Environmental Engineering Reference
In-Depth Information
Table 6.2
Summary of Results Obtained from NY05 and VP44
Growth Studies
Witconol
SN-120
Tween
80
Tergitol
NP-15
Surfactant alone
No growth
Growth
No growth
Surfactant + biphenyl
No growth
Growth
Growth
Surfactant + 4-CB
No growth
Growth
Growth
Initially, solutions of 4-CBA and 2-CBA were prepared by dissolving each
acid in 1
N
KOH. The pH of the solution was brought below 7.0 by addition
of 2
N
H
2
SO
4
. A growth flask was then prepared with approximately 100 ml
of K1 nutrient solution, and 2-CBA or 4-CBA was added to reach a concen-
tration of approximately 2 m
M
. The flasks were inoculated with engineered
strains of RHA1, NY05, and VP44 provided by investigators at Michigan
State University (MSU), to which 2 ml of 100 m
M
CBA solution was added
each day. Once the solution in each flask reached an optical density of 1.0
or greater, 10 ml of the solution was used to inoculate a second flask con-
taining the same K1 and CBA solution, and the process was repeated.
After the second flask reached an optical density of 1.0, the solution was
centrifuged and the supernatant was discarded. The biomass was resus-
pended in K1 media and centrifuged for a second time. This process was
repeated two more times in an attempt to remove all of the CBA from
solution. After the final centrifugation, the biomass was resuspended to a
concentration of 0.2 g biomass/ml. This solution was equally divided and
used to inoculate three growth flasks, the first containing a solution of K1
and BP, the second containing a solution of Tergitol NP-15 and BP, and the
third containing a solution of Tween 80. A sequential plating method was
used, with five plates being produced for each instance with the hope that
the fifth plate would produce individual colonies. These plates were labeled,
sealed with parafilm, and forwarded to laboratories for PCR analysis. A
summary of the PCR analysis for RHA1(
fcb
) is given in Table 6.3. Here, note
that under all of the growth conditions evaluated, the plasmid gene was
detected by PCR analysis.
Table 6.3
Representative Results of Plasmid Stability Tests
Plasmid
Gene
Initial
Substrate
PCR
Analysis
Substrate
Biphenyl + K1
RHA1 +
fcb
4-CBA
5/5
Tween 80 + K1
RHA1 +
fcb
4-CBA
5/5
Tergitol NP-15 + K1
RHA1 +
fcb
4-CBA
5/5
