(86b) Chloroform Aerobic Cometabolic Biodegardation in a Continuous-Flow Reactor

Chlorinated Aliphatic Hydrocarbons (CAHs)
are common contaminants of groundwaters and industrial wastewaters. Lab-scale
assays and field pilot tests showed that aerobic cometabolism with aliphatic
and aromatic hydrocarbons as growth substrates can lead to the rapid and
complete dechlorination of a wide range of CAHs, including high-chlorinated
compounds such as 1,1,2,2-tetrachloroethane. Aerobic cometabolism can therefore
be regarded as a promising technology for the treatment of CAH-contaminated
sites and wastewaters.

However, with specific regard to the implementation of this technology
for the remediation of CAH-contaminated sites, several issues still need
to be addressed. Of these, two deserve particular mention: i) it is important
to avoid the risk of a complete consumption of the supplied growth substrate
within a short distance from the injection wells: in other words, the bioreactive
zone must be sufficiently long to allow the complete biodegradation of
the target contaminants; ii) aquifer clogging near the injection well can
occur as a result of an excessive biomass growth on the growth substrate
supplied.

The supply of alternated pulses of growth substrate and oxygen represents
an interesting solution, potentially effective both in the creation of
a long bioreactive zone and in the control of aquifer clogging: as a result
of hydrodynamic dispersion and substrate sorption, the over-lapping of
substrate and oxygen occurs at low concentration, over a wide aquifer portion
and, in each point, in a discontinuous way.

This study, focused on chloroform (CF) cometabolism by butane-grown
bacteria, was conducted in a 2-m continuous-flow column reactor simulating
a portion of saturated aquifer. The main goals were: a) to investigate
the pulsed injection of growth substrate and oxygen as a tool to control
clogging of the porous medium and to attain a wide bioreactive zone; b)
to determine the minimum substrate/CAH ratio required to sustain the cometabolic
process; and c) to determine the most suitable kinetic and fluid-dynamic
model to fit the experimental data of butane utilization and CF cometabolism.

A preliminary group of fluid-dynamic tests was aimed at determining
the main fluid-dynamic parameters required for the modeling of the process:
the reactor effective porosity n_eff and longitudinal dispersivity
a_L,
and the retardation factors R relative to butane and CF (assuming R = 1
for oxygen). These tests consisted of pulses of only oxygen (with no butane
or CF in the column), only butane or only CF. For each compound, different
pulses at different interstitial velocities were operated. The results,
elaborated by means of a PDE solver (Comsol Multiphysics) yielded the following
best-estimates: n_eff = 50%, a_L=
0.35 mm, R_butane = 1.08, R_CF = 1.

A kinetic model of aerobic cometabolism, together with a set of kinetic
parameters, was derived from a previous batch study conducted with the
Rhodococcus bacterial strain that resulted the prevailing species in the
soil utilized in this work, after a long period of butane utilization and
CF degradation (Frascari et al., Appl. Microbiol. Biotechnol. 73 (2006)
421-428).

The complete fluid-dynamic/kinetic model was utilized to run a series
of simulations solved with Comsol Multiphysics, with the goal to design
three types of injection of alternated pulses of growth substrate (butane)
and oxygen, characterized by different values of the ratio of butane utilized
to CF degraded (B/CF ratio). Indeed, in a previous batch study conducted
with the same soil utilized in this study (Frascari et al., Process. Biochem.
42 (2007) 1218-1228), the minimum B/CF ratio required to sustain the cometabolic
process resulted equal to 1.6 mg_B/mg_CF. In a process
of CAH cometabolism, the supply of growth substrate is necessary not only
to re-produce or re-activate the biomass killed or inactivated by the toxic
CAH degradation products, but also to produce the reducing energy (NADH)
consumed but not re-generated by the CAH transformation process. Therefore,
the above-mentioned three types of butane and oxygen pulsed injection were
designed to attain, in comparison with the minimum B/CF ratio estimated
in the batch tests, a first ratio largely higher (17 mg_B/mg_CF),
a second ratio slightly lower (1.0 mg_B/mg_CF) and
a third ratio slightly higher (2.3 mg_B/mg_CF), so
as to validate in a continuous-flow saturated reactor the estimate of the
B/CF ratio obtained by means of batch slurry assays.

The column reactor was then run for three consecutive periods, characterized
by the three above-mentioned pulsed injections. In these tests, the interstitial
velocity was set to 0.5 m/d (corresponding to a 2-day hydraulic retention
time), and the CF inlet concentration to about 0.4 mg/L.

The first phase (butane consumed / CF degraded = 17, corresponding
to a ratio of butane supplied / CF supplied = 27) was run for 35 days,
and yielded and average CF removal equal to 50%. The cometabolic process
proved sustainable under these conditions. The designed schedule of pulsed
injection (butane pulse: 21 mg/L, 7.5 h; 3 h without butane or oxygen;
oxygen pulse: 20 mg/L, 11 h; 2.5 h without butane or oxygen) allowed the
development of a bioreactive zone over the entire length of the column
(2 m) and prevented any measurable clogging of the porous medium.

The second phase (butane consumed / CF degraded = 1.0, corresponding
to a ratio of butane supplied / CF supplied = 2.4) was run for 46 days.
The corresponding sequence of butane and oxygen pulsed injection was based
on a 3.5-day cycle. During the first 20 days the average CF removal was
equal to 80%, and the cometabolic process appeared sustainable. A typical
representation of the concentration profiles of butane, oxygen and CF over
the column during this period is provided in Figure 1, together with the
corresponding model simulation performed with the kinetic parameters derived
from the single-strain kinetic study. It can be observed that, at a given
instant, CF cometabolic degradation occurred in the reactor zones characterized 
- as a result of the pulsed injection - by the presence of oxygen. Due
to convection and dispersion, these zones shifted through the reactor.
We therefore obtained a CF outlet concentration characterized by a fluctuating
trend. However, after 20 days, the CF degradation rate decreased and the
cometabolic process rapidly halted. The most likely interpretation for
this event is that, as a result of the excessively low ratio of butane
consumed to CF degraded, the cellular storage of NADH was progressively
consumed. A model interpretation of this hypothesis, based on the work
of Chang and Alvarez-Cohen (Environ. Sci. Technol. 29 (1995) 2357-2367)
is in progress.

The third phase (butane consumed / CF degraded = 2.3, corresponding
to a ratio of butane supplied / CF supplied = 2.0) was run for 49 days.
The corresponding sequence of butane and oxygen pulsed injection was based
on a 3.5-day cycle. Under these conditions, the cometabolic process resumed
rapidly and remained stable over time, with an average 80% CF removal.
No sign of aquifer clogging was observed.

In conclusion, the experimental and modeling results show that:

a) the pulsed injection of growth substrate and oxygen is an effective
tool to prevent aquifer clogging as a result of an excessive biomass growth,
and to attain a long bioreactive zone;

b) in the specific case of our process of CF cometabolism with butane,
the minimum ratio of substrate utilized / CAH degraded ranges between 1.6
and 2, with a good agreement between the results obtained in batch slurry
assays and those deriving from the column tests; the corresponding ratio
of substrate supplied / CAH supplied depends on several factors, among
which the number of substrate and oxygen pulses in each cycle plays an
important role;

c) the kinetic parameters previously estimated in batch, single-strain
assays, combined with the fluid-dynamic parameters evaluated in the first
part of this work, allowed the development of an effective modeling tool
for the design of the pulsed injection and for the interpretation of the
experimental data.

Overall, this work provides encouraging indications on the successful
application of aerobic cometabolism for the in-situ remediation of sites
contaminated by a wide range of CAHs.