Biomedical Engineering Reference
In-Depth Information
concentrated by passing the broth over a custom-built cell concentrator constructed with six
Kerasep
TM
tubular ceramic membranes (Novasep, Inc., Boothwyn, Pennsylvania; refer to
Figure
3.10
in Section
3.4.3
) contained within stainless steel piping to prevent oxygen intrusion.
Concentrated cells are stored at 4
C in 18.5-L stainless steel soda kegs (refer to Figure
3.12
in
Section
3.5.2
) that are pressurized with nitrogen.
For seed culture production, revised anaerobic mineral medium (RAMM) medium (Shelton
and Tiedje,
1984
) without sodium bicarbonate (NaHCO
3
) and sodium sulfide (Na
2
S) is added to
the 20-L reactor and steam sterilized at 121
C and 15 pounds per square inch (psi) for 45 minutes
(min). After sterilization, the reactor is connected to a nitrogen tank to maintain a positive
pressure of nitrogen in the vessel during cooling to 30
C. After the temperature in the reactor
reaches the set point temperature of 28-30
C and anaerobic conditions are achieved (measured
DO
0 mg/L), nitrogen flow is stopped and NaHCO
3
solution is added aseptically to the
medium. The reactor is then inoculated with 2 L of SDC-9
TM
or other culture containing
approximately 10
10
-10
11
Dhc
/L. The final volume of medium in the 20-L reactor is 16-18 L.
After inoculating the reactor, sterile 10% yeast extract (YE) solution is added to a final
concentration of 0.1% YE (weight per volume [w/v]), vitamin B12 (0.03 mg/L) and PCE or TCE
is added to a final concentration of 10 mg/L. No lactate is added at the beginning of the culture
process because the YE provides sufficient carbon to support the initial cell growth and
the addition of lactate at this point in the process results in significant methane formation
in the reactor. The reactor is operated at 28-30
C with an agitator speed of 100 revolutions per
minute (rpm). pH is maintained at 6.4-7.2 by the addition of an anoxic solution of NaOH (2 N).
Alternatively, to increase pH during bacterial growth, the reactor is sparged with nitrogen
to remove dissolved carbon dioxide (CO
2
). After 1 day of culture growth, sodium lactate
(60% solution) is added continuously to the reactor at a flow rate of 0.02-0.04 mL/hour (h) L
of medium. A second addition of PCE or TCE (10 mg/L) is added to the reactor only after
complete dechlorination of PCE/TCE, but before complete dechlorination of
cis
-DCE. Typi-
cally, PCE/TCE is added to the medium when the concentration of
cis
-DCE in the medium is
reduced to 1-3 mg/L. When the culture reaches an OD
550
of approximately 1.0, it is transferred
anaerobically to the 750-L reactor.
ΒΌ
3.2.2.2 550-L Scale
Intermediate size batches (to 550 L) of
Dhc
cultures are prepared in a 750-L stainless steel
reactor. The 750-L reactor is prepared with 540 L of RAMMmedium containing 0.1-0.2% (w/v)
YE, but without NaHCO
3,
and sterilized as previously described. After sterilization and cooling,
vitamin B12 (0.03 mg/L) and NaHCO
3
(660 grams [g]) dissolved in 10 L of deionized (DI) water
is added to the reactor through a sterile filter, and neat PCE/TCE is added to a final
concentration of 10 mg/L. The reactor is connected to a nitrogen tank to maintain anoxic
conditions, and is operated under the same conditions as described for the 20-L reactor except
the agitator speed is set at 60 rpm. The automatic pH control system on the reactor is
inactivated to avoid addition of excess sodium ion (as NaOH).
Once the appropriate temperature (28
C) is reached in the reactor, the seed culture is
aseptically transferred to the larger reactor while maintaining strict anaerobic conditions. After
1 day of culture growth, a continuous feed of sodium lactate (60% solution) is initiated with a
flow rate of 0.02-0.04 mL/h
L. Periodically, samples are taken from the reactor and
analyzed for the presence of chlorinated products, volatile fatty acids (VFAs) and
Dhc
concentration. After complete dechlorination of the first addition of PCE/TCE, chlorinated
solvent is again added to a final concentration of 10 mg/L. Subsamples (25 mL) of the culture
are periodically removed from the reactor to measure cell density and to perform bottle assays
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