PNNL-13501 - Pacific Northwest National Laboratory

triangular configuration. Once the tubes and shield are in
place, liquid nitrogen is injected into the tubes for
approximately 20 to 30 minutes (Figure 1, top).
Immediately after the liquid is injected, a chain hoist is
used to lift the frozen tear-drop-shaped core (Figure 1,
bottom) from the bed of the river. This method is capable
of extracting 70 cm cores that weigh, on average, about
24 kg. Once the core is extracted, it is photographed,
bagged, and placed on dry ice in a sample transport box
for further processing and analysis at the laboratory.
Figure 1. Filling hollow tubes with liquid N 2 to freeze river
sediments in situ (top). Intact frozen core obtained from the
Columbia River using the freeze core technique (bottom).
Although the focus of this project is on the development
and evaluation of approaches for collecting samples from
the hyporheic zone, select microbiological and
geochemical analyses were applied to the collected
samples to assess the effectiveness of the methods
described above and to gain preliminary insights into the
characteristics of this environment. Frozen and unfrozen
samples were analyzed using microbiological cultivation
techniques and direct extraction and characterization of
nucleic acids using methods that have been developed and
employed by our Laboratory for analyses of microbial
communities in subsurface sediments and soil. Pore
waters were extracted from sediments by centrifugation
and analyzed for total cations and anions, dissolved
organic and inorganic carbon, and trace metals.
Results and Accomplishments
Freeze-Core Sampling
Intact frozen cores of Columbia River sediment were
successfully collected from near the 100 H area of the
Hanford Site in March and again in May 2000. Three
intact frozen blocks of sediment were removed on each
occasion and treated as replicates. Unfrozen sediment
was collected adjacent to the freeze core location and
used as controls to assess the impact of freezing on viable
microbial populations, metabolic activity, and on pore
water geochemistry. Collected samples were returned to
laboratories in the 331 Building. Freeze-cores remained
frozen during transport and subsequent sampling.
Microbiological Analyses
Microbial characterization of Columbia River sediments
revealed an unexpectedly large and diverse microbial
community, whereas, previous experience with saturated
subsurface sediments from the Hanford confined aquifer
suggested the opposite. Direct community diversity
analysis based on terminal restriction fragment
polymorphism analyses of 16S ribosomal RNA genes
showed between 10 and 15 major ribotypes, indicative of
significant microbial diversity. Populations of viable
aerobic heterotrophic bacteria in unfrozen sediment
ranged from 10 6 to 10 7 colony-forming units per gram; in
the frozen sediments, the populations were lower,
between 10 5 to 10 6 colony-forming units g -1 (Figure 2).
These results suggest that freezing does have an impact
on bacterial cell viability, reducing populations by
approximately 10-fold or less. Although the results
shown in Figure 2 are from the May sampling, identical
trends were also observed in the March samples.
Log CFU g -1 dry sediment
7
6
5
4
3
2
1
0
HA1 HA2 HA3 FC1 FC2 FC3
Sample
Medium
10% PTYG
FS R2A
Dilute R2A
Figure 2. Populations of viable aerobic heterotrophic
bacteria in hyporheic ambient (HA) and freeze-core (FC)
Columbia River sediments
Earth System Science 201