SAMPLING THE DEEP BIOSPHERE
The fascinating possibility that microbial activity may inhabit oceanic crust as old and as deep as that at Site 801 provided the motivation for sampling the subseafloor in the search for extremophilic life. Water, sediment, and rock samples were collected to determine cell abundance and community composition. Samples were examined microscopically and used to establish cultures and to extract and characterize DNA. Sample protocols were established and tracer tests conducted to determine the extent of microbial contamination introduced during drilling and sample preparation.
Shipboard Sampling
Water samples were collected to determine background microbial
populations introduced in the drilling process. Undisturbed water
in Hole 801C was viewed as a potentially excellent in situ
microbial culture, and was thus sampled using the water-sampling
temperature probe (WSTP) before the hole was disturbed; ~300 mL of
water was recovered from a depth of 540 m in the hole. Water
samples were also collected from the drill pipe and from the sea
surface (from the z-boat) upwind of the JOIDES Resolution.
A series of samples of different rock types were collected for
culturing, DNA and Adenosine triphosphate (ATP) analyses, and
total cell counts. Figure 17 shows an example of a bacterial
population contaminated with fluorescent microspheres. Whole-round
samples of basement rocks, including glass samples, were (1) taken
and transported to an anaerobic chamber where they were cracked
open and interior pieces were used to inoculate cultures and (2)
also used for shore-based microscopic analyses and DNA extraction.
Cultures have also been started at in situ pressure, and some
samples have been stored under pressure for shore-based analyses.
Hole 801C, and to a lesser extent volcanic rocks from Site 1149
preserve small amounts (several grams to a few grains) of fresh
basaltic glass. These samples were studied microscopically for
traces of microbial activity. Several samples preserve filament
like textures that are identical to textures attributed to
microbes in deep-sea basalt from young oceanic pillow basalt.
Whether these textures are trace fossils of past microbial
activity or signs of extant microbes living in this high-pressure
seafloor environment remains a fascinating subject of shore-based
study.
Tracer Tests
The issue of core contamination often makes interpretation of
observations in deep subsurface microbiology difficult because
nonindigenous microorganisms can be introduced during the drilling
and sampling processes. This concern is amplified for extremely
deep environments where the abundance of microorganisms is
generally very low and the potential for contamination is great.
Because the Ocean Drilling Program ventures into the realm of deep
biosphere research, the issue of sample contamination, especially
as it pertains to the drilling process on board the JOIDES
Resolution, must be addressed.
The drilling fluids (mud and/or surface seawater), the drill
string itself, and sample handling procedures on deck are all
possible sources of contamination. Quantitative contamination
tests have been conducted in other drilling operations using a
variety of chemical, microbiological, and particulate tracers (see
review by Griffin et al., 1997). An ideal chemical tracer is
inert, not found naturally in the environment, and easily detected
at extremely low concentrations. One of the goals during Leg 185
was to establish shipboard procedures on the JOIDES Resolution to
deliver perfluorocarbon tracers (PFTs) and fluorescent
microspheres 0.5-1.0 µm in size during drilling and to detect the
tracers in recovered cores. Sediment and igneous rock require
different drilling techniques, and tracer experiments were
performed in both types of formations.
PFT data, along with the measured concentration of microbial
cells in surface waters, can be used to estimate the extent of
potential microbial contamination. The measured number of cells in
the surface water at Site 801 was 4.2 x 102 cells/µL. Based on this
value, microbial contamination is less than one cell per gram when
there is less than ~0.0025 µL of drilling fluid contamination per
gram of sample. For interior samples, this is the case for five of
the 12 unconsolidated sediments cored by APC, one of the five
basalt analyses, and one of the four consolidated sediments
analyzed that were cored by RCB.
Caution must be used in the interpretation of the PFT
contamination tests. Although low PFT values can be taken as proof
of minimal contamination, high PFT levels do not unequivocally
prove microbial contamination. There are two reasons for this.
First, the PFT is able to penetrate much smaller pores than
contaminating microorganisms. Second, perfluoro
(methylcyclohexane) is volatile at room temperature and can cause
contamination during handling via gas-phase transfer of the tracer
from drilling fluid on the exterior sediment and rock surfaces to
the interior samples.
The microspheres are not a perfect mimic of microorganisms.
Whereas microspheres are similar in size to microorganisms, they
have different surface properties and, therefore, may behave
differently as they migrate through the formation or attach to
mineral surfaces. Microspheres are dispersed in the core barrel
when rock passes through the core catcher, so the rocks are
exposed to the tracer only after it has been saturated with
drilling fluid and the physical disturbance of the formation by
drilling. Thus, microspheres are less likely to be introduced into
cracks than PFTs.
The rapid analysis and high sensitivity of PFTs makes it
possible to quickly check samples for the possibility of
contamination before performing the microbiological manipulations
and, therefore, avoid wasting resources on contaminated samples.
In addition, if samples are routinely taken, it may be possible to
build up a database correlating the probability of contamination
with different types of sediment and rock formation
characteristics (e.g., porosity, percent veins, and mineralogy)
and type of drilling. As little as 10-12g of PFT is detectable.
Higher sensitivity may possibly be achieved by using a smaller
bore column on the gas chromatograph. This should sharpen the
peaks and improve the signal to noise ratio. The use of a less
volatile PFT may also improve the reliability of the method as an
indicator of microbial contamination.
The processing of hard rock relies on the use of water as a
lubricant and cooling fluid, both during subseafloor drilling and
sample preparation in the laboratory. Water can introduce
microbial contamination to the core as well as transfer surface
contaminants into the interior of a sample during the processing
of wet samples. For this reason, to sample core interiors core
surfaces should be dried before handling and broken with a rock
splitter, which does not require water, instead of a rock saw.
Because rock saws and polishing grits are required to make thin
sections, it is critical that contamination is controlled during
these processes as well. Establishing a protocol for the cutting
and polishing of thin sections, without the introduction of
microbial contaminants, is essential.
On average, the sediments cored with the APC showed less
susceptibility to contamination, and several core interiors were
entirely free of contaminants. RCB coring resulted in the presence
of chemical, but not particulate, tracers in the interior of the
cores. This difference is probably, though not unequivocally,
because of the nature of the coring. The APC core barrel is fired
into the sediment in less than a second, whereas the RCB can take
several hours to cut a 9.5-m core with drilling fluid continuously
flowing through the bit.
As more research on microbial activity in the deep biosphere is
conducted, it is important to remain cognizant of the issue of
contamination. Although the absolute quantity of the contaminant
seems very small, unlike chemical contamination, microbial
contamination can be amplified after the sample has been taken.
This is a concern with nonindigenous microorganisms growing in
cultures inoculated with material from the deep biosphere, as well
as in samples where the DNA of nonindigenous microbes may be
amplified using the polymerase chain reaction. Therefore,
continued, routine testing for microbial contamination during
drilling on board the JOIDES Resolution will provide the
scientific community with quality data on deep biosphere
microbiology.