Using Fluorine-Induced Chemiluminescence To Detect Organo-Metalloids
in the Headspace of Phototrophic Bacterial Cultures Amended with Selenium
and Tellurium
Verena Van Fleet-Stalder and Thomas
G. Chasteen
Department of Chemistry and
Texas Regional Institute for Environmental Studies
Sam Houston State University, Huntsville,Texas, USA
Abstract
Brief Introduction of Chemiluminescence Methods
Analytical methods based on chemiluminescence have taken their strong position
among the more mundane analytical techniques because of a triumvirate of
strengths: sensitivity, selectivity, and in many cases, a wide linear detection
range. This is true even though chemiluminescence is not as widely applicable
as absorption, emission, or even fluorescence methods of detection since
so few molecules undergo chemiluminescent reactions. Because chemiluminescence,
light emission generated from a chemical reaction, requires no light source
for excitation, the analytical signal appears out of an essentially black
background, and the only background signal is that of the photomultiplier
tube's (PMT) dark current. Therefore light source warm-up and drift and
interference from light scattering are absent. In the case of systems where
red and near infrared light are observed in analytical detection, red sensitive
PMT's dark current can be minimized by cooling; with blue light emission
detection, cooling is not required. Detection limits routinely orders of
magnitude lower than fluorescence methods are achievable. In addition,
interfering molecules are often less of a problem too since chemiluminescence
reactions can be so selective.
The initial reports in the scientific literature of lophine (2,4,5-triphenyl
imidazole) and then lucigenin (N,N'-dimethyl-9,9'-diacridinium nitrate)
chemiluminescence in the last quarter of the nineteenth century [1]
blossomed in this century into reports involving many different chemiluminescent
reagents. The most common or well known solution phase systems involve
luminol (or its derivatives), oxalate esters, lucigenin (or its derivatives),
ruthenium tris-bipyridine, and luciferin. Gas phase examples include the
ozone- and fluorine-induced, sodium vapor, and chlorine dioxide chemiluminescence
detectors for gas chromatography. Because many of the solution phase systems
use hydrogen peroxide or organic peroxides as oxidant and these can be
generated many ways in liquid systems and because many of the solution
phase reagents mentioned above can be tagged onto a large variety of analytes,
high performance liquid chromatographic (HPLC) solution phase chemiluminescence
is more common and variously applied than gas phase chemiluminescence reactions.
In a very general way, the requirements for the analytically useful
production of light from a chemical reaction are: 1) excess chemical energy
produced by the reaction must be relatively efficiently used to populate
the excited state of the emitter and 2) the excited species must have few
mechanism of deactivation except light emission. In many systems, the initially
excited state molecule is used as a conduit of energy to excite a second
or third molecule which, instead, is the actually emitting species. In
solution-phase systems, pH and catalysts must also be considered; however,
in gas phase systems reaction cell pressure and temperature can be important
factors. A more detailed description of these phenomenon and their applications
can be found in the literature in a number of places [1,
2, 3, 4,
5, 6, 7,
8, 9].
The use of chemiluminescence as a detection method following analytical
separation makes up a significant share of its application. For example,
liquid phase chemiluminescence has been applied to high performance liquid
chromatography [10, 11]
and very recently to capillary electrophoresis [12,
13, 14].
Gas phase analytical chemiluminescence reactions have in the main been
employed with gas chromatography (GC) to detect trace chemical species
or target analytes in complex matrices [15,
16, 17, 18,
19, 20, 21,
22]. Other
workers have recently employed "separationless" chemiluminescence methods
to determine total sulfur content in gasoline [23]
and coal [24] and
nitrate and nitrite in flow injection analysis [25].
A very recent supercritical fluid chromatographic (SFC) interface to a
chemiluminescent nitrogen detector has also been reported for the examination
of polymers and pharmaceuticals [26];
although SFC/chemiluminescence techniques have appeared before [27,
28, 29, 30].
This glancing survey of liquid and gas phase chemiluminescence is not
meant to imply that these are the only roles for this method. Researchers
have, for instance, recently used in vivochemiluminescence initiated
by UV-A irradiation of mouse skin as a means of determining skin oxidative
stress processes [31].
Others have used chemiluminescence as a means of following antioxidant
evaluation in mouse kidney and brain [32]
and plasma [33],
as a means of DNA detection and sequencing, detection of polymerase chain
reaction-derived nucleic acids, and alkaline phosphatase determination
[34].
Finally, chemiluminescence has long been used as a means of measuring
concentrations of short lived species in gas mixtures and in the atmosphere
and to that end chemiluminescent techniques have been used to determine
ozone [35], NOx
[36], and hydrogen
peroxide [37] in
the atmosphere, to detect the possible emitter in the reaction of tetrakis(dimethylamino)ethylene
with oxygen [38],
and to map out the "hot bands" of HNO produced in the reaction of NO with
HCO [39] among many
other gas phase applications [40,
41]. The Journal of Bioluminescence and
Chemiluminescence (John Wiley & Sons Publisher) is obviously an excellent
source in this field and periodically publishes literature searches sorted
by year and author [2]. |
Description of a Fluorine-Induced Chemiluminescence
Detector
The experimental results described below were obtained using a gas chromatographic
detection system based on the low pressure, gas phase chemiluminescence
of the reaction mixture of molecular fluorine with organo-sulfur, -selenium,
and -tellurium compounds separated from (gas phase) headspace samples.
This detector was originally developed in the research group of John Birks
at the University of Colorado, USA [42,
43] and was manufactured and sold by Sievers
Instruments (Boulder Colorado, USA). This system can be divided up into
three parts: the chromatograph, transfer line, and reaction cell; PMT and
photon counting electronics; and the molecular fluorine generator (See
Figure 1). |
|
Figure 1. Schematic of the Fluorine-Induced Chemiluminescence
Detector |
Chromatograph and transfer line
A Hewlett Packard 5890 II gas chromatograph (GC) fitted with a 30 m,
0.32 mm i.d. capillary column (DB-5, 0.25 µm film) was used for all
chemiluminescence and flame ionization detector (FID) work reported here.
The GC injector was maintained at 275°C while the oven was temperature
programmed using liquid nitrogen as the oven cryogen. A typical temperature
program involved a subambient period to cryogenically trap gaseous samples
as a thin band at the head of the column (-20°C) for 1 min, followed
by a fast temperature ramp (20°C/min) to a final temperature 25 or
50°C above the highest boiling analyte (usually dimethyl trisulfide,
b.p. ~170°C).
Instead of being plumbed into our gas chromatograph's normal detector
(an FID), the chromatographic column in this system passed through the
wall of the GC oven inside a heated transfer line (a 4 mm i.d. nickel tube
wrapped with heat tape, thermostatically maintained at ~150°C). The
end of the transfer line was attached directly to the cylindrical reaction
cell and the column terminated within a mm or so inside that stainless
steel cell (reactor volume ~15 cm3). Experimentation has shown
that a transfer line temperature greater than 150°C does not improve
the chromatographic integrity even for compounds that boil at substantially
higher temperatures; however, an increased transfer line temperature does
contribute to the warming of the (red sensitive) PMT housing which is adjacent
to the reaction cell, and thereby, increases dark current. |
PMT and photon counting electronics
Figure 2 depicts a schematic of the reaction cell
and PMT housing. The PMT used in this work was a red-sensitive Hamamatsu
#R2228, end-on photomultiplier tube maintained at a voltage of 1300 V (peak
sensitivity ~650 nm, range 300~900 nm). The detector's photon counting
electronics have a integration interval, from 2 seconds--adjustable by
factors of 2--down to 0.0156 seconds. Signals collected for a chosen period
were integrated by a HP 3396 integrator. |
|
Figure 2. Schematic of Reaction Cell and Photomultiplier
Tube Housing of the Fluorine Induced Chemiluminescence Detector |
Molecular fluorine generator
One of the most unique parts of this system is the method of generating
molecular fluorine, F2. Early developmental research with this
system by Birks and coworkers involved a microwave discharge of sulfur
hexafluoride (SF6) as molecular fluorine source [44],
but high backgrounds led the workers to use an alternative source: 5% F2
in He [43]. The inconvenience
and safety consideration of maintaining a tank of F2 in an analytical
laboratory ultimately lead to another solution: electrical discharge of
SF6 (purity 99.996%). Figure 3 shows the
physical arrangement of the parts of the small in-line SF6 discharge
used in the work reported here. Not depicted in that figure is the flow
restricter placed in-line between the sulfur hexafluoride tank and the
discharge chamber: at a SF6 delivery pressure of ~15 psi the
restricter allows about 1 mL/min SF6 flow into the discharge
chamber. |
|
Figure 3. Sulfur Hexafluoride Electrical Discharge |
Comparison of a detection limit for a standard
organosulfur compound using either 5% F2 in He or the electrical
discharge yields essentially the same detection limit; however, the 5%
F2 in the He system is about 25 to 50% noisier than the discharge
while producing 25 to 50% greater signals for the same mass organosulfur
species; ergo, the same detection limit for both.
Mechanism for chemiluminescence
The mechanism of chemiluminescence light production in the fluorine-induced
chemiluminescence detector is not well understood and certainly not as
clearly as that for the sulfur/ozone-induced chemiluminescence detector
which has been recently studied in detail and in which one major emitter,
SO2*, has been detected [20,
17]. This could be because the O3
reaction sequence may be somewhat simpler than the F2-induced
chemiluminescent reactions; however, in regards to the O3-induced
chemiluminescence system, Burrow and Birks have recently published an intriguing
set of experiments which "imply that an unknown sulfur species, X, is formed
in the combustion zone and/or transfer line of the instrument and converted
to SO upon reaction with ozone in the chemiluminescence detection cell"
[45] so maybe things
in that system are not as simple as once thought.
The hypothesized reaction of molecular fluorine with organosulfur species
yields electronically and vibrationally excited HF whose emission signature
can be seen in low pressure reaction mixtures of molecular fluorine and
methanethiol, CH3SH [46]
or ethanethiol, CH3CH2SH [47].
Two mechanisms for the production of HF in this system have been proposed
[43, 4,
48]. One possible mechanism can be seen
in Figure 4 in which hydrogen fluoride is shown to
be the emitting species. This involves a 5-centered reaction intermediate. |
Figure 4. Animation of One of the Proposed
Gas Phase Mechanisms of Chemiluminescence in the F2-Induced
Chemiluminescence Detector
(Click Esc or Command-period to stop the animation in
most browsers.) |
|
Probably more important [48]
is a mechanism involving a charge transfer intermediate in which molecular
fluorine accepts an electron from the less electronegative element, sulfur.
The resulting fragmentation process following the formation of the charge
transfer complex subsequently yields various fragments (including excited
HF) which contribute to the chemiluminescence emissions [49,
46, 47]. Therefore
the actual reaction sequence is not simple, and the light actually measured
by a photomultiplier tube focused on a gas phase mixture in this detector
comes from multiple emitting chemical species.
Apparently one of the requirements for the efficient production of detectable
light in this reaction is an analyte containing a so-called beta hydrogen
which can be abstracted by fluorine as F2 approaches the relatively
electropositive heteroatom in detectable analytes (S, Se, Te, etc.). In
Figure 4, this b hydrogen
is part of the adjacent methyl group. The absence of a b
hydrogen in a particular analyte confers little or no detectability for
that compound in this detector; therefore, H2S and di-tert-butyl
sulfide, for example, exhibit little or no response.
Initially applied to organosulfur species [42,
43], this GC detector was later used to
determine biogenic organo-selenium and -tellurium [50,
5, 52] and organo-phosphines
and phosphinate esters [53].
More recently we have used this system to detect biological organo-antimony
production in a monoculture of a Pseudomonassp.
and in soil enrichment cultures [54]. |
Materials and Methods
The following work involves the use of the gas chromatographic/fluorine-induced
chemiluminescence detector for the determination of organo-S, -Se and -Te
metabolites in the headspace above phototrophic bacteria which were amended
with salts and elemental forms of these metalloids. In addition, one of
Frederick Challenger's proposed biological intermediate compounds [55],
dimethyl selenone (CH3)2SeO2 , was added
in parallel experiments to determine if that chemical species would undergo
bioreduction to yield dimethyl selenide and dimethyl diselenide.
Reagents
All reagents were used as received. Sodium selenate (Na2SeO4),
sodium selenite (Na2SeO3), and sodium tellurate (Na2TeO4)
were obtained from Strem Chemicals (Newburyport, MA, USA). Dimethyl sulfide
(DMS), dimethyl disulfide (DMDS), dimethyl selenide (DMSe), and dimethyl
diselenide (DMDSe), were purchased from Aldrich Chemical Co. (Milwaukee,
WI, USA). Dimethyl telluride was procured from Alfa Products (Danvers,
MA, USA).
Dimethyl selenone (DMSeO2) was synthesized following the
method of Krief et al. [56]
with slight modifications that we have been reported elsewhere [57].
The minimal growth medium used (hereafter, Sistrom medium) has also been
described elsewhere [58],
but briefly, has well-defined buffer and vitamin components and 20 mM succinate
as a carbon source at a pH of 6.8 adjusted before autoclaving.
Bacterial cultures
Six different phototrophic strains (Deutsche Sammlung von Mikroorganismen,
Göttingen, Germany) were investigated: Rhodocyclus tenuis (DSM
#109), Rhodobacter sphaeroides 2.4.1 (DSM #158), Rhodobacter
capsulatus (DMS #1710), Rhodospirillum rubrum S1 (DSM #467)
and G9 (DSM #468), and Rhodopseudomonas blastica (DSM #2131). In
addition, in one series of experiments the type strain of Rhodobacter
sphaeroides 2.4.1 from the American Type Culture Collection (Rockville,
MD, USA) was also investigated. Initially made available as stabs (thanks
to Professor Reinhard Bachofen, Institute for Plant Biology and Microbiology,
University of Zürich, Switzerland) 10 milliliter bacterial cultures
were anaerobically grown (Sistrom medium) in 16 mL test tubes sealed with
Teflon® coated septa in incandescent light of 10 W/m2
intensity. After inoculation of liquid medium the cultures were left in
the dark overnight to deprive them of oxygen before incubation in incandescent
light. Ten mg portions of metallic selenium and tellurium were filled into
the test tubes prior to autoclaving. These cultures were sampled 7 days
after inoculation. The water soluble selenium and tellurium compounds were
added from sterile-filtered stock solutions in Sistrom medium. Amendments
were added to 10 mL cultures that had grown for two to three days; fresh
stock solutions of recrystallized dimethyl selenone were made on the days
of amendment [59, 57].
After the amendment these cultures were left in the dark again, and their
headspace was analyzed after six to seven days of photoheterotrophic growth.
The optical density at 660 nm was used as a measure of biomass.
Headspace analyses
One mL gas phase samples were removed from the headspace of the test
tubes (kept at approximately 25°) by piercing the septum with the needle
of a 1 mL gas tight syringe (Alltech Associates, Inc., Deerfield, IL, USA),
extracting 1 mL headspace, and immediately injected that gas sample into
the hot injector of the GC (275°C). The chromatograph's temperature
program was immediately initiated.
Chromatography
Analysis of volatile, methylated selenium, tellurium and sulfur compounds
was carried out by capillary gas chromatography coupled with fluorine-induced
chemiluminescence detection using the instrumentation and conditions described
above. Retention times of commercial standards were used to identify DMS
(CH3SCH3), DMDS (CH3SSCH3)
DMSe (CH3SeCH3), DMDSe (CH3SeSeCH3),
and DMTe (CH3TeCH3).
Flame Ionization Detection
A flame ionization detector was used in a few experiments to compare
the response of that detector to the response of the fluorine-induced chemiluminescence
detector to volatile, biologically produced compounds in bacterial headspace.
Cultures of Rhodobacter sphaeroides 2.4.1 were cultured as described
above and their headspace gases sampled after 7 days growth. The instrumental
conditions (hydrogen, air, and N2 makeup gas settings) of the
FID were those specified by the manufacturer. The temperature of the detector
was 275°C. The same chromatographic column used in the chemiluminescence
work was deinstalled from that detector and reconfigured to the FID for
these experiments. The head pressure of the carrier gas (helium) in the
FID work was adjusted to yield almost identical retention times for the
major organo-sulfur and -selenium analytes (that is, as compared to the
retention times in the chemiluminescence chromatography). Different column
head pressures are required to achieve similar retention times in chromatography
with these two detectors because the FID is basically at atmospheric pressure;
while the reaction cell of the chemiluminescence detector where the capillary
column ends is approximately 1 torr. |
Results
Comparison of an FID and an SCD chromatogram
The response of the flame ionization
detector--the most common capillary GC hydrocarbon detector--to organosulfur
and organoselenium compounds is substantially less than that of the fluorine-induced
chemiluminescence detector. Figure 5 show a comparison
of the headspace analyses of replicate, live bacterial cultures amended
with 10 mM sodium selenate as determined by each detector. These phototrophic
bacteria were grown for seven days before sampling. Peaks routinely quantifiable
in the low to middle ppbv range via gas phase chemiluminescence are basically
undetectable using FID. |
|
Figure 5. Alternating FID and Chemiluminescence
Chromatograms
of the Headspace of Replicate Bacterial Cultures
Amended with 10 mM Na2SeO4
(Click Esc or Command-period to stop the animation
in most browsers.)
|
|
Selenium amendments
The six bacterial strains investigated were grown
for 6-7 days in the presence of 1 mM selenate, 1 mM selenite, or 1 mg/mL
metallic Se. Figure 6 displays the results from these
selenium experiments. Using optical density as a measure of cell population,
some strains were inhibited by the presence of selenium and others enhanced.
While all strains produced varying amounts of DMS and DMSe, two of the
six strains (R. capsulatus and R. blastica) produced no detectable
DMDSe (low parts per billion by volume). Except for cultures of R. tenuis
the presence of 1 mM selenate had an inhibitory effect on the production
of DMS. |
|
Figure 6. Culture Population and Organosulfur and Organoselenium
Production by Six Phototrophic Bacterial Cultures Amended with Selenate,
Selenite or Selenium Metal |
Dimethyl selenone amendments
As the data in Figure 7 denote, the addition of
1 mM dimethyl selenone to growing cultures of these microbes had no significant
effect on growth except in the case of R. blastica and R. sphaeroides.
The production of DMS was in all cases positively affected by DMSeO2
amendment at this concentration. Furthermore, as we have seen before [59]
organo-selenium production was greatly increased over the organo-Se production
by selenate or selenite additions to these same microorganisms in analogous
experiments. |
|
Figure 7. Production of Organosulfur and Organoselenium
Compounds by Phototrophic Bacteria Amended with Dimethyl Selenone |
Tellurium amendments
The biological production of dimethyl telluride was a less common result
of adding tellurate to these six microbes (Figure 8).
Only R. tenuis, R. rubrum S1, and R. rubrum G9 yielded
detectable amounts of DMTe in culture headspace after 7 days incubation.
Furthermore, most of this DMTe production came from elemental Te addition
(see below). Also of interest is the large amount of DMTe produced by R.
tenuis although only selenite yielded headspace DMDSe in the analogous
Se experiments. |
|
Figure 8. Production of Dimethyl Telluride by Phototrophic
Bacteria Amended with Sodium Tellurate and Tellurium Metal |
Elemental Se and Te
Possibly most surprising, elemental selenium (Se0) and tellurium
(Te0) amendments--the additions of insoluble solids which basically
remained as insoluble powders at the bottom of the test tube cultures--yielded
reduced and methylated Se and Te in four cultures' headspace as Figure
9 shows. Three of these same four strains produced both DMTe and DMSe:
R. tenuis and R. rubrum S1 and G9, while R. capsulatus
and R. blastica, although growing quite well in the presence of
these solids, produced no methylated metalloids in the cultures studied.
In the two strains that did not contain detectable amounts of dimethyl
selenide or dimethyl telluride, the presence of the elemental metalloids
had opposite effects on the production of dimethyl sulfide: In cultures
of R. capsulatus more DMS was produced than in the control, whereas
in R. blastica less DMS was found than in the control. |
|
Figure 9. Production of Dimethyl Selenide and Dimethyl
Telluride by Phototrophic Bacteria Amended with Selenium and Tellurium
Metal |
Mixed selenate/tellurate amendments
Table 1 shows the summary of experiments involving
all strains studied. Also reported here are the first mixed metalloid amendments
to microbial cultures of which we know. Expected was the lack of organometalloids
in cultures without metalloid salt amendments. DMS was determined in all
culture headspace. Cultures amended with sodium tellurate turn black due
to elemental Te bioproduction; this even in cultures which did not produce
any detectable dimethyl telluride. The presence of 1 mM tellurate in 0.1
mM and 1 mM selenate amended cultures inhibited production of organoselenium
dramatically. On the other hand, the combination of 1 mM selenate and 0.1
mM tellurate enhanced the tellurate volatilization (organo-Te production)
significantly.
Discussion
The addition of heteroatoms (non-carbon and -hydrogen atoms) to alkanes
decreases the response of the flame ionization detector to those heteroatom-containing
species. In the case of formaldehyde (CH2O) and carbonyl sulfide
(COS) the FID response is basically zero. The reason for this poor FID
response is not clearly understood but probably stems from the mechanism
of ion formation in the FID flame that involves the formation of CH fragments
[60, 61].
All of the analytes reported here contain one or two sulfur or selenium
atoms and methyl groups. The differences between these two detectors' response
to a mixture of organo-sulfur and -selenium analytes, as seen in Figure
5, is striking and is, in fact, a graphic representation of the reason
why specialized detectors are required in GC chromatography.
In 1992 Moore and Kaplan reported the high level resistance of proteobacteria
to rare-earth oxides and oxyanions [62].
One year later McCarty et al. [63]
found dimethyl selenide in the headspace of several strains of purple nonsulfur
bacteria, after amending them with sodium selenate. Based on these results
we started to investigate six strains of purple nonsulfur
bacteria with regard to their capability of methylating and reducing selenium
and tellurium compounds. The purple nonsulfur bacteria studied are resistant
to metalloid oxyanions at the concentrations investigated here. Cell populations,
as measured by optical density, were seldom extensively inhibited. The
mutant, Rhodospirillum rubrum G9, however, was an exception. This
phototroph is missing a photosynthetic pigment and appears green instead
of red like the other strains studied here. The key to this resistance
seems to lie in the ability of these organisms to reduce and in some cases
methylate the toxic compounds to which they are exposed. Many reduced,
methylated metalloids are volatile (boiling points <200°) and in
this work were specifically and very sensitively analyzed by fluorine-induced
chemiluminescence detection after separation by capillary gas chromatography.
Table 2 displays physicochemical data for a few relatively
volatile organ-selenium and -tellurium compounds. Compounds with much higher
boiling points and small aqueous Henry's Law constants would have vanishingly
small presence in culture headspace while still being produced in culture
solution.
These six strains of purple nonsulfur bacteria were grown under photo-heterotrophic
conditions and exposed to varied concentrations of selenite, selenate,
and dimethyl selenone, a proposed intermediate of (Challenger's) selenium
oxyanion reduction /methylation pathway [55].
After one week the headspace of these cultures was tested for dimethyl
selenide, dimethyl diselenide, and dimethyl selenenyl sulfide. In all six
cases the highest amounts of volatile selenium compounds were found in
cultures amended with dimethyl selenone (Figure 7)
and the lowest amounts in cultures doped with selenate (Figure
6).
The Challenger mechanism has also been expanded by Reamer and Zoller
[64], Doran [65],
and Chasteen [66].
Doran's mechanism suggests selenite first be reduced to elemental selenium
and then methylated to dimethyl selenide. Thus we also exposed the six
bacterial strains to elemental selenium and tellurium to test this hypothesis.
In four of the six strains, volatilization was observed upon incubation
with elemental selenium (Figure 6). This is a surprising
result since the elemental forms of most metals are considered to be biologically
stable and relatively inert (Doran's mechanism aside) and therefore to
not take part in the cycling of these elements in the biosphere in any
significant way. However, storage of elemental sulfur in phototrophic sulfur
bacteria (visible as large granules within the cells), e.g. Chromatium
okenii, and its possible use as a pool of electron accepting as well
as electron donating species has been shown [67].
Our results for Te0 and Se0 suggest that some microbes
might be able to reduce and mobilize elemental forms to a greater extent
than is presently thought.
It has also been put forth that reactions of biologically-produced organoselenium
and organosulfur species could exchange and/or disproportionate and thereby
produce a mixed organo-S/Se compound, dimethyl selenenyl sulfide (CH3SeSCH3).
This compound has been detected in anaerobic and phototrophic headspace
[59, 52]
and is also reported for 4 of the phototropic strains examined here (Figure
7).
Selenium and tellurium belong to group VIB of the periodic table and
form very similar compounds with oxygen, hydrogen and methyl groups. Therefore,
the bacteria were also amended with tellurate and selenate at the same
time in an effort to investigate any synergistic effects of these metalloids.
It is known that cultures of purple nonsulfur bacteria amended with tellurium
oxyanions turn coal black from elemental tellurium formed by microbial
activity [62]. In
two of the six strains investigated, dimethyl telluride was found in the
headspace after 7 days exposure to sodium tellurate and these two strains
also produced dimethyl telluride when they were grown in the presence of
elemental tellurium (Figure 8). Furthermore, if selenate
and tellurate were added together to cultures of purple nonsulfur bacteria,
elemental tellurium was formed; however, in some strains that had not
produced DMTe when amended with tellurate alone, mixed SeO42-/TeO42-
amendments yielded headspace concentrations of both DMSe and DMTe (Table
1). This suggests to us that the biological response of many of these
phototrophs to tellurate to yield DMTe was somehow "turned on" by the co-presence
of selenate. Furthermore it appears that the tellurate reduction and methylation
to produce DMTe is mainly biological in nature and not simply chemical,
that is, caused by the presence of other (reducing) organo-sulfides or
-selenides in culture albeit biologically produced themselves. We assert
this hypothesis because
|
1) organosulfur biologically produced in 1 mM tellurate experiments did
not produce DMTe in the presence of tellurate in those experiments and
|
|
2) organoselenium and organosulfur both present in 0.01 mM selenate/1 mM
tellurate experiments also did not produce DMTe from the tellurate present.
|
DMTe was most strongly produced (and in all six strains under discussion
here) in experiments with the mixed metalloid amendments of 0.1 mM tellurate
and 1 mM selenate (Table
1).
Conclusions
Fluorine-induced chemiluminescence detection following headspace sampling
and separation by capillary gas chromatography is a viable method of determining
the effects of toxic metalloid amendment to live bacteria grown in photosynthetic
and anaerobic cultures as measured by headspace components. All of the
phototrophic bacteria studied here reduced and methylated one or the other
oxyanions of Se used and three strains reduced and methylated elemental
Te and elemental Se. All strains responded to the addition of dimethyl
selenone by producing dimethyl selenide and, in 3 of the 6 strains examined,
dimethyl diselenide was also detected. Finally, some of the phototrophic
bacteria studied increased their release of dimethyl telluride--produced
by their bioreduction and methylation of tellurate--in the presence of
selenate. And furthermore the control experiments showed that this synergism
was biological and not merely caused by the presence of other organo-sulfides
or -selenides in culture.
Acknowledgments
This research was supported by a Cottrell College Science Award of Research
Corporation, the Swiss National Foundation, the Texas Regional Institute
for Environmental Studies, Sam Houston State University Research Enhancement
Funds, and the Robert A. Welch Foundation.
Bibliography |
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