, with a sampling frequency of one resi-
dence time. Samples were centrifuged at 16,000g for 1 to 2 min. The super-
natant was stored at –18°C in preparation for acetone, butanol, ethanol, and
glucose analysis. Comparisons of the media were made in the same reactor
(not in parallel reactors). The pH of the reactor effluents was not measured.
The reader is advised that the pH inside the bioreactor was not controlled.
Solventogenic clostridia regulate pH at approx 5.0. A schematic diagram of
the reactor is shown in Fig. 1.
Analyses
Cell concentration in the reactor effluent was estimated by optical den-
sity (OD) and cell dry weight method using a predetermined correlation
between OD at 540 nm and cell dry weight. Acetone-butanol-ethanol (ABE)
and acids (acetic and butyric) were measured using a 6890 Hewlett-Packard
gas chromatograph (Hewlett-Packard, Avondale, PA) equipped with a
flame ionization detector and 6 ft × 2 mm glass column (10% CW-20M,
0.01% H3PO4, support 80/100 Chromosorb WAW). Productivity was calcu-lated as the average ABE concentration multiplied by dilution rate (h–1
).
Dilution rate was defined as feed flow rate per reactor volume and is based
on the total volume of the reactor. ABE yield was defined as grams of ABE
produced per gram of glucose utilized.
Glucose concentration was determined using a hexokinase- and glu-
cose-6-phosphate dehydrogenase– (Sigma, St. Louis, MO) coupled enzy-
matic assay. The fermentation broth was centrifuged (microfuge centrifuge)