WITH the recent focus on biological
warfare agents that can cause diseases such as anthrax or plague,
the myriad ways that bacteria can benefit people are easily overlooked.
In the area of cleaning up pollution, researchers are discovering
the extraordinary capabilities of some bacteria to grow on and
degrade substances, such as chlorinated solvents and benzene, that
are quite toxic to humans. Many of these bacteria have been found
in contaminated aquifers, leading to
the discovery that naturally occurring bacteria can actually promote
At Lawrence Livermore, environmental microbiologists Harry Beller
and Staci Kane are interested in the natural role of bacteria in
breaking down compounds of environmental concern. They head up
an applied research team that is part of the Environmental Protection
Department’s efforts to understand how these unusual bacteria
can naturally degrade the compounds that escape from leaking underground
fuel tanks (LUFTs). To get a better handle on how these bacteria
work in the subsurface, researchers are using two advanced techniques
for rapidly and reliably detecting the bacterial degradation of
toxic compounds in soil and groundwater samples collected at LUFT
technique, real-time polymerase chain reaction (PCR), is widely
used in biomedical research and has also been applied
at Livermore to detect bioterrorism agents. The other technique,
liquid chromatography/tandem mass spectrometry (LC/MS/MS), is also
used to detect chemical warfare
agents and in the biomedical and pharmaceutical industries. Both
techniques show great promise for monitoring the activity of bacteria
that are cleaning up groundwater naturally.
benzylsuccinate synthase (BSS) reaction, which anaerobic bacteria
use to attack (a) toluene and (b) xylenes as the first step
of the biodegradation process. The BSS enzyme catalyzes the
addition of toluene or xylene to fumarate, a compound that
is typically present in many bacteria.
What Lurks Below the Surface
Groundwater contamination at LUFT sites is a pervasive problem
at federal and commercial facilities throughout the U.S. By 2002,
more than 427,000 releases from LUFTs had been confirmed nationwide,
and according to the U.S. Environmental Protection Agency, the
cleanup backlog totaled more than 142,000 sites. Among the compounds
in gasoline that are of greatest regulatory concern are benzene,
toluene, ethylbenzene, and the three xylene isomers. (Isomers
are compounds that have the same atomic composition but differ
in structural arrangement.) BTEX is the acronym for this mixture
of hydrocarbons. BTEX are among the most toxic and water-soluble
constituents of gasoline.
is one of several accepted methods for restoring BTEX-contaminated
aquifers to environmentally satisfactory conditions.
Intrinsic bioremediation (also called natural attenuation) relies
on indigenous bacteria to degrade contaminants in place and is
a cost-effective approach favored by the LUFT owners responsible
for cleanup. However, regulatory agencies and the public are sometimes
of intrinsic bioremediation, viewing it
as a “do nothing” approach.
key to acceptance of intrinsic bioremediation at a given LUFT site
is the ability to demonstrate in a substantial, scientifically
credible manner that biodegradation of the contaminants is occurring.
As Beller notes, “One of the roadblocks to the widespread
acceptance of intrinsic bioremediation in groundwater is the difficulty
of proving that measured decreases in BTEX concentrations are due
to bacterial degradation and not to nondestructive processes such
as dilution or dispersion. The monitoring methods that we have
developed are designed to be capable of providing incontrovertible
evidence of the biodegradation of BTEX compounds.”
Basics of the Real-Time
Polymerase Chain Reaction
a way to rapidly identify DNA by the real-time polymerase
chain reaction (PCR) was a breakthrough event in the
mid-1990s that launched Livermore’s biodefense
program. At the time, PCR was a well-established technique
for identifying specific regions of DNA. PCR works
by making multiple copies of a particular segment (referred
to as the amplicon) of the DNA in the sample. When
the sample is heated, the double-helix of DNA separates
into two single complementary strands. When the sample
is cooled, single, short (18- to 25-nucleotide) strands
of DNA called primers attach to the ends of the target
region to be amplified. Subsequently, a heat-stable enzyme (Taq DNA polymerase from Thermus
aquaticus, a bacterium isolated from hydrothermal
vents) replicates the region of DNA bracketed by the
primers. With each
heating–cooling cycle, the amount of DNA doubles.
Eventually, after 20 cycles, a single target would
be amplified a millionfold.
dramatic advance in PCR technology was
the development of real-time PCR, which
allows for rapid quantification of specific
genes. In addition to the specific primers
used in conventional
PCR, real-time PCR also includes a probe (typically
20 to 35 nucleotides long) that specifically binds
to a region of the target DNA that is bracketed by
the primers. The probe is labeled with fluorescent
dyes at each end. One dye quenches the fluorescence
of the other when the probe is intact. The real-time
PCR method relies on the exonuclease activity of Taq DNA polymerase that cleaves the probe, resulting in
fluorescence. The amount of fluorescence
is proportional to the amount of replication, which in turn is
proportional to the number of initial target DNA copies. By performing
real-time PCR with specific DNA standards, a calibration curve
is obtained to calculate the amount of target
DNA in the environmental DNA extract.
real-time PCR technique is fundamental to the Livermore-developed
Handheld Advanced Nucleic Acid Analyzer (HANAA) and Biological
Aerosol Sentry and Information System (BASIS), which are used to
identify microorganisms that present a biological threat.
more information about PCR and its biodefense applications,
see S&TR, January/February
2002, Rapid Field Detection of Biological Agents, and June
1998, Reducing the Threat of Biological Weapons.
underlying LUFT sites is typically oxygen-depleted (anaerobic)
because oxygen-respiring (aerobic) bacteria rapidly
use up the available oxygen. Once the oxygen in the groundwater
environment is depleted, intrinsic bioremediation can only work
if anaerobic bacteria are present that can degrade BTEX. As recently
as 15 years ago, notes Beller, conventional wisdom held that anaerobic
BTEX-degrading bacteria didn’t exist because degradation
of BTEX compounds in the absence
of oxygen was an insurmountable biochemical challenge.
research in the past seven years not only showed that such bacteria
exist, but it also identified the key enzyme,
benzylsuccinate synthase (BSS) that carries out the first step
of anaerobic degradation of toluene and xylenes. During this time,
researchers discovered the gene sequences that lead to the production
of this enzyme. The BSS reaction is found
in diverse anaerobic, toluene-degrading bacterial cultures that
represent the range of bacteria one would expect to find in aquifers
under LUFT sites.
and Kane decided to leverage this new understanding of anaerobic
BTEX degradation to develop methods that would unequivocally demonstrate
whether intrinsic BTEX bioremediation was occurring at a given
LUFT site. Their methods focus on two aspects
of the metabolic process.
The first monitoring method focuses on the bssA gene. This method
gives scientists a tool for counting the bacteria that harbor a
gene specific to anaerobic toluene and xylene degradation. The
second method focuses on the unique metabolic products of the BSS
reaction, which are benzylsuccinate and methylbenzylsuccinates.
This method sensitively detects these metabolites, which have no
known sources other than the anaerobic degradation of toluene or
explains, “The power of these so-called signature
metabolites is that their mere presence in groundwater definitively
demonstrates the degradation of specific compounds. There is no
other way that these compounds could appear in the groundwater.”
of the similarity of bssA gene sequences in four different
bacterial strains. This figure shows the genetic code as “translated” into
amino acids, which make up the enzyme. Only selected portions
of the benzylsuccinate synthase enzyme are shown, specifically,
portions of the largest of three subunits that make up the
enzyme. Among the different bacterial strains, yellow indicates
identical amino acids, and green indicates similar amino acids.
Monitoring Bacteria Genetically
first method quickly and accurately counts the number of copies
of a specific bacterial gene, bssA, in samples of aquifer sediment.
Because each bacterial cell typically contains only one copy of
the bssA gene, the number of gene copies is equivalent to the population
of bacteria that contain bssA. Thus, this method quantifies the
number of bacteria that are genetically capable
of anaerobic toluene or xylene degradation in a
the BSS pathway is the only one to date that has been identified
with anaerobic toluene degradation, measuring populations
containing bssA probably is inclusive
of most anaerobic toluene- and xylene-degrading bacteria,” says
does counting the copies of bssA relate to intrinsic bioremediation?
Kane explains that if anaerobic bacteria at a LUFT site are metabolizing
BTEX and proliferating, it is reasonable to expect that the populations
of these bacteria should be higher within BTEX-contaminated areas
than in nearby, uncontaminated areas. “This method allows
us to compare bacterial populations containing bssA over distance
or time,” she adds.
researchers use analysis based on the real-time PCR—also
known as quantitative PCR or TaqMan® PCR—to
quantify copies of the bssA gene by targeting DNA sequences
that are unique to this gene. The same technology has been used
by scientists at Livermore to develop real-time PCR methods for
targeting specific bioterrorist agents such as Bacillus anthracis, which causes anthrax.
team, including microbiologist Tina Legler, began by comparing
the bssA genes in four different toluene-degrading bacterial strains.
At the time this study started, bssA sequences were only available
for two strains, so bssA sequences were determined for two additional
strains to provide a better assessment of the diversity of bssA sequences
among toluene-degrading bacteria. In this and later studies, the
team found a high degree of similarity in this gene sequence
among different organisms. After
aligning four DNA sequences chosen from their studies, the researchers
on a stretch of DNA—about 130 base pairs—for development
of their real-time PCR method. To target this region, they designed
degenerate primers that corresponded to sequences from all four
strains and an internal probe that complemented bssA from all four
Real-time PCR of bssA successfully
tracked toluene-degrading bacteria under denitrifying conditions.
The relationship between toluene degradation and increases
in numbers of bssA copies is apparent.
test the technique, the team took samples from four sites with
different histories of BTEX exposure, including three LUFT sites
and an uncontaminated site. They spiked the sediments with BTEX,
incubated them in the laboratory under various conditions, and monitored
the BTEX degradation activity.
For real-time PCR analysis, they extracted and purified the total
DNA from more than 100 5-gram sediment samples of these laboratory
incubations. Using real-time PCR, the researchers successfully tracked
bacterial population trends that were consistent with observed anaerobic
toluene degradation activity.
also discovered that, of all the environments studied, the ones with
denitrifying conditions—that is, where nitrate was being
respired by bacteria in the degradation process—had the most
rapid toluene degradation and the largest abundance of bssA. In the
samples with the most rapid toluene degradation, the numbers of bssA copies increased 100- to 1,000-fold during the first 4 days of incubation,
the time when most of the toluene was being consumed. The team validated
its method by comparing
its results with those produced by traditional hybridization-based
methods that do not use PCR amplification and by analyzing the sequences
of PCR products to confirm the method’s specificity.
PCR technique, Kane notes, has many advantages over other bacteria-counting
methods that require cultivating the bacteria
in the laboratory and then calculating the original populations. “Cultivating
anaerobic bacteria can be a difficult, sometimes seemingly impossible,
task. Because they reproduce slowly, cultivation is
also time-consuming,” she says. “Getting results can
take from days to months, whereas the real-time PCR method does the
job in less than an hour.”
plus is the method’s sensitivity. PCR can detect
as few as five copies of a gene per analysis. It is also highly
selective—an important quality, since it must avoid false-positive
Concentrations over time of selected
BTEX hydrocarbons and their corresponding benzylsuccinate
metabolites in groundwater from a site in Seal Beach, California.
In this experiment, BTEX and bromide were added to the
groundwater at the sampling site. All concentrations are
normalized to bromide (an inert tracer that corrects for
the effects of dilution) and to the maximum concentrations
of the compounds themselves. (Copyright 1995 by the American
Chemical Society. Reused with permission from Environmental
Science and Technology, 1995 29, 2869.)
Detecting Key Signatures
The second method for tracking intrinsic bioremediation of BTEX
in groundwater was developed by Beller, using LC/MS/MS to detect
signature metabolites of BTEX degradation.
Instead of counting the populations of bacteria harboring a gene
associated with BTEX degradation, this method measures distinctive
metabolites (benzylsuccinate and methybenzyl-succinate isomers)
that are uniquely associated with anaerobic toluene and xylene
successfully used the signature metabolite approach in a controlled-release
field study before coming to work at Livermore.
However, he didn’t have access
to LC/MS/MS then, and the more traditional analytical methods he
used were labor-intensive and time-consuming. Traditional methods
for such analysis typically involve extraction of a large (1-liter)
water sample with organic solvent, concentration of the solvent
to a small volume, chemical treatment called derivatization that
makes the benzylsuccinates more amenable to further analysis, and
gas chromatography/ mass spectrometry analysis.
the isotope-dilution LC/MS/MS method, Beller can skip
the extraction, concentration, and derivatization steps and analyze
a groundwater sample in less than 10 minutes. The method is highly
sensitive, accurate, and precise, and it requires modest samples—less
than 1 milliliter of groundwater. The detection limits for the
LC/MS/MS technique are about 0.3 microgram of benzylsuccinate or
methylbenzylsuccinate per liter (roughly the equivalent of a thimbleful
of water in an Olympic-size swimming pool).
test this method, Beller turned to a fuel terminal with contaminated
groundwater. Since 1911, the
terminal has been in the business of blending and distributing
petroleum products, such as gasoline and diesel fuel. The team
collected groundwater samples quarterly for a year from 12 wells
located in the highly anaerobic aquifer. Methylbenzylsuccinates
were detected in the three wells with the highest BTEX concentrations.
The methylbenzylsuccinate concentrations ranged from less than
0.3 to 205 micrograms per liter. Beller found a strong and consistent
correspondence between concentrations of methylbenzylsuccinates
and their parent compounds, xylenes, throughout the most contaminated
portion of the aquifer.
the LC/MS/MS method proved to be a rapid, selective, and sensitive
method for detecting benzylsuccinates, which are prime
indicators of anaerobic bacteria hard at work degrading hydrocarbons.
Highly correlated concentrations
of methylbenzylsuccinates (signature metabolites of xylenes)
versus xylenes in groundwater from three different wells
at four sampling times at a contaminated, fuel-distribution
terminal. (Copyright 2002 by the American Chemical Society.
Reused with permission from Environmental Science and
Technology, 2002 36, 2727.)
Future of “Natural” Bioremediation
A major challenge for the regulatory acceptance of intrinsic bioremediation
is to provide evidence that decreases in the concentrations of
groundwater contaminants truly represent biological metabolism
of these contaminants rather than nondestructive, natural processes
such as dilution. Beller and Kane have developed two independent
methods to meet that challenge. They applied these techniques
to contaminants at LUFT sites and are extending their use to
other classes of contaminants, such as nitrate and high explosives.
Despite the significant strides that researchers have made in developing
new methods for monitoring intrinsic bioremediation, a remaining
challenge is to progress from qualitative evidence (is biodegradation
occurring?) to quantitative evidence (what is the rate of in situ
biodegradation, and what proportion of contaminant decreases can
be attributed to biodegradation?). Beller and Kane are investigating
ways to adapt their methods to yield more quantitative data.
additional long-term goal of this research is to gain a better
understanding of how ethanol—which
is a strong contender to replace MTBE in gasoline—could affect
the population of anaerobic BTEX-degrading bacteria and, therefore,
the rates of intrinsic BTEX biodegradation in the subsurface. Before
this project, notes Kane, researchers had no way to quickly assess
the populations of anaerobic BTEX-degrading bacteria in a given
environment. The real-time PCR technique, with its ability to quantify
the abundance of the bssA gene, provides a tool for doing just
Key Words: benzene, toluene, ethylbenzene, xylene (BTEX); biodegradation;
groundwater; hydrocarbons; intrinsic bioremediation; leaking
underground fuel tank (LUFT); liquid chromatography/tandem mass
(LC/MS/MS); natural attenuation; polymerase chain reaction (PCR).
For further information contact Harry Beller (925) 422-0081
(email@example.com) or Staci Kane (925) 422-7897 (firstname.lastname@example.org).
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