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doi:10.2204/iodp.proc.310.103.2007

Microbiology

During Expedition 310, various methods were applied to quantify bacterial abundance, diversity, and activity. This combined approach included cell counting by 4′,6-diamidino-2-phenylindole (DAPI) staining, adenosine 5′-triphosphate (ATP) activity measurements, and cultivation of microorganisms. To guarantee that we were indeed investigating indigenous microorganisms, samples were obtained only from selected attached biofilms that showed a high degree of activity as measured by ATP.

Core handling and sampling

Samples for microbial studies were collected based on microbial activity measurements using the ATP test, which is described in “Activity test by ATP monitoring.”

Because of the nature of modern environments, special sampling precautions against oxygen, temperature, and pressure changes were not necessary because the physiochemical conditions on deck were similar to in situ conditions. Microbiological samples were taken systematically from cavities in the core material wherever high activities could be measured by the ATP test.

Because of the drilling approach and the nature of the sediments, it was difficult to collect sterile samples. In order to quantify the degree of contamination of the drilling fluid (seawater), water samples collected from the core liner were measured by the ATP test and compared with sterile water and seawater (see Table T6). Results show extraordinarily low activity of microorganisms in the drilling fluid compared with those of the control samples (tap water and seawater).

During the first part of Expedition 310, samples were obtained from small windows opened in the plastic liners (Fig. F14). Only biofilms attached to pieces of coral or microbialites were used for cultivation, identification, activity experiments, and deoxyribonucleic acid (DNA) analysis. In order to avoid microbial degradation, samples were immediately chemically fixed and/or frozen at –60°C. Because of technical problems with ruptured plastic liners, split metal liners were used instead. This change exposed the cores to more sources of microbial contamination on deck. ATP measurements and sampling were therefore completed before the cores were transported to plastic liners for curation.

Precautions, such as the use of sterile spatulas, forceps, pincers, small chisels, and gloves, were taken during sampling to reduce external contamination. The nature of the samples (biofilm, unconsolidated sediment, and crusts) dictated the best technique to use to obtain aseptic samples. Samples of unconsolidated sediment were taken immediately after core recovery; the underlying 0.3–0.5 cm of sediment was removed with a sterilized spatula, leaving behind an uncontaminated surface.

Activity test by ATP monitoring

ATP is the universal energy-transferring intermediate molecule in all organisms. ATP is stable inside cells but becomes unstable outside living organisms; thus, the presence of ATP acts as a marker molecule for living cells. This is affirmed by the fact that ATP is not known to form abiotically. ATP can be easily detected with high sensitivity and high specificity using an enzymatic assay (luciferase):

ATP + luciferin + O₂ → AMP + oxyluciferin + PPi

+ CO₂ + photon,

where

• AMP = aminomonophosphate, and
• PPi = pyrophosphoric acid.

A photon is emitted as a result of the reaction, which is detected by a photomultiplier. In principle, the detection limit is a photon produced by a single molecule of ATP. Typical sensitivity (significant above background) of commercially available instruments is 0.01 attomoles/mL (0.01 × 10^–18 mol/mL) water, corresponding to about five Escherichia coli cells.

Detection of ATP during Expedition 310 was used for two purposes: (1) detection of living biofilms in the reef framework and (2) assessment of microbial contamination by drill waters, equipment, and other sources.

In order to test microbial activity immediately after coring, a quick activity test, which is commercially available for industrial hygiene monitoring, was applied. The Uni-Lite NG luminometer (Biotrace International Plc., Bridgend, UK), in combination with the “Clean-Trace” and “Aqua-Trace” swab kits, measures ATP concentrations by a firefly enzyme-based test. The sensitivity of the test is on the order of 20–40 microbes, expressed in relative light units (RLU). Previously, tests carried out in the Geomicrobiology Laboratory, Swiss Federal Institute of Technology (ETH), Zurich (Switzerland), proved that this method could be applied on geological material, such as rock surfaces and environmental biofilms. Additionally, application of this test to water samples can aid evaluation of the degree of contamination of the drilling water, which percolated inside the core liner.

The following procedures were used to measure microbial activity. Using the Clean-Trace surface test kits, ~1 cm² of the rock surface was sampled, which provided results within a 30 s analysis. For water samples, an Aqua-Trace test stick utilizes a test tube with a 0.1 mL water sample, providing an activity measurement using the same method as above and also expressed in the same units (RLU). The ATP test can be calibrated using a sample with a known density of microbes. However, the ATP content of microbes can vary according to their feeding status.

Measurements for ATP were performed on the surfaces of sedimentary rocks, in the cavities of the reef framework, on percolated drilling water, and on seawater collected 10 m below sea surface. Additionally, purified water for analysis and chemical solutions from the shipboard geochemical laboratory were monitored for the degree of contamination.

Exoenzyme activity

Hydrolytic exoenzymes are indicators of metabolically active bacteria. Exoenzyme activity can be measured very sensitively by fluorescent-labeled substrate analogs (Coolen and Overmann, 2000). The result gives an idea of which substrates are used by microbial communities living in the reef subsurface. The activity of three exoenzymes was measured during Expedition 310:

• Alkaline phosphatase, cleaving inorganic phosphate from organic molecules such as phospholipids;
• β-glucosidase, cleaving sugar molecules from polysaccharides; and
• Aminopeptidase, cleaving proteins and peptides into smaller fragments.

As the measurement of exoenzyme activity is a common method in marine seafloor environments, data can be compared with existing databases.

Exoenzyme activity in sediment samples was determined by the fluorogenic substrate analogs for the hydrolytic exoenzymes alkaline phosphatase (EC 3.1.3.1), β-glucosidase (EC 3.1.21), and leucine aminopeptidase (EC 3.4.1.1). Alkaline phosphatase and β-glucosidase were assayed with MUF-phosphate (4-methylumbelliferone-phosphate) and MUF-β-D-glucoside (4-methylumbelliferone-glucoside, obtained from Sigma). Aminopeptidase activity was measured with MCA-labeled leucine (7-amino-4-methylcoumarin-leucine, obtained from Sigma).

Fresh samples (~0.5 g) were weighed and incubated in 2 mL reaction vials with 1.5 mL sterile filtered seawater. Substrate analog solution (5 µL), required to yield a final concentration of 33 µM, was added to initiate the enzymatic reaction. Samples were incubated for 30 and 90 min at a temperature of 28°C. For each substrate analog, a control of the same sediment material was inactivated by boiling in distilled water for 20 min to assess the nonenzymatic hydrolytic cleavage of the substrate analogs. This procedure represents a reliable control for abiotic cleavage of fluorogenic substrates in various samples.

After the incubation time, NaOH was added to increase the pH of the solution to 11, together with 1.7M (final concentration = 0.1M) of tetrasodium ethylenediamine tetraacetic acid (EDTA) to prevent carbonate precipitation. The assays were centrifuged for 5 min, and the concentration of free dissolved fluorophores was determined fluorometrically by a LS-5B luminescence spectrometer (PerkinElmer), with excitation wavelength of 360 nm and an emission wavelength of 450 nm. Depending on exoenzyme activity, samples were diluted 1:10 to 1:100.

For calibration, MUF and MCA standards in artificial seawater at concentrations of 10–2000 nM were used. The exoenzymatic activity is expressed as nanomol per hour per gram of substrate analogue cleaved.

Microscopy: DAPI staining

Microscopic cell counting by DAPI is a standard technique to stain DNA of an entire microbial community in environmental samples (Hobbie et al., 1977). This technique was applied to attached biofilms recovered in Tahiti reef cores to quantify microbial communities.

Samples were obtained directly from cores with aseptic tools and transferred into sterile 2 mL reaction vials. Samples were fixed with 1 mL 3% formaldehyde solution and washed with marine PBS buffer (phosphate buffered saline + 2.7 g/L NaCl).

To remove fine carbonate debris and detach cells from mineral surfaces, 1.5 mL of EDTA solution was added (0.3M EDTA; pH 7.3; 2.7 g NaCl/L), mixed and incubated for 1–2 h. Following this procedure, samples were placed in an ultrasonic bath for 3 s and vortexed. Supernatants were transferred to another vial and incubated for 1 h to obtain a solution that is nearly free of carbonate particles. The EDTA solution was removed from the solid phase using a 10 min centrifugation. The supernatant was discharged and the pellet suspended in 0.1 mL saline PSB (NaCl, 2.7 g/L) concentration. A 10 µL aliquot of each sample was spread on gelatin-coated multiwell microscope slides (3 g gelatin; 0.5 g CrKSO₄/L) and left to dry for 10 min. DAPI (250 ng/mL; 5 µL) staining was applied to each sample, and samples were incubated for 5 min. Slides were washed in a PBS NaCl solution for 15 min. To avoid bleaching, 5 µL “Citifluor” was added to each well and covered with a slide cover.

Samples were examined using a Zeiss AxioskopII microscope with a 100× objective, equipped with a 50 W ultraviolet lamp and appropriate filter. Images were recorded by a Zeiss MRc5 digital camera.

Because of the vibration caused by the ship’s engines, it was often impossible to obtain sharp pictures with 100× objective.

Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) can be used to identify and quantify specific microbial cells in environmental samples. Oligonucleotides are used to stain selective microbial cells according to their phylogenetic affiliation. Samples were fixed onboard for future application of specific probes during shore-based studies.

Cultivation of microorganisms

In order to identify the microorganisms occurring in biofilms and understand their function by investigating their metabolism, aerobic microorganisms were cultivated. Subsamples of biofilms growing on reef material were incubated on board. Biofilm material was diluted in a sterile PBS saline solution, inoculated on agar plates, and incubated at in situ temperature (24°C). Unfortunately, only a small fraction of the in situ microbes can be cultivated on a selective medium.

Isolation of microorganisms

After 15–30 days incubation, individual colonies were sampled and inoculated on new agar plates. These cultures were identified by 16S ribonucleic acid (RNA) analysis and could be preserved for further investigations concerning microbially mediated mineral precipitation.

The 16S ribosomal ribonucleic acid gene

Molecular phylogenetic analyses are based on 16S ribosomal ribonucleic acid (rRNA) sequence analysis. The sequence of the 16S rRNA molecule is highly preserved among all organisms. Thus, rRNA is an excellent molecule to detect evolutionary relationships among prokaryotic organisms. Ribosomal RNA is phylogenetically ancient, functionally constant, universally distributed, and preserved across broad phylogenetic distances.

Scanning electron microscopy: fixing of samples

For scanning electron microscopy (SEM) studies, a fresh sample (~0.5 g) was taken from the core material, transferred in a sterile reaction vial, fixed with a 3.5% glutaraldehyde solution, washed two times in marine PBS, and stored in a 80% ethanol:20% PBS solution at –70°C.

Culturing

From selected samples where a high cell density and a high ATP signal was observed, culturing of aerobic microbes was done on marine cell preserved solution (CPS) agar plates. The composition of the CPS agar was artificial seawater supplied with soluble starch and peptone.

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