and 2-ethylbutanol (96% purity) were obtained from Aldrich
Chemical Co. (Milwaukee, WI). TBOS (98% purity) and
tetrapropoxysilane (TPOS) (98% purity) were obtained from
Gelest, Inc. (Tullytown, PA). TKEBS was synthesized in the
lab by adopting Von Ebelman’s synthesis for tetraalkoxy-
silanes (1, 6). Stoichiometric quantities of silicon tetrachloride
soning with mercuric chloride (HgCl
25 mg/ L.
2
) at a concentration of
Aerobic biodegradation experiments were conducted in
batch bottles prepared under aseptic conditions by auto-
claving all implements used for the construction of the batch
bottles. The batch bottles were handled under a laminar flow
hood to avoid microbial contamination. Autoclaved synthetic
buffered medium (198 mL) and nutrient mixture (2 mL) were
added to the batch bottles, and the remaining 110 mL of
headspace was purged with helium (99.99% purity) to remove
(
semiconductor grade, Aldrich Co.) were reacted with a slight
excess of 2-ethylbutanol in the presence of pyridine as an
acid acceptor in benzene solvent. The products were vacuum-
distilled to yield approximately 95% pure TKEBS. The above
compounds were used both as culture substrates and in the
preparation of analytical standards.
nitrogen prior to the addition of 22 mL of pure O
procedure facilitated accurate measurement of O
incomplete separation of O and N by the GC column.
2
. This
2
due to
Analytical Methods. TBOS, TKEBS, 1-butanol, and 2-eth-
ylbutanol concentrations were quantitatively determined by
liquid-liquid extraction of 1 mL aqueous samples with 0.5
mL of dichloromethane and agitation for 5 min on a vortex
mixer. After complete separation of the two immiscible
phases, 2 µL of the dichloromethane extract was injected
into the GC/ MS, a HP-5890 GC connected to a HP-5971 mass-
selective detector. The chromatographic separation was
carried out with a Rtx-20 column (30 m × 0.25 mm, 1.0 µm
film) from Restek, Inc. (Bellefonte, PA). The mass spectrom-
eter was operated in the selective ion monitoring mode for
the quantitative analysis of the compounds. The ions
monitored were m/ z 56 for 1-butanol, m/ z 70 for 2-ethyl-
butanol, m/ z 235 for TPOS (internal standard), m/ z 277 for
TBOS, and m/ z 361 for TKEBS. The concentrations were
normalized with TPOS (10 mg/ L) as an internal standard
that was added to dichloromethane prior to extraction.
Because TBOS and TKEBS have low aqueous solubility (less
than 0.5 mg/ L) (19, 20), samples that contain higher than
soluble amounts of TBOS and TKEBS form an emulsion with
water. Prior to sampling, the sample containers were placed
laterally on a high-speed reciprocating shaker table and
agitated vigorously for at least 20 min to maintain homo-
geneity of the compounds in solution. Shaking of the
containers was continued until just before the samples were
collected, to obtain consistent results. The relative standard
deviations of the TBOS and TKEBS measurements were
approximately 10% and 15%, respectively.
2
2
A buffer medium designed to maintain a constant pH 7
was used as the aqueous matrix for the aerobic biodegrada-
tion studies. The buffer medium was prepared by adding
2 4 2 4
KH PO (15 g) and Na HPO (20 g) to 1 L of deionized water
and adjusting the pH to 7 with 6 M NaOH. The nutrient
medium used was prepared by combining concentrated stock
solutions of major nutrients with a concentrated stock of
trace nutrients at a 10:1 ratio. The major nutrient stock
solution was comprised of 9 g of NaNO and 0.5 g of MgSO
3 4
in 1 L of deionized water, and the minor nutrient stock was
comprised of the following in 100 mL of deionized water:
ZnSO
g; MnSO
4
‚H
2
O, 0.303 g; FeSO
‚H O, 0.302 g.
4
‚7H
2
O, 1.2 g; CoSO
4
2
‚7H O, 0.102
4
2
The TCE and c-DCE cometabolism experiments were
conducted in batch bottles prepared in a manner similar to
that explained above. The desired solution concentrations
of TCE and c-DCE were obtained by spiking appropriate
volumes of distilled water saturated with TCE and c-DCE.
Control batch bottles were prepared by autoclaving and/ or
chemical poisoning with mercuric chloride (HgCl
bottles were incubated in an environmental chamber at 30
C with continuous shaking at 150 rpm. The total mass of
2
). The batch
°
TCE or c-DCE in the microcosms was determined by
measuring gas phase concentrations and calculating aqueous
phase concentrations assuming equilibrium Henry’s law
partitioning (21). From the volumes of liquid and headspace
in the microcosms and the concentrations of TCE and c-DCE
in these two compartments, the total mass balances in the
microcosms were verified.
Gas phase TCE and c-DCE concentrations were measured
by injecting 100 µL of the headspace sample into a HP-5890
GC connected to a photoionization detector (PID) followed
by a flame ionization detector (FID). Chromatographic
separation was carried out with a 30 m megabore GSQ-PLOT
Bacterial Culture. The microbial culture used for the
aerobic biodegradation studies and the cometabolism ex-
periments was stimulated from activated aerobic sludge
obtained from the Corvallis, OR, wastewater treatment plant.
The activated sludge was acclimated to high concentrations
of TBOS (500 mg/ L) in a 125 mL batch bottle for a period of
over 10 months before the stimulation of the culture occurred.
This microbial culture was enriched by repeatedly centrifug-
ing the cells and transferring them into a different batch
bottle with buffered medium with regrowth on TBOS. The
enriched culture was subsequently used as an inoculum for
a 2 L(1 Lliquid volume) batch reactor that was continuously
fed TBOS. The reactor utilized the same nutrient media as
described for the batch bottles. The reactor was equipped
with a fixed magnetic stir rod and stir plate for continuously
mixing the contents of the reactor. The reactor was continu-
ously fed neat TBOS at a rate of 80 µL/ day with a syringe
pump. Everyday, 200 mL of solution with cells was wasted
from the reactor, and 200 mL of fresh buffer medium and
nutrients were added. The mean cell residence time in the
reactor was approximately 5 days. The microbial culture from
this reactor was ultimately used for the subsequent experi-
ments.
column from J&W Scientific (Folsom, CA). The gases O
CO in the batch bottle headspace were measured with a
2
and
2
HP-5890 GC connected to a thermal conductivity detector.
The method involved direct injection of a 0.1 mL gas sample
from the headspace of the batch bottle into the GC with a
gas-tight syringe (Hamilton Co., Reno). Chromatographic
separation was carried out with a Supelco Carboxen 1000
packed column (15 ft × 0.125 in., S.S Support).
The dichloromethane extract prepared for the TBOS
measurements was also used for the determination of c-DCE
epoxide with TPOS as the internal standard. The GC/ MS was
operated in the selective ion mode, and c-DCE epoxide was
measured by monitoring the ions m/ z 48 and 112. Normalized
area ratios were used to quantify c-DCE epoxide, as analytical
standards for this compound were not available.
Experim ental. Batch bottles were constructed with 310
mL serum bottles (Wheaton Industries, Millville, NJ) fitted
with rubber-lined caps and butyl rubber septa. Abiotic
hydrolysis studies were conducted in batch bottles with 250
mL of phosphate buffer and 60 mL of headspace. The
phosphate buffer was prepared by mixing varying quantities
Inhibition experiments were carried out by addition of
acetylene, which is an inhibitor of monooxygenase enzymes
(13, 14). Pure acetylene gas (20% of headspace volume) was
added to the batch bottles to ensure maximum inactivation.
3
-
of Na
2
HPO
4
and KH
2
PO
4
(approximately 22 mM total PO
4
)
to obtain a range of pH between 5 and 9. These experiments
were conducted under sterile conditions by chemical poi-
1
0 7 8
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. 7, 1999