Professional Documents
Culture Documents
BY HIGHER PLANTS
W.F. Mueller1, G.W. Bedell1, S. Shojaee1 and P.J. Jackson2, 1New Mexico State University,
Department of Chemistry and Biochemistry, Las Cruces, NM 88003 and 2Los Alamos
National Laboratories, Life Sciences Division, Los Alamos, NM 87545
ABSTRACT
The uptake and biotransformation of TNT was studied in cell suspension cultures and in
whole plants of Datura innoxia and Lycopersicon peruvianum. In cell culture, TNT was
rapidly removed from the growth medium and recovered from the cell extract in the form
of a variety of biotransformation products resulting from nitroreduction, deamination, N-
acetylation and side chain oxidation to aldehyde and carboxylic acid metabolites. Whole
plants of the same species grew well in soils contaminated with TNT up to 750 ppm; at
1000 ppm TNT the Datura plants showed some signs of phytotoxicity, while the
Lycopersicon plants were severely affected. Both species removed TNT from soil and
stored its metabolites at levels up to thirty times higher than the TNT soil concentrations.
After a two week growth period, only 4 to 9.2% of the applied TNT was found in the soils.
KEY WORDS
bioremediation, 2,4,6-trinitrotoluene (TNT), soil, explosives, plants
Isolated metabolite fractions were analyzed ter TNT addition to the culture; the majority
by gas chromatography-mass spectrometry of the total radioactivity consisted of more
(GC-MS) either underivatized or after polar metabolites eluting at shorter rete n-
methylation with diazomethane. A Varian tion times than TNT. The chromatograms
Saturn ion trap GC-MS system was used show the presence of a transient metab o-
with a 30 m x 0.25 mm DB-5 column te m- lite which is less polar than TNT (Rt 12
perature-programmed from 40 to 280ºC. min.) and has its highest concentration at
2.5 hours.
Results
GC-MS analysis of the isolated metabolite
TNT and total radioactivity disappeared fractions yielded a number of mass spectra
rapidly from the cell culture medium. Figure indicative of TNT biotransformation pro d-
1 shows the radio-HPLC chromatograms of ucts. The primary products of nitroredu c-
medium samples taken at 0.5, 2.5 and 4.5 tion, 4-amino-2,6-dinitrotoluene and 2-
hours after addition of TNT- 14C to the cul- amino-4,6-dinitrotoluene, had been d e-
ture. The TNT peak at retention time 10 scribed earlier as TNT metabolites in other
has almost disappeared by 4.5 hours, and organisms [4, 5]; the published mass spe c-
little other radiolabeled material was found tra were matched well by the spectra of two
in the medium. components of the more non-polar group of
metabolites shown in Figures 3 and 4. The
The wash buffer, which was collected after
spectra show the molecular ion at m/z 197,
washing the intact cells separated from the
a loss of 17, which is characteristic for ar o-
medium, contained very little radioactivity,
matic nitro-compounds, resulting in an i n-
indicating that little TNT remained adsorbed
tense peak at m/z 180 and consecutive
to the outside of the cells.
losses of 46 or 47 as the two nitro-groups
are lost as NO 2 or HNO 2, respectively.
The radio-HPLC chromatograms of the cell
extracts at 0.5, 2.5 and 4.5 hours are
After methylation, one of the more polar
shown in Figure 2. Little TNT was found in
fractions yielded the mass spectrum shown
the extracts even as early as 0.5 hours a f-
in Figure 5, with a molecular weight of 211,
two consecutive losses of 17 producing the ions common to benzoyl compounds with
fragment ions at m/z 194 and 177, losses the general formula C 6H5CO-R. A search of
of 46 from either the molecular ion or the the spectra against the 49K NIST mass
fragment ion at m/z 194, and a base peak spectral library showed a good match with
at 118, which can be explained by a loss of N-acetylamino benzamide. This structure
59 (-COOCH 3) from m/z 177. The proposed was excluded by the fact that it can not
structure of this metabolite is a diamino- have any structural isomers, but the fra c-
nitrobenzoic acid which was converted into tion contained two isomeric substances r e-
the methyl ester by diazomethane trea t- solved by gas chromatography. The stru c-
ment. Figure 6 shows the fragmentation tures assigned to the two metabolites are
scheme of this compound. 2-acetylamino benzaldehyde and 4-
acetylamino benzaldehyde; their molecular
Another fraction gave a mass spectrum weight is 163, and loss of 58 (-NHCOCH 3)
with a molecular ion at m/z 206, a base yields the benzoyl ion at m/z 105, which
peak at m/z 149, and a strong tropylium ion can expel CO to form the phenyl ion at m/z
at m/z 91 (Figure 7). The structure a s- 77.
signed to this metabolite is one of the two
possible (2,4- or 2,6-) di-N- Discussion
acetylaminotoluenes. The two major fra g-
ments arise from the consecutive losses of The initial uptake studies show that both
57 (-NCOCH 3) and 58 Datura and Lycopersicon cell cultures ab-
(-NHCOCH 3). sorb TNT readily from the nutrient medium
and internalize and biotransform it rapidly.
The gas chromatogram of another m e- Within the 24 hour time period of these e x-
tabolite fraction contained two well-resolved periments, only small quantities of biotran s-
peaks with practically identical mass spe c- formation products were released back into
tra, one of which is shown in Figure 7. The the medium, and TNT was removed co m-
molecular ion is 163, and the two major pletely. Biotransformation appears to be
fragments dominating the spectra are at initiated by reduction of one nitro-group,
m/z 105 and 77, the benzoyl and phenyl resulting in the two isomeric amino- dinitr o-
toluenes found by other researchers in
bacteria, fungi and animals. This initial step nated soil and how the plants handle TNT
is followed by further nitroreductions, after absorption. Two greenhouse studies
cleavage of C-N bond, most likely by ox i- were performed: in the first, TNT soil levels
dative deamination, and acetylation of the of 100, 150 and 250 ppm were used. As all
amino groups remaining on the ring. The plants grew very well and no signs of phyt o-
methyl group is subject to oxidation resul t- toxicity were seen, a second set of exper i-
ing in the benzaldehyde and benzoic acid ments was added, in which concentrations
derivatives that were identified. The comb i- of 500, 750 and 1000 ppm TNT in soil were
nation of these biotransformation reactions used.
produces a variety of polar metabolites that
can be seen as broad unresolved peaks in Procedures
the radio-HPLC chromatogram of the cell
Seeds of Datura innoxia were kept in run-
extract (Figure 2).
ning water for 15 days before planting into
unpasteurized peat potting soil to improve
UPTAKE, BIOTRANSFORMA- germination. For Lycopersicon peruvia-
TION AND DISTRIBUTION OF num, shoot cuttings of adult plants were
TNT FROM SOIL BY WHOLE dipped in Ferti-Lome rooting powder (0.1%
PLANTS indole-3-butyric acid) and planted into peat
potting soil. The young plants were allowed
After the studies with cell suspension cu l- to grow an average height of 15 cm before
tures had shown that growing cells of Da- transplantation into the TNT-spiked soils.
tura and Lycopersicon can rapidly absorb
TNT from the nutrient solution and metab o- To simulate southwestern desert soil, a
lize it to a variety of biotransformation mixture of sand and fine gravel was used
products, we decided to investigate to for the TNT uptake studies. Batches of soil
which extent whole plants of the same were treated with solutions of TNT in d i-
species can take up TNT from contam i- chloromethane to produce TNT soil co n-
centrations of 100, 150, 200, 250, 500, 750 The methanol extracts were analyzed by
and 1000 ppm. After the spiking, the so l- HPLC with UV detection for residual TNT
vent was evaporated under a stream of n i- and by radio-HPLC for TNT and metab o-
trogen, then the batches were tumbled in lites.
the porcelain container of a ball mill for 30
minutes to ensure even distribution of the Results
TNT. HPLC analysis of triplicate soil sa m-
ples taken from the prepared batches Phytotoxicity
showed a distribution of ±5% around the Compared with control plants grown in u n-
target levels. At each level, 1 kg of soil was spiked sandy soil, all plants grew well in
spiked with 9 µCi of uniformly ring-labeled soils with TNT levels up to 500 ppm. There
14
C-TNT to allow the quantitative determ i- was no reduction in growth or discoloration
nation of uptake, distribution and biotran s- of leaves. At a TNT concentration of 750
formation of TNT by the plants. Additional ppm, Datura and Lycopersicon appeared
plants were grown in soils with the same to be slightly affected, showing some ye l-
TNT concentrations, but without radiol a- low spots on the leaves, but no decrease in
beled TNT; their purpose was to increase flowering or significant loss of leaves. At
the number of plants for phytotoxicity o b- 1000 ppm TNT in soil, the Lycopersicon
servation and to provide an additional plants showed moderate stress, with r e-
source of TNT metabolites. duced flowering and drying and loss of
some leaves and flowers. The Datura
The plants were transplanted individually plants still grew quite well and looked
from the potting soil into containers with healthy, with the exception of some yellow
100 g of TNT-spiked soil. They were kept spots on the leaves.
for 14 days in the greenhouse at temper a-
tures ranging from 25 to 35ºC and were TNT uptake and biotransformation
watered daily to replace the evapotransp i-
ration loss by approximate weight. After Tables 1 and 2 list the concentrations of
transplantation, the plants appeared TNT and/or metabolites in roots, stems and
healthy and continued to grow to an ave r- leaves of Datura innoxia and Lycopersicon
age of 20 cm. peruvianum plants grown in soils contai n-
ing 100 to 1000 ppm 14C-labeled TNT. The
On the 15th day the plants were removed ppm values were calculated from counts of
from the soils and separated into roots, total radioactivity, using the specific activity
stems and leaves. Plant sections from re p- (mCi/mmol)of the TNT used for the exper i-
lications were composited (e.g., the roots of
ment and the molecular weight of TNT (227 whole plant extracts.
g/mol).
At the end of the studies, only 4 to 9.2% of
At the lower concentrations of TNT in soil, the TNT added to the soils was recovered,
more of the radiolabeled material was based on analysis of soil extracts by UV-
translocated from the roots into stems and HPLC and on direct liquid scintillation
leaves than at TNT soil levels of 500 ppm counting of soil samples.
or more. Analysis of methanol extracts of
the plant sections showed that no TNT was Discussion
translocated into the above-ground parts of
The studies with whole plants showed that
either species; all radioactivity was present
both Datura and Lycopersicon plants can
in the form of more polar metabolites. Even
grow in soils contaminated with TNT up to
in the roots, most of the radioactivity was
at least 750 ppm. The plants absorb TNT
present as metabolites, and only 0.3 to 1%
through the roots and metabolize it readily
of the residual radioactivity was found as
into more polar products, which then are
TNT. Initial HPLC and GC-MS studies ind i-
partially translocated into stems and
cate that the same metabolites found in the
leaves. The extent of translocation appears
cell culture studies are also present in the
to be dependent on the soil concentration nation, oxidation of the methyl group to the
of TNT; considering the short duration of corresponding aldehydes and carboxylic
the greenhouse studies, it is possible that acids, and N-acetylation of amino groups
the plants just did not have enough time to remaining on the ring. The results show
move the larger amount of metabolite m a- clearly that in plants the biotransformation
terial from the higher TNT levels from the does not stop at the amino-dinitrotoluene
roots into the upper plant parts. After the level, but goes on to form a variety of pro d-
14-day growth periods, the TNT levels in ucts with greatly reduced toxicity.
the soils were depleted to less than 10% of
the starting concentrations; however, the The studies with whole plants in TNT-
total of the radiolabeled material recovered contaminated soils show that the two plant
from soils and plant parts was only 10 to species can tolerate TNT soil levels in e x-
25%. Since leaching as a source of label cess of 750 ppm, absorb the TNT through
loss could be excluded by the fact that the the roots, metabolize it and translocate the
planter cups had no drain holes, brea k- biotransformation products to the stems
down or volatilization are the only possible and leaves. Under the greenhouse cond i-
explanation for the loss of TNT from the tions of the experiments, TNT levels in the
soils. experimental soils were reduced to less
than 10% in two weeks.
CONCLUSIONS
Bioremediation of TNT-contaminated soils
The studies conducted so far with Datura therefore appears to be a cleanup option at
and Lycopersicon peruvianum indicate that sites with low to intermediate levels of
both plant species would be well suited for contamination as they exist at military sites
removal of TNT from contaminated soils at such as shooting or bombing ranges, or at
levels below 1000 ppm. The mass spe c- experimental blast sites. Plant bioremedi a-
trometry data from the cell culture studies tion also may be an attractive approach to
have shown that in the plant cells TNT u n- further reduce or completely remove resi d-
dergoes nitroreduction, removal of nitrogen ual TNT after composting of highly co n-
from the ring, probably by oxidative deam i-
REFERENCES
1. U.S. EPA, Office of Drinking Water,
Health Advisory on 2,4,6-trinitrotoluene,
PB90-273566, 1989