SEPA
United States
Environmental Protection
Agency
EPA/540/S-93/505
October 1993
SUPERFUND INNOVATIVE
TECHNOLOGY EVALUATION
Emerging Technology
Summary
Pilot-Scale Demonstration of a
Two-Stage Methanotrophic
Bioreactor for Biodegradation of
Trichloroethene in Groundwater
BioTrol, Inc., developed a two-stage,
methanotrophic, bioreactor system for
remediation of water contaminated with
trichloroethylene (TCE) and other chlo-
rinated, volatile, aliphatic hydrocarbons.
The first stage was a suspended-growth
culture vessel with a bubbleless meth-
ane transfer device. The second stage
was a plug-flow reactor fed with con-
taminated groundwater and effluent
from the culture vessel. The system
was tested at bench- and pilot-scale.
When operating optimally, 89% of the
influent TCE was degraded. Reactor ki-
netics were consistent with first-order
biodegradation kinetics. Actual meth-
ane use in the pilot-scale reactor re-
sulted in projected methane costs of
$0.33 per 1000 gal of water treated.
This cost could be reduced by modifi-
cations to the system. Calculated theo-
retical minimum methane costs were <
$0.05 per 1000 gal. Variability in the
degree of TCE degradation and diffi-
culty in maintaining the activity of the
microbial culture during continuous
operation were noted. Sustained use of
the technology will require modifica-
tions to culture conditions.
This Summary was developed by
EPA's Risk Reduction Engineering
Laboratory, Cincinnati, OH, to announce
key findings of the SITE Emerging Tech-
nology program that is fully docu-
mented in a separate report (see Project
Report ordering information at back).
Introduction
Chlorinated, volatile, aliphatic hydrocar-
bons (Clx-VOCs) are the most commonly
reported contaminants of groundwater. The
reason for their widespread occurrence in
the environment is their widespread use
as solvents and degreasers. Since this
problem came to light as recently as the
early 1980s few approaches have been
developed for remediating TCE-contami-
nated sites. Currently available remediation
methods for subsurface environments in-
clude air sparging of the groundwater,
vacuum extraction of contaminants from
the vadose zone, and extraction of con-
taminated water for air-stripping. These
techniques transfer contamination from the
subsurface environment to either the air
or to activated carbon, which must then
be landfilled or incinerated. Landfilling the
contaminated activated carbon transfers
the contamination to another environment,
and incineration is costly and requires con-
siderable energy and capital equipment to
completely oxidize volatile chemicals.
Treatment systems based on oxidation of
contaminants that use ultraviolet radiation
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in combination with a chemical oxidant
(peroxide or ozone) are also available,
but these methods are energy intensive
and require addition of expensive chemi-
cals.
A number of particularly promising new
approaches rely on bacterial cooxidation
of the Clx-VOCs during growth on another
(primary) carbon source. One such bacte-
rium is the obligate methanotroph
Methylosinus trichosporium OB3b (here-
after, M.t. OB3b). This organism produces
soluble methane monooxygenase (sMMO)
when grown on single-carbon substrates
(methane, methanol, or formate). SMMO
is an enzyme of low substrate specificity
capable of catalyzing a variety of oxida-
tion reactions in addition to the oxidation
of methane. Among those reactions are
the oxidations of several Cl -VOCs (see
Table 1 for list)-reactions often resulting
in stoichiometric quantities of mineral end
products (carbon dioxide, water, and chlo-
ride ion).
The full report addresses the use of
M.t. OB3b for remediation of TCE-con-
taminated groundwater with the use of a
two-stage bioreactor. In this system, cells
produced at high concentration in a cul-
ture medium contacted contaminated
groundwater in a plug-flow reactor. The
objectives of the study were:
. to determine reactor design parameters
at bench scale and to operate a pilot-
scale reactor to achieve degradation
of TCE and
. to determine operating values for
parameters that influence the
economic competitiveness of the
system.
Since the economic viability of the sys-
tem was dependent on the efficiency of
methane utilization in the culture vessel,
an innovative methane transfer method
was used to increase methane transfer
efficiency.
Procedure
In bench-scale experiments, the con-
ceptual design was evaluated and starting
values for the operational parameters of
the bioreactor were determined. After the
concept was confirmed at the bench, a
pilot-scale reactor that used the design
criteria established during the bench tests
was constructed.
For the bench-scale system, cells were
grown in a 2000 ml chemostat vessel in
1000 ml of culture medium and fed to
fabricated glass columns where they con-
tacted contaminated water. The total flow
rate was adjusted by adding make-up wa-
ter. Influent and effluent TCE concentra-
tions were measured over the plug-flow
reactor before and after initiation of cell
culture flow to the column. Thus, conser-
vation of TCE was established before in-
troducing the cells.
For the pilot test, groundwater treated
by air-stripping to remove TCE was ob-
tained from a nearby army munitions facil-
ity and carried in a stainless-steel tank
truck to BioTrol's pilot testing facility. The
water was then piped from the truck to a
500 gal, polyethylene surge tank and me-
tered into a stainless-steel plug-flow reac-
tor at a controlled rate. A high-concentra-
tion TCE solution (prepared in degassed
distilled water) was metered into the influ-
ent groundwater upstream from the influ-
ent sampling port. The TCE solution was
held in a Teflon gas-sampling bag that
collapsed as the TCE solution was pumped
out. TCE and cells were added within the
Table \ Summary of Compounds Degraded by Methylosinus trichosporium 0636
Methanes
dichloro (methylene chloride}
trichloro (chloroform)
Ethanes
1,1-dichloro
1,2-dichloro
1,1,1-trichloro
Ethenas
chloro (vinyl chloride)
1,1-dichloro (vinylidene chloride)
t-1,2-dichloro (DCE)
c-1.2-dichlom(DCE)
trichloro (TCE)
Other
1,3-dichioropropene (-propylene)
2,2,2-tr/chloroacetaldehyde (chloral hydrate)
closed reactor system to avoid TCE losses
by volatilization. Once again, TCE conser-
vation was established before bacteria
were introduced to the plug-flow reactor.
Culture medium containing a high den-
sity of cells (Asoo = 1.8, or approximately
32 mg dry ceils/L) was pumped into the
plug-flow reactor at a rate equivalent to 1/
10 the rate of groundwater flow. The total
flow to the plug-flow reactor was 1 L/min.
(See Table 2 for operating parameters.)
The TCE concentration was measured
over the full length of the plug-flow reactor
(at approximately 20-ft intervals). Reactor
performance was determined on the basis
of TCE concentrations throughout the re-
actor. Growth and activity of the microor-
ganisms within the culture vessel were
evaluated on the basis of culture density,
color (visually), and sMMO activity with
the use of a colorimetric assay. All materi-
als contacting the contaminated water were
either stainless steel, Teflon, or glass.
Results and Discussion
A flow diagram of the reactor system is
provided in Figure 1, and the operational
parameters for the bench tests are shown
in Table 2. The intention of the bench test
was to determine parameters that would
provide for stable, continuous treatment
of TCE by M.t. OB3b. A culture-vessel
dilution rate of 0.02/hr was established
experimentally to maintain a steady-state
M.t. OB3b concentration based on the
growth rate of the bacteria in the
chemostat. Growth of the organisms is,
however, a function of methane availabil-
ity, which is, in turn, a function of gas
transfer efficiency. Gas transfer efficiency
is variable based on the chemostat's char-
acteristics (aerator and impeller dimen-
sions, etc.) and, thus, will change during
scale-up.
TCE biodegradation over several hours
of treatment with the use of the optimized
bench-scale reactor system is illustrated
in Figure 2. Average influent and effluent
TCE concentrations were 563 and 63 parts
per billion (ppb), respectively, which cor-
respond to an 89% TCE reduction.
Although these results signify an out-
standing potential for TCE treatment
through the reactor, instability of the pure
culture of A/I. f. OB3b was noted. This sug-
gests that ultimate modifications to the
culture system would be needed to achieve
stable, long-term treatment. The instability
was noted as a sharp decrease in sMMO
activity by colorimetric assay followed by
change in the color of the culture from
yellow to dull green color. A mixed culture
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that included M.t. OB3b, various other
morphologically diverse bacteria, and an
abundance of ciliates 'was observed in the
culture medium by phase-contrast micros-
copy. To achieve continuous treatment of
TCE, this problem must be addressed;
however, to determine the feasibility of
the treatment concept before added effort
was given to culture development, the pi-
lot demonstration proceeded.
The primary objectives of the pilot-scale
demonstration were (1) to determine
whether TCE degradation activity similar
to that observed during the bench test
could be induced at pilot scale and (2) to
evaluate the costs of operating the reac-
tor system to treat TCE-contaminated wa-
ter. Because the cost of methane gas was
a primary concern, a bubbleless, gas trans-
fer device was added to the reactor sys-
tem to increase the methane transfer effi-
ciency (Figure 1). Culture medium was
circulated through the device for methane
saturation and then returned to the culture
vessel.
When operating optimally, 88% of the
influent TCE was biodegraded by using
the operating parameters shown in Table
2. TCE concentrations were measured at
various distances down the plug-flow re-
actor to compare actual data to a first-
order kinetic model. The results are shown
in Figure 3. Clearly, with optimal perfor-
mance, the first-order model adequately
describes TCE removal from the contami-
nated water.
TCE disappearance was monitored
through the reactor system during two
separate operations. Operations 1 and 2
lasted 10 and 8 days, respectively. Some
degree of TCE treatment (minimum 14%)
was accomplished on each day of each
operation. Typically, 5 or 6 days of near-
optimal reactor performance (and approxi-
mately first-order reactor kinetics) were
followed by a few days of decline before
sMMO activity was essentially eliminated.
This was true for both bench- and pilot-
scale systems. Day-to-day treatment effi-
ciency, however, changed considerably,
even though the growth rate (and thus,
the cell concentration in the culture ves-
sel) remained constant. This implicates
the physiological conditions of the bacte-
rial culture, which are probably affected
by fluctuating concentrations of metabolic
byproducts (such as methanol) or by com-
petition from other organisms in the cul-
ture vessel.
During the pilot demonstration, meth-
ane was used at a rate of 240 ml/min at
standard temperature and pressure. The
apparent yield based on this flow rate and
cell production rate was 3 x 10~3 g cells/g
theoretical oxygen demand. Since meth-
ane is a highly usable substrate for these
Table 2. Bioreactor Operational Parameters for Bench and Pilot-Scale Systems
Units
i1" i i set:
Volume L
Cell dilution rate h-r'
Cell density Asfg
Methane flow rate L-ftr'
Air flow rate L*hr''
Plug Flow Contactor:
Length rn
I.D.' cm
Volume L
Culture medium flow/ (qj L-hr'
Groundwatar flow (q,J L*hr1
Total bioreactor flow (Qb) L-hr1
HRT hr
Bench Unit
1
0,02
6,1
1.05
21
0,61
2,54
0.31
0.0280
0.252
0.280
1.1
Pilot Unit
300
0.02
1.8
14.4
30
5
80
6
SO
66
0.91
organisms and since typical yields on car-
bon substrates are >10'' g cells/g BOD, a
high degree of methane stripping was sus-
pected. Based on methane cost of $0.507
hundred ft3, the calculated cost of meth-
ane during the pilot demonstration was
approximately $0.33/1000 gal of water
treated.
Although methane was added via the
gas transfer device, air was still introduced
through sparging devices within the cul-
ture vessel. The efficiency of methane use
would probably be improved by adding
both air and methane through the gas
transfer device and, thus, sparging would
be avoided. In addition, during this test,
the vessel was stirred by an agitator that
could be avoided or at least minimized.
These improvements would increase the
efficiency of methane use and, thus, re-
duce the cost associated with methane
supply. (Theoretically, minimum rates of
bacterial methane consumption would re-
sult in methane costs of < $0.05/1000 gal
of treated water.) Thus, it is expected that
using this technology would result in lower
treatment costs than would either ad-
vanced oxidation or carbon adsorption/
disposal.
Conclusions and
Recommendations
It was concluded that:
. methanotrophic TCE degradation in
this two-stage bioreactor system is
feasible, and
. the cost of methane necessary to
support TCE biodegradation is not
excessive in relation to the costs of
other technologies available for
destructive TCE removal from water.
The extent of degradation of TCE from
day to day was highly variable. Conversely,
the culture xlfinsijv jf^A was relatively
stable at approximately 1.8. Although the
culture was not axenic, M.t. OB3b con-
centrations in the culture medium remained
high through the end of the test runs.
Thus, the fluctuations in TCE degradation
activity more likely resulted from changes
in the expression of sMMO by the culture.
Future studies should focus on stabiliza-
tion of the culture for consistent, long-term
treatment of chlorinated, volatile, aliphatic
hydrocarbons.
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\Uathanm
Effluent
Figure 1. Schematic diagram of the bioreactor system. In the bench-scale system methane and air were introduced to the culture vessel through a glass
air diffuser. The diagram includes a representation of the bubbleless gas-saturating device ("Methane Exchg") that was used in the pilot-scale
system.
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I
LU
600
500
400
300
200
100
n INFL
O EFFL
0123
HRT (hours)
Figure 2. Bench-scale TCE concentrations in plug-flow reactor influent and effluent streams. Hour zero corresponds to the first TCE measurement
after the flow of bacterial culture to the reactor began.
1500
1000
O
Ul
500
0
10
20
50
Figure 3.
30
HRT (mm)
TCE concentrations at various distances down the length of the plug-flow reactor as a function of the hydraulic residence time to thatport. Results
are from the third day of operation of the pilot reactor, showing approximation of reactor performance to first-order kinetics. The parameters
(estimated by nonlinear regression ± S. E. of the estimate) were Sg = 1896± 40 ppb and K, = 3.43 ± 0.14 x HWmin (2.06 ± 0.08/hr).
concentration in the plug flow reactor was 3.6 mg dry weight/L.
5 'U.S. GOVERNMENT Printing OFFICE: 1968- 750-071/80080
The cell
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