SUMMARY: ATP, an obligate constituent of all living organisms, is extracted from viable microorganisms in boiling TRIS buffer following sample concentration by vacuum filtration. The extracted ATP is analyzed in a photometer by the firefly bioluminescence reaction, and the ATP content is related to total living (biomass) microbial carbon by the application of a laboratory- derived extrapolation factor.

1. Principle

In field studies it is often desirable to determine the total amount of living cellular material (biomass). Conventional methods (i.e., fresh or dry weight determinations, rate of increase of cell numbers, etc.) usually cannot be used owing to (a) lack of sensitivity in the analytical procedures, (b) the presence of a heterogeneous assemblage of organisms, (c) the presence of dead cells and (d) the presence of detrital (non-living) organic material which is not associated with the living cells. Estimation of cellular biomass by measurement of adenosine triphosphate (ATP) is not limited by any of these considerations.

The rationale for using ATP to estimate biomass is the ubiquitous distribution of ATP in all living cells, the rapid loss of ATP from dead cells and the fairly uniform concentration of ATP in the protoplasm of all microbial cells. Data on ATP concentrations can thus be extrapolated to biomass parameters, such as cellular organic carbon or dry or fresh weight (Holm-Hansen, 1973). ATP is extracted from cells using boiling TRIS buffer and is stored frozen (-20 °C) prior to analysis by firefly bioluminescence.

2. Precautions

ATP samples must be processed as rapidly as possible, because the ATP content of microorganisms can change rapidly when cells are stressed. Furthermore, a phenomenon referred to as the "filtration effect" causes a loss of ATP when cells are exposed to dessicating conditions immediately after the water is drawn through the filter pad (Karl and Holm-Hansen, 1978). For this reason it is very important that the samples are filtered immediately upon sampling and extracted immediately upon filtration; any delay will cause a decline in ATP content. Because the firefly bioluminescence assay is inhibited by metals, it is also important to use clean stainless steel forceps when handling the filters. It is also essential that the extraction buffer (TRIS) is boiling (100 °C), as inefficient extraction results at temperatures (<95 °C). TRIS buffer boiling must be confirmed before starting the filtration process.

3. Sampling, Filtration, Extraction and Storage

3.1. Samples for ATP determinations are collected in clean Niskin bottles attached to the rosette/CTD unit.
3.2. As soon as the samples arrive on deck, water is prefiltered through a drawing tube containing an in-line 202 µm Nitex mesh prefilter to remove large zooplankton and particles which might otherwise affect the precision and accuracy of microbial biomass determinations. Samples are drawn into 4-liter polyethylene bottles that are rinsed 3 times with approximately 100-200 ml seawater from the appropriate depth. Filtration is begun immediately (be sure heating block is on and has achieved a temperature >110 °C; time required ~1 hour).
3.3. Filter triplicate samples at each depth through 47 mm GF/F filters. Total volume required per sample will depend on depth: between 0-150 m use 1 liter per sample, below 150 m use 2 liters per sample.
3.4. As soon as the last few drops of water have passed through the filter, remove the filter tower from the base, fold the filter in half then in half again and plunge the folded filter into 5 ml of boiling TRIS buffer (pH 7.4; 0.02 M) which is kept partially covered to eliminate evaporative volume loss. Try to avoid "bumping" of the TRIS buffer (caused by superheating of the buffer).
3.5. After a 5 minute extraction period, remove the tubes from the heating block, allow to cool to approximately room temperature, secure the rubber stoppers and freeze (-20 °C) in upright position.
3.6. In order to minimize sample cross-contamination it is best to start with deepest sample, which, in most cases, contains the lowest concentration of ATP.

4. Analysis

4.1. Prepare enough firefly lantern extract (Sigma Chemical Co., FLE-50) to process all samples and at least ten external ATP reference standards. Lyophilized FLE-50 should be reconstituted in 5 ml distilled water and allowed to "age" at room temperature for at least 6 hours (but no longer than 24 hours) in order to reduce the background luminescence. Approximately 1 hour before starting the assay, dilute each 50 mg vial of reconstituted FLE-50 with 15 ml of sodium arsenate buffer (0.1 M, pH 7.4) and 15 ml of MgSO4 (0.04 M). Immediately before use, filter the FLE-50 mixture through a GF/F filter.
4.2. Turn on ATP photometer at least 30 minutes before use.
4.3. Prepare set of ATP reference standards ranging from 0.1-100 ng ATP ml-1 (see Chapter 16, section 5).
4.4. Using the automatic injector and computer-assisted photometer, analyze the peak height of light emission (0-15 seconds) for each sample and standard.

5. Preparation of ATP Standards

A primary (reference) ATP standard is prepared by dissolving exactly 10 mg of high purity (99.9%) ATP (sodium salt) into exactly 10 ml of sterile distilled water. Be sure to weigh out an amount equivalent to 10 mg of free acid form of ATP (not 10 mg of hydrated Na-ATP) taking into account the cation contribution and average hydration state of the molecule. This latter information is supplied by the manufacturer. Immediately prepare 1/10 and 1/100 dilutions of the primary stock, place a portion of each solution into a 1 cm quartz spectrophotometercuvette and measure the absorbance at 259 nm. Calculate the exact concentration as follows:

A = Elc

where: A = absorption at 259 nm
E = ATP molar extinction coefficient (15.4 x 103)
l = path length (cm)
c = concentration of ATP in moles per liter

After calculating the precise concentration of ATP in the primary standard, dilute the stock (gravimetrically) into sterile-filtered (0.2 µm) 0.02 M TRIS buffer pH 7.4 to yield an ATP standard solution of exactly 1 µg ml-1. Compare with "old" standard and if different by >|1%|, repeat the dilution step. Store frozen in 1 ml aliquots.

Working standards, ranging from 0.1-100 ng ATP ml-1, are prepared by diluting a vial of the primary standard with freshly-prepared TRIS buffer (0.02 M) just prior to use. These working standards are discarded at the end of the day.

6. Data Reduction and Calculations

6.1. Plot ATP standard data and calculate linear regression statistics for the standard curve. Use LOTUS spreadsheet to calculate ATP concentration (ng l-1) from data on peak height (relative to standards), volume of extract and volume of sample filtered.
6.2. ATP concentrations can be related to total biomass by applying the extrapolation factor; 250 x ATP = C (Karl, 1980). This relationship is based upon direct laboratory and field analyses performed over the past two decades. While there is a range in the C/ATP ratio for microorganisms grown under a variety of environmental conditions, the relationship of C/ATP = 250 appears to be reasonable for samples collected from the oligotrophic North Pacific Ocean (Laws et al., 1987).

7. Equipment/Supplies

8. Reagents

9. References