Phospholipid fatty acid analysis as part of the Yucca Mountain Project. Final report Page: 2 of 16
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members of the extant microbiota4. Based on the above traits, the PLFA analysis is the
appropriate assay for the characterization of the microbiota in the subsurface sediments
surrounding the proposed high level nuclear waste repository.
Methods: Sediment samples of approximately 100 gram weights were received
via overnight mail shipped on dry ice. The sediments were thawed and homogenized in a
mill prior to extraction in a Bligh and Dyer (chloroform:methanol) solvent system
modified to contain a phosphate buffers. After an extraction period of approximately 3
hrs. additional aliquots of chloroform and water were added resulting in the separation of
the organic lipid containing phase from the aqueous phase5. The organic phase was
further separated on a column of silicic acid into neutral-, glyco- and polar-lipids6. The
polar lipid fraction was then subjected to a mild alkaline methanolysis resulting in the
recovery of polar-lipid (phospholipid) fatty acid methyl esters (PLFA)6. The PLFA were
then further separated, quantified and identified by capillary gas chromatography coupled
to a mass selective detector7.
Diglyceride fatty acids were recovered from the glyco-lipid fraction after isolation
on a thin layer chromatography plate8. These fatty acids were then derivatized and
identified in the same manner as the PLFA. The whole cell assay entailed an acid
hydrolysis of a portion the organic phase9 followed by a strong acid methanloysis9 with
subsequent identification by capillary gas chromatography with mass selective detection7.
Bacterial isolate identifications were made using the MIDI system developed by
Microbial ID, Inc., Newark, DE.
The multivariate statistical analyses were performed on arcsin transformed mole
percentages using the software package Einsight by Info-Metrix, Seattle, WA.
Results and Discussion (biomass): PLFA and whole cell results are provided in
appendices A and B, respectively. In figure 1, a plot of the biomass estimates is made for
both of these lipid fractions. The figure illustrates an average over a particular sample set
of only those values above a background level of 0.60 pmole gram-1 for the PLFA and
7.00 pmole gram-1 for the whole cell analysis, see appendix C. A PLFA background of
0.60 pmole gram-1 is roughly equivalent to 1.5 x104 cells granr1 assuming 1 pmole of
PLFA is equivalent to 2.5x104 cells10.
A general decrease in viable biomass occurred moving from the Tiva Canyon to
the Topopah Spring horizons. This decrease in biomass was also positively correlated
with depth. The lower biomasses occurred at the greater depths. Of the three geologic
horizons represented in the figure, the Topopah Spring horizon showed, on average, the
lowest viable biomass and the Tiva Canyon horizon the highest. In all of the sediments
analyzed, viable biomass estimates were low (compared to a surface soil which typically
shows 109 cells gram-1) with the highest level of PLFA occurring in sample #3951c at
11.20 pmole gram-1 or 2.8x105 cells gram-1.
Whole cell lipid biomass was considerably greater than that of the viable biomass.
The two to three order magnitude discrepancy between the two lipid classes suggests that
the whole cell assay recovered lipid material not only from microbial cells but from
sedimentary material (organics) as well. In a number of the samples amide and hydroxy
fatty acids were detected comprising anywhere from 1 to 73% of the whole cell profile2
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Ringelberg, D.B. & White, D.C. Phospholipid fatty acid analysis as part of the Yucca Mountain Project. Final report, report, September 1, 1996; New Mexico. (https://digital.library.unt.edu/ark:/67531/metadc688543/m1/2/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.