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By Paul B.
Hatzinger, Robert J. Steffan, and Scott R. Drew
Although
methyl tert-butyl ether (MTBE) has been used as a fuel
additive since the late 1970’s, the microbial degradation of
this compound received little attention until a few years ago
when reports concerning its prevalence as a groundwater
contaminant began to emerge. Initial studies suggested that
MTBE was resistant to microbial degradation, but more recent
research has shown that MTBE can be biologically degraded by
specific types of microorganisms. Mixed cultures of bacteria
capable of MTBE degradation were initially isolated from
industrial sludge. These consortia contained several
microbial species whose roles in MTBE degradation were not
well defined. Later studies revealed that the oxygenate could
be partially or fully degraded by certain pure cultures of
bacteria and fungi, but that these organisms could not utilize
MTBE as a sole substrate for growth. Rather, each organism
utilized a second substrate, such as propane, n-butane, or
pentane, for growth and co-metabolized MTBE after growth on
that substrate. Most recently, pure cultures of bacteria
capable of growth on MTBE have been isolated by scientists at
the University of California, Davis (Strain PM1), Equilon (Rhodococcus
sp.), and Envirogen (Hydrogenophaga flava ENV735). Each of
these strains has been shown to grow on MTBE as a sole carbon
source, and each is currently being tested in the field for in
situ remediation of MTBE.
Envirogen is
developing and testing in situ and ex situ technologies for
the bioremediation of MTBE in groundwater based upon several
years of laboratory research. These technologies are also
applicable for tert-butyl alcohol (TBA), a production
byproduct of MTBE that is often present in groundwater
contaminated with MTBE. The first approach relies on
stimulating the growth and activity of propane-oxidizing
bacteria that co-metabolically degrade MTBE after growth on
propane. The second approach employs a novel bacterium that
utilizes MTBE as a growth substrate. This organism is
currently being tested for both in situ and bioreactor
applications. The potential effectiveness of propane
biostimulation, bioaugmentation with a pure culture, and a
membrane bioreactor for MTBE and TBA remediation are discussed
in this article.
Biostimulation with Propane
Propane-oxidizing bacteria are found as part of natural
microbial communities in many environments, including soils,
sediments, and subsurface aquifers. These bacteria have the
ability to utilize propane as a substrate for growth.
Research conducted in our laboratory beginning in the
mid-1990s revealed that many propane-oxidizing cultures are
also able to degrade MTBE to CO2. The organisms do not use
the oxygenate as a growth substrate, but rather co-metabolize
MTBE (i.e., degrade it without gaining energy or cell mass)
after initial growth on propane. It appears that the
microbial enzyme that catalyzes the oxidation of propane for
cell growth, propane monooxygenase, also initiates the
oxidation of MTBE. Based on this observation, Envirogen has
conducted laboratory and field studies to evaluate the
potential use of propane addition to stimulate MTBE
biodegradation by indigenous microorganisms or to maintain the
activity of exogenous propane-oxidizers in groundwater
aquifers. Envirogen was issued a patent for this technology
in 1998 (US Patent # 5,814,514).
Laboratory
tests of in situ propane biostimulation have been conducted
using aquifer materials collected from several locations in
the United States. In some of these samples, the addition of
propane was followed by complete degradation of MTBE. For
example, the addition of propane to microcosms containing
sediments and groundwater from a site in California resulted
in the degradation of MTBE from approximately 8 mg/L to below
detection. Additional MTBE was added to the samples several
times to show that degradative activity can be maintained in
an aquifer through propane addition. These samples also were
amended with inorganic nutrients (nitrogen and phosphorus) and
oxygen to promote growth of the propane-oxidizing strains. In
some cases, aquifer samples contained very low numbers of
naturally-occurring propane-oxidizing bacteria, and propane
addition alone did not enhance MTBE degradation. However, the
addition of an exogenous propane-degrading strain (ENV425) as
a seed culture followed by propane addition to maintain this
culture successfully promoted MTBE degradation at such sites.
The enhanced degradation of MTBE at a site in New Jersey where
inoculation of ENV425 was required.
There are
several advantages of using propane biostimulation for
degrading MTBE in situ. A general distribution system is
required to supply low levels of propane and air (or oxygen)
throughout the MTBE containing region. However, the
technology can be applied in a number of configurations, some
utilizing existing systems, depending on site characteristics
and treatment needs. Potential application designs include
the following: 1) a multi-point biosparging system; 2) propane
and air delivery points arranged to form a permeable treatment
wall to prevent off site migration of MTBE; 3) permeable
treatment trenches designed with air and propane injection
systems; 4) in situ recirculating treatment cells to capture
and treat migrating contaminant plumes; and 5) propane and
oxygen injection through bubble-free gas injection devices to
minimize off-gas release and contaminant stripping.
Furthermore, propane is widely available, transportable even
to remote sites, and relatively inexpensive. Therefore,
propane biostimulation has the potential to be an attractive
remediation option at a wide variety of MTBE-contaminated
sites.
Propane
biostimulation is currently being demonstrated by Envirogen as
a remediation technology for MTBE at the Port Hueneme National
Environmental Technology Test Site (NETTS) in California.
This demonstration is funded through the Environmental
Security Technology Certification Program (ESTCP) of the
Department of Defense. The technology is also undergoing
verification under the EPA’s MTBE Treatment Technology
Evaluation Program. Results from the field demonstration in
Port Hueneme are expected in fall of 2001.
Bioaugmentation
In addition
to propane biostimulation for MTBE remediation, Envirogen has
conducted studies with a recently isolated bacterium of the
species Hydrogenophaga flava (ENV735) which utilizes MTBE as a
growth substrate. This is one of only a few pure cultures
known to grow on MTBE as a sole source of carbon and energy,
and thus require no co-substrate (e.g., propane). Strain
ENV735 also grows on tert-butyl alcohol (TBA), a compound that
is frequently found in conjunction with MTBE in subsurface
plumes. Results from laboratory studies indicate that ENV735
may have broad application for treatment of MTBE in the
subsurface. The bacterium degrades MTBE effectively over the
pH values (5 to 7) and at the temperature (15oC) expected in
many groundwater aquifers across the United States. In
addition, ENV735 is unaffected by the presence of BTEX
compounds (benzene, toluene, ethylbenzene, xylenes) up to a
concentration of at least 100 mg/L. These results suggest
that the organism is well adapted to degrade MTBE in many
subsurface environments.
Studies
conducted with field samples from eight different MTBE sites
confirm the potential of this bacterium for in situ MTBE
bioremediation. Inoculation of ENV735 in aquifer microcosms
yielded positive results in samples from six of the eight
sites tested to date. For example, MTBE was degraded from
approximately 20 mg/L to below detection in aquifer samples
from a site in southern California. Samples that did not
receive the bacterium showed no decline in MTBE levels.
Strain ENV735 also degraded MTBE to below detection in site
samples from northern California, New Jersey, and
Pennsylvania. MTBE degradation was not observed in aquifer
samples from two other sites. However, each of these sites
also contained substantial levels of high-molecular-weight
organic contaminants. It is likely that one or more of these
contaminants was toxic to bacteria. This hypothesis is
supported by the fact that BTEX, present at low mg/L levels in
one set of samples, also did not decline appreciably during
the study. This is unusual because these gasoline
constituents are usually readily degraded by indigenous
bacteria under the conditions of the assay (aerobic, nutrients
added). Further studies are underway to fully assess the
impact of specific non-BTEX organics on ENV735, and to define
sites where this technology will and will not be applicable.
Overall, the
results from the initial aquifer microcosm studies with ENV735
have been very promising. The aquifer samples collected
represent the geochemical conditions at actual sites, and the
microcosm assays were conducted at representative site
temperatures. In addition, the samples were not sterilized to
provide strain ENV735 with a selective advantage. The
inoculated bacterium had to compete with native microorganisms
for nutrients and oxygen as would occur after inoculation to
the subsurface. Thus, the data suggest that strain ENV735 has
the potential to survive and degrade MTBE in subsurface
environments under a variety of conditions. A field
demonstration of MTBE remediation using strain ENV735 is
planned to begin in October, 2001. This demonstration will be
funded through a research grant received this year by
Envirogen from the National Science Foundation.
Membrane
Bioreactors
In some
cases, remediation of MTBE-contaminated groundwater or
wastewater will require the use of an ex situ treatment
system. Typical remediation scenarios where pump-and-treat
systems and ex situ MTBE treatment are required include source
reduction, mitigation of plume migration, or geological
settings where in situ approaches are not feasible. Although
several technologies have been able to treat the BTEX
components of gasoline and MTBE, many of these approaches are
expensive when treating high concentrations of MTBE. In
addition, two of the most common options, carbon adsorption
and air stripping are not feasible when both MTBE and TBA
occur together and require treatment. Because most MTBE-degrading
cultures grow slowly and with low cell yield on the oxygenate,
reactors must be designed to maximize the quantity of cell
biomass retained in the system. Otherwise, the size and
capital cost of the treatment system will be prohibitive.
To maximize
biomass retention and minimize reactor size for this
application, Envirogen selected a membrane bioreactor (MBR)
that employs microfiltration membranes as separators to retain
high levels of microbial cells. The MBR system consists of a
suspended growth reactor fitted with internal microporous
hollow fiber membranes. Effluent water is removed from the
reactor through the membrane system to minimize losses of
biomass. MBR reactors are well suited for treating higher
strength waste streams and waste streams containing slowly
biodegraded organics, such as MTBE, because of their ability
to retain biomass and generate high volumetric efficiencies.
These reactors are also simple to operate and produce effluent
with low solids (TSS <1 mg/L).
Studies to
evaluate MBR performance under different operating conditions
are ongoing. Initial studies used 85-L suspended growth
reactors fitted with hollow fiber membranes. The MBRs were
seeded with the MTBE-degrading strain ENV735. In one test, a
15 mg/L feed concentration of MTBE was added to an MBR. The
hydraulic retention time (HRT) was maintained at 3 hr and the
solids retention time (SRT) was 120 hr. BTEX and tert-butyl
alcohol (TBA) were also added in this test at 5 mg/L each.
Effluent MTBE concentrations were less than 0.05 mg/L during
the test, representing a greater than 99.6% removal
efficiency. BTEX and TBA were completely degraded in the MBR.
To evaluate MBR performance at higher concentrations of MTBE,
the oxygenate was added to influent water at a concentration
of 1,000 mg/L. The HRT was initially maintained at 3 days
(1.2 L/hr into the 85-L reactor), and after a period of
successful operation, the HRT was reduced to 1 day,
effectively tripling the MTBE loading. MTBE removal rates
reached 42 mg/L/hr during operation with a 1-day HRT,
representing a removal efficiency of >99.9%.
The MBRs
seeded with strain ENV735 efficiently removed MTBE from
groundwater at concentrations ranging from 15 – 1,000 mg/L.
TBA and BTEX were also degraded in the reactors, and MTBE
removal efficiency was not affected by these compounds.
Furthermore, cell yields in the reactors with MTBE as the sole
feed source were remarkably high (40%) relative to cell yields
observed with other cultures (typically <20%). These results
suggest that full-scale MBR systems will be able to operate
effectively to treat groundwater with MTBE alone or MTBE with
BTEX and/or TBA contamination. An MBR can be economically
coupled to a post-treatment polishing system, such as
activated carbon adsorption, to achieve MTBE levels below
detection from most waste streams.
Conclusions
Few remedial
technologies are currently available for in situ or ex situ
remediation of MTBE-contaminated sites. The results of our
work suggest that propane biostimulation is a low cost in situ
remediation technology for MTBE. The technology can be
applied in a variety of configurations, including existing
sparging systems, permeable treatment walls or trenches, or
recirculation systems, depending on site conditions. In cases
where natural propane-oxidizers do not exist in sufficient
numbers, or do not respond to propane injection, exogenous
strains such as ENV425 can be added as seed cultures to
facilitate MTBE degradation. Bioaugmentation with strain
ENV735, an organism that utilizes MTBE as a growth substrate,
also shows great promise. Experiments are currently underway
to isolate strains of ENV735 which move readily through
subsurface aquifers, so that these bacteria can be easily
distributed in areas containing MTBE. Application of adhesive
cells of ENV735 in cut-off trenches to prevent off-site
migration of MTBE plumes is another treatment option. Field
demonstrations of both of these in situ technologies are
underway. When groundwater must be extracted to prevent
migration of MTBE and TBA or to reduce the contaminant source,
MBRs seeded with strain ENV735 can be employed to treat the
recovered groundwater. Results from pilot-scale MBR testing
suggest that this reactor design holds great promise for
treatment of MTBE in many concentration ranges. Reactors may
also be combined with conventional technologies to provide a
variety of cost-effective solutions. The task of cleaning up
MTBE-contaminated groundwater in the Unites States is a large
one. However, new bioremediation technologies promise to
provide an economically attractive option for achieving
remedial goals.
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