# Alkaline phosphatase activity of marine bacteria studied with ELF97 substrate: success and limits in the P-limited Mediterranean Sea

France van Wambeke, Jiri Nedoma, Solange Duhamel, Philippe Lebaron

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France van Wambeke, Jiri Nedoma, Solange Duhamel, Philippe Lebaron. Alkaline phosphatase activity of marine bacteria studied with ELF97 substrate: success and limits in the P-limited Mediterranean Sea. Aquatic Microbial Ecology, 2008, 52, pp.245-251. 10.3354/ame01238. hal-00321319

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# Alkaline phosphatase activity of marine bacteria studied with ELF 97 substrate: success and limits in the P-limited Mediterranean Sea

France Van Wambeke¹*, Jiří Nedoma², Solange Duhamel¹, Philippe Lebaron³,⁴

¹Laboratoire de Microbiologie, Géochimie et Ecologie Marines, CNRS, UMR 6117, Case 901,
Centre d'Océanologie de Marseille, Université de la Méditerranée - Campus de Luminy, 13288 Marseille Cedex 9, France

²Biological Centre of the Academy of Sciences of the Czech Republic, Hydrobiological Institute, Na sádkách 7,
37005 České Budějovice, Czech Republic

³Université Pierre et Marie Curie-Paris 6, and ⁴CNRS, UMR7621, 66650 Banyuls-sur-Mer, France

**ABSTRACT:** The fluorogenic substrate Enzyme-Labeled Fluorescence 97 (ELF-P) is hydrolyzed by the P-cleaving enzyme phosphatase, producing ELF 97 alcohol (ELFA), a fluorescent-insoluble product. This reaction is used for monitoring phosphatase activity at the single-cell level. Most frequently, ELF-P has been used to determine the P-limitation status of microphytoplankton, but rarely of heterotrophic bacteria. We incubated ELF-P on filters to monitor marine bacterial cultures and oligotrophic Mediterranean Sea samples. Results were compared to classical measurements of bulk alkaline phosphatase activity using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P). A high percentage of the cultured cells were labeled with ELFA (the ratio of ELFA spots to total DAPI counts in P-limited cultures ranged from 26 to 100%, depending on the strain). In contrast, this ratio never exceeded 0.01% in Mediterranean samples, even when P was demonstrated to be a significant limiting factor. This protocol is useful for application on cruises and with cultures, but was not sufficiently sensitive to detect P-stressed bacterial cells in oligotrophic marine environments.

**KEY WORDS:** Marine bacteria · Alkaline phosphatase · Cell specific activity · Ectoenzyme · ELF97 phosphatase substrate · Phosphorus limitation

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Developing new techniques for detecting phosphorus (P)-stress in bacteria at the cell level from natural environments is a challenge in microbial ecology. Such developments are important in understanding how P-limitation can affect both the structure and the functional role of bacterial communities. The molecule 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (Enzyme-Labeled-Fluorescence 97 phosphatase substrate [ELF-P], Molecular Probes) is a soluble substrate, which, when cleaved by the cell's enzyme (phosphatase), produces ELF 97 alcohol (ELFA), a bright fluorescent yellow-green precipitate labeling the site of en-

zymatic activity (Gonzalez-Gil et al. 1998). In aquatic systems, this technique has mainly been used as a tool for identifying P-limited phytoplankton cells. To date, only 4 published studies have focused on natural bacterial communities. This includes studies on activated sludge (Van Ommen Kloeke & Geesey 1999), an acidified lake (Nedoma & Vrba 2006), and bacterial colonies and biofilms (Huang et al. 1998, Espeland & Wetzel 2001). In most of these studies, however, the abundance and activity of bacteria were fairly high.

The aims of the present study were (1) to test an easy protocol (suitable for research expeditions) on marine isolates, and (2) to test this technique in the Mediterranean Sea under P-limited conditions.
## MATERIALS AND METHODS

Two marine species were used: *Alteromonas infernus* and *Pseudomonas denitrificans*. Cells were grown in minimum requirement medium (Lyman & Fleming 1940) with a single source of carbon (C) and energy and a single source of P, plus trace amounts of iron and vitamins. The concentration of organic C (as glucose or pyruvate) was low (Table 1) in order to obtain less dense cultures. Different concentrations of inorganic P were added to produce diverse initial C/P ratios of the provided substrates (Table 1, Expts a and c). *A. infernus* was also grown in natural seawater amended with pyruvate (Table 1, Expt b). Seawater was aged in the dark for 1 mo at 4°C to remove most nutrients. This seawater was 0.2 µm filtered, and the filtrate was supplemented with excess nitrogen (0.1 mM NH₄Cl), carbon (0.33 mM C-pyruvate), and trace amounts of iron and vitamins. Phosphate (16.5 µM) was added only in the +P treatment.

The station DYFAMED (Marty et al. 2002) was studied for ELF application at the end of March 2003. Soluble reactive P (SRP) and dissolved organic P (DOP), determined as the difference between filtered samples before and after wet-oxidation, were measured according to standard colorimetric techniques (Raimbault et al. 1999).

Alkaline phosphatase (AP) activity was measured fluorometrically using 4-methylumbelliferyl phosphate (MUF-P). The linear increase in fluorescence in seawater with added MUF-P was followed over time (excitation at 365 nm and emission at 460 nm) with a Kontron SFM 23B spectrofluorometer. For *in situ* and culture samples, we used MUF-P concentrations of

1 and 50 µM, respectively, which were found to be saturating in preliminary experiments. MUF-P tracks both dissolved AP activity and particle-associated (mostly microorganisms) AP activity. Dissolved-associated AP activity was negligible in cultures (<3%) and could reach up to 36% of measured phosphatase activity at the DYFAMED station (results not shown).

Contrary to MUF-P, ELF-P substrate provides information on particle-associated AP. The Endogenous Phosphatase Detection Kit (Molecular Probes E 6601) was used. Component A ($A_{kit}$) was diluted 1/20 in component B ($B_{kit}$) of the kit in accordance with the manufacturer's instruction to generate the solution applied to the slides. Immediately after filtration of a water sample (live sample) onto a 0.2 µm pore size black polycarbonate filter, the filter was transferred onto a glass slide, and 40 µl of the diluted solution $A_{kit}/B_{kit}$ were applied to the surface of the filter and gently spread with a plastic tip. The slide was then put into a horizontal, capped 50 ml plastic tube to which wet paper was added to prevent evaporation. Following a 1 h incubation at room temperature in the dark, the filter was removed, put briefly on a piece of absorbing paper, and then transferred into a 47 mm Petri dish containing a 47 mm absorbent pad (AP100 Millipore) soaked with a solution of phosphate buffered saline (PBS, 10 mM, pH 7.5) and 1% formaldehyde to stop the activity. The filter was transferred successively onto 3 PBS-formalin soaked pads, and left 5 min on each one. The filter was then air-dried on a piece of absorbent paper before being transferred onto a glass slide. Forty µl of a DAPI solution (25 µg ml⁻¹) were added, and the filter was incubated for 10 min in the dark. The filter was dried again on absorbent paper and mounted on a glass slide. The yellow

<table><caption>Table 1. *Pseudomonas denitrificans*, *Alteromonas infernus*. Summary of alkaline phosphatase (AP) activity in bacterial cultures, including initial conditions and the main results (bacterial numbers, ELFA spots, MUF-P based activities, and specific activities). BN: bacterial numbers (DAPI counts)</caption><thead><tr><th></th><th>Code of experiment:<br>Medium:<br>Carbon enrichment:</th><th colspan="2">Pseudomonas denitrificans</th><th colspan="2">Alteromonas infernus</th></tr><tr><th></th><th></th><th>a<br>Artificial seawater<br>Glucose</th><th>b<br>Natural seawater<br>Pyruvate</th><th>c<br>Artificial seawater<br>Pyruvate</th></tr></thead><tbody><tr><td>Concentration of C (mM C)</td><td></td><td>3.3</td><td>0.33</td><td>0.55</td></tr><tr><td>NH<sub>4</sub>Cl addition (mM N)</td><td></td><td>6.7</td><td>0.1</td><td>6.7</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub> addition (µM P)</td><td></td><td>165</td><td>0 (in situ 0.61)</td><td>5.8</td></tr><tr><td>Conditions</td><td>Excess P</td><td>8.25</td><td>16.5</td><td>Equilibrium</td></tr><tr><td>Duration of the culture (d)</td><td>13</td><td>Low P</td><td>Low P</td><td>8</td></tr><tr><td>Period of max. BN (d)</td><td>8–13</td><td>7–11</td><td>5–8</td><td>3–7</td></tr><tr><td>Max. BN (ml<sup>-1</sup>)</td><td>1.2–1.5 × 10<sup>8</sup></td><td>3.1–4.6 × 10<sup>6</sup></td><td>1.2–1.7 × 10<sup>7</sup></td><td>3.2–4.5 × 10<sup>7</sup></td></tr><tr><td>Period of max. AP activities (MUF-based) (d)</td><td>11–13</td><td>6–8</td><td>8–13</td><td>8</td></tr><tr><td>Max. MUF-based AP activities (nM h<sup>-1</sup>)</td><td>22–38</td><td>43–154</td><td>1400–2000</td><td>37 000</td></tr><tr><td>Period of max. specific activities (d)</td><td>0–13</td><td>6–8</td><td>2–13</td><td>8</td></tr><tr><td>Specific activities (amol cell<sup>-1</sup> h<sup>-1</sup>)</td><td>0.1–6.5</td><td>16–33</td><td>10–160</td><td>1300</td></tr><tr><td>Period of max. ELFA spots (d)</td><td>8–12</td><td>7–11</td><td>2–13</td><td>1.2–5</td></tr><tr><td>Max. ELFA spots (ml<sup>-1</sup>)</td><td>3–5 × 10<sup>4</sup></td><td>3.4–7.0 × 10<sup>5</sup></td><td>4.8 ± 3.0 × 10<sup>6</sup></td><td>2–7 × 10<sup>7</sup></td></tr><tr><td>Ratio ELFA spots / BN (%)</td><td>0.02–0.05</td><td>9–17</td><td>46 ± 28</td><td>172 ± 114</td></tr></tbody></table>
fluorescence of ELFA spots and the blue fluorescence of
the DAPI stain were examined (Fig. 1) using an Olym-
pus BH2 microscope equipped with a long pass
dichroic mirror, type U (DM 400, barrier filter L420).

Variations of this protocol were tested. Following the
filtration step, the filter was cut into pieces and incubated
over different time periods or with different concen-
trations of ELF-P. In addition, a comparison was made
between the abundance of ELFA spots and the rate
of ELF-P hydrolysis measured in a spectrofluorometer
calibrated with standard dilutions of ELFA (ref E 6578).
This comparison was possible only in cultures, due to the
very low sensitivity of ELFA measured spectrofluoro-
metrically. In this attempt, 1.8 ml of *Alteromonas infer-
nus* culture were incubated with 200 µl of a 100 mM Tris-
HCl (pH 7.5) solution and 4 to 40 µl of ELF-P (ref E 6588)
stock (5 mM) or diluted solutions in Milli-Q water
(0.5 mM), giving final ELF-P concentrations of 2 to
100 µM. The increase in fluorescence (excitation 350 nm,
emission 530 nm) was followed over time. At the end of
the incubation, 500 µl were sub-sampled, fixed with 1 %
final concentration of formaldehyde, and filtered
through 0.2 µm polycarbonate filters for epifluorescence
microscopy and counting of ELFA spots.

<figure><img src="image_2.png" /><figcaption>Fig. 1. *Alteromonas infernus*. ELFA labeled culture (yellow-green) counterstained with DAPI (blue) as observed with a dichroic mirror type U. Note that the ELFA signal is much more intense than DAPI</figcaption></figure>

RESULTS AND DISCUSSION

Protocol

There are several important features in our protocol
(see Table 2 for a comparison to previously published
protocols). First, in order to work on live and unmodified
samples, the ethanol pre-treatment step was omitted,
since ethanol may damage cell membranes and/or in-
duce the bleaching of pigments (Nedoma et al. 2003,
Dignum et al. 2004). Second, to concentrate the cells, we
used filtration because centrifugation requires high
speeds and a lengthy processing time, making it less
convenient when working on vessels. Third, we used the
Endogenous Phosphatase Detection Kit, which is com-
monly used (Table 2), although only partial information
regarding its composition is available (see Dignum et al.
2004, Nedoma et al. 2007). The ELF-P has been used
omitting component B of the kit during incubation in the
liquid phase (Nedoma et al. 2003), and ELFA precipita-
tion was still possible (Fig. 2c,d). However, this is only
feasible in cell-rich water samples with high phos-
phatase activity (Nedoma et al. 2003, Table 2).

Finally, we used DAPI as a counterstain because
both dyes are visible (DAPI-stained bacteria in blue,
ELFA spots in yellow-green) and cyanobacteria
(orange) and chloroplasts (red) in natural seawater
samples are easily discriminated. This also makes the
technique compatible with future efforts to combine
the method with fluorescent *in situ* hybridization
(FISH) techniques. However, the fluorescence of the
ELFA spots is much higher and fades more slowly than
DAPI fluorescence (Fig. 1). Consequently, with only
visual observation on the microscope, it is impossible
to confirm that DAPI-stained cells are associated with
an ELFA spot, since their similar size prevents seeing
the DAPI stain in the same location as an ELFA spot.
However, this discrimination is possible using narrow
band filters and image analysis (Nedoma & Vrba 2006)
or flow cytometry (Duhamel et al. 2008).

One of the first assumptions we tested was the pres-
ence of a false positive response. We showed that num-
ber of cells labeled with ELFA was higher in situations
where P-limitation occurred (Table 1 [Expt a], Fig. 3c).
Indeed, there was no significant labeling in C-limited
cultures of marine bacteria, even at the stationary phase,
suggesting that even if the substrate enters senescent
cells, intracellular phosphatase cannot significantly react
with ELF-P. However, P-sufficient *Alteromonas infernus*
also expressed phosphatase activity (Table 1 [Expt b]),
suggesting that for this strain, a fraction of phosphatase
activity seemed to be constitutive. These bacteria are
thus able to exert significant hydrolysis of phospho-
mono-ester bonds of organic molecules even in the pres-
ence of notable sources of inorganic phosphate. In that
<table><caption>Table 2. Summary of ELF labeling procedures in different studies. Phytop: phytoplankton, CENT: centrifugation, ETH: treatment with 70% ethanol, PREFILT: prefiltration of the sample (followed by treatment of the filtrate), FILT: filtration of the sample (followed by treatment of the filter), WASH: rinsing procedure, FORM: formaldehyde, GLU: glutaraldehyde, PBS: phosphate buffered saline solution, EM: epifluorescence microscopy, TLM: transmitted light microscopy, CFM: flow cytometry. A<sub>kit</sub>, B<sub>kit</sub>: component A (ELF-P concentrate) or B (detection buffer) of the Endogenous Phosphatase Detection Kit (Molecular Probes E6601), respectively. ELP<sub>pure</sub>: ELP-P 5 mM solution of Molecular Probes (E 6585). csDAPI, csPI, csAO: use of a counterstain DAPI, PI, and AO, respectively</caption><thead><tr><th>Sample</th><th>Initial volume treated</th><th>Pre-treatment of the sample</th><th>Support for incubation</th><th>Incubation conditions</th><th>Stop reaction</th><th>Observation</th><th>Source</th></tr></thead><tbody><tr><td>Marine phytop cultures</td><td>5 ml</td><td>CENT-ETH</td><td>Pellet</td><td>100 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>Washes in 10 mM PBS</td><td>CFM</td><td>Gonzalez-Gil et al. (1998)</td></tr><tr><td>Bacterial colonies</td><td>Colony on filter</td><td></td><td>Filter</td><td>Filter on cellulose pad saturated with A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>Filter on pad with 1% FORM 5 min</td><td>EM - csPI</td><td>Huang et al. (1998)</td></tr><tr><td>Biofilm</td><td>Biofilm on a slide</td><td></td><td>Slide</td><td>Slide immersed in A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>1% FORM cryo-embedding and sectioning</td><td>EM - csPI</td><td></td></tr><tr><td>Photosynthetic biofilm</td><td>Suction of the biofilm in liquid phase</td><td>CENT</td><td>Pellet</td><td>20 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>4% GLU-FILT O.2 µm</td><td>EM- csDAPI</td><td>Espeland &amp; Wetzel (2001)</td></tr><tr><td>Activated sludge flocs</td><td>1 ml</td><td>CENT-ETH</td><td>Liquid</td><td>100 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>0.1 ml drop onto a microscope slide</td><td>TLM/EM- csAO</td><td>Van Ommen Kloeke &amp; Geesey (1999)</td></tr><tr><td>Eutrophic lake water-phytop</td><td>150 ml</td><td>CENT-ETH</td><td>Pellet</td><td>100 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>WASH 0.1 M PBS</td><td>TLM / EM</td><td>Rengefors et al. (2001)</td></tr><tr><td>Mesotrophic freshwater pond-phytop</td><td>500 ml</td><td>FILT 10 µm ETH-CENT</td><td>Pellet</td><td>100 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>WASH 0.1 M PBS</td><td>TLM/EM</td><td>Rengefors et al. (2003)</td></tr><tr><td>Phytop cultures (dinoflagellate)</td><td>9 ml</td><td>CENT-ETH</td><td>Pellet</td><td>100 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>CENT-WASH</td><td>TLM/EM</td><td>Dyhrman &amp; Palenik (1999)</td></tr><tr><td>Field samples</td><td>11</td><td>FILT 0.8 µm ETH-CENT</td><td>Pellet</td><td>100 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>CENT-WASH</td><td>TLM/EM</td><td></td></tr><tr><td>Trichodesmium cultures and seawater colonies</td><td>Concentrate in a 130 µm net</td><td>Isolated colonies WASH FSW</td><td>2 colonies</td><td>100 µl A<sub>kit</sub>/sterile seawater 1/20 45 min</td><td>Colonies on a 5 µm filter-WASH FSW-10% FORM-transfer on filter</td><td>EM</td><td>Dyhrman et al. (2002)</td></tr><tr><td>Coastal - seawater phytop</td><td>11</td><td>FILT 0.8 µm ETH - CENT</td><td>Pellet</td><td>100 µl A<sub>kit</sub>/low P-seawater 1/20 1 h</td><td>CENT-WASH</td><td>TLM/EM</td><td>Ruttenberg &amp; Dyhrman (2005)</td></tr><tr><td>Oligotrophic lake phytop-bacteria</td><td>5 ml</td><td>FILT 0.4 µm in tissue culture inserts</td><td>Concentrate in inserts</td><td>200 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 1 h</td><td>FILT-RINSE</td><td>EM- csDAPI</td><td>Carlsson &amp; Caron (2001)</td></tr><tr><td>Eutrophic reservoir phytop</td><td>5 ml</td><td>PREFILT 200 µm</td><td>Liquid</td><td>20 µm ELF<sub>pure</sub> 1 h to 5 h</td><td>FIX 4 mM HgCl<sub>2</sub>-FILT 1 µm</td><td>EM</td><td>Strojová et al. (2003)</td></tr><tr><td>Lake and reservoir phytop</td><td>1 to 5 ml</td><td>PREFILT 200 µm</td><td>Liquid</td><td>20 µm ELF<sub>pure</sub> 30 min to 5 h</td><td>FIX 4 mM HgCl<sub>2</sub>-FILT 1 µm</td><td>EM</td><td>Nedoma et al. (2003)</td></tr><tr><td>Cyanobacterial cultures</td><td>14 ml</td><td>0.1 % GLU or 0.01 % FORM-CENT</td><td>Pellet</td><td>50 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>100 times dilution in flow cytometry sheath fluid</td><td>CFM</td><td>Dignum et al. (2004)</td></tr><tr><td>Marine samples</td><td>0.25 to 1 1</td><td>FILT 0.4 µm-10% DMSO-ETH</td><td>Filter</td><td>400 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 45 min</td><td>Filter back on tower RINSE FSW</td><td>EM</td><td>Lomas et al. (2004)</td></tr><tr><td>Lake bacterioplankton</td><td>7 ml</td><td>PREFILT 200 µm</td><td>Liquid</td><td>20 µm ELF<sub>pure</sub> 0 to 40 min</td><td>2% FORM-DAPI-FILT</td><td>EM</td><td>Nedoma &amp; Vrba (2006)</td></tr><tr><td>Bacterial cultures marine samples</td><td>0.5 ml (culture), 10 to 30 ml (field)</td><td>FILT 0.2 µm</td><td>Filter</td><td>30 µl A<sub>kit</sub>/B<sub>kit</sub> 1/20 30 min</td><td>Pads with 10 mM PBS + 1% FORM</td><td>EM- csDAPI</td><td>This study</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>Fig. 2. Typical curves showing the effect of time of incubation (a) and concentration of ELF-P (b,c,d) on the abundance of ELFA spots and ELF-P hydrolysis rates. Data in (a) and (b) used Mediterranean Sea water samples (incubations made on filters). Data in (c) and (d) used pure cultures. ELF-P hydrolysis rate (detected by spectrofluorometry) was measurable only in P-deficient *Alteromonas infernus* cultures. Error bars are ±SE within different fields of observation or transects. A<sub>kit</sub>, B<sub>kit</sub>: component A (ELF-P concentrate) or B (detection buffer) of the Endogenous Phosphatase Detection Kit, respectively</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 3. Vertical profiles of bacterial production (BP), soluble reactive phosphorus (SRP) concentration and dissolved organic phosphorus (DOP) concentration (a); abundances of bacteria (bact), *Synechococcus*-like cells (cyano), and phototrophic nanoflagellates (pnan) (b); and maximum hydrolysis rate of MUF-P (V<sub>max</sub> hydrolysis rate) and abundances of ELFA spots (c). All samples were collected at the DYFAMED station, NW Mediterranean Sea, sampled on 30 March 2003</figcaption></figure>

case, this capacity could give access to the C or N com-
pounds of the molecule hydrolyzed in environments de-
ficient in labile sources of organic matter, like the deep
sea (Hoppe & Ullrich 1999).

The Time 0 sample (acting as an abiotic control, be-
cause it was fixed with formalin) only exhibited a low fluo-
rescence signal, thus confirming the absence of signifi-

cant abiotic labeling with ELF-P. Development of ELFA
spot abundances over time in the incubations confirmed
the presence of a lag phase that was apparent in cultures
as well as in natural samples (Fig. 2a). This lag time was
also reported for freshwater phytoplankton (Nedoma et
al. 2003, Dignum et al. 2004) and can vary with activity,
temperature, and cell size (J. Nedoma unpubl. results). A
maximum concentration of label was obtained generally within 1 h in natural samples. Both the number of ELFA spots and ELF-P hydrolysis rates (measured by fluorometry) increased with the concentration of ELF-P added. The shape of these curves was not reproducible from one sample to another (Fig. 2c,d), suggesting that the range of concentrations that we tested was not always sufficient to reach maximum hydrolysis rates, or that multiphasic kinetics were present (presence of different enzymes: Hoppe 2003, Nedoma & Vrba 2006). These tests confirmed that the typical 1/20 dilution of the solutions in the kit we used (i.e. 250 µM) in the 1 h incubation is generally sufficient to optimize cell labeling.

## Bacterial cultures

All tests carried out using cultures are summarized in Table 1. Maximum bacterial abundances were higher under excess-P than under low-P conditions. However, bulk activity and cell-specific activities varied depending on the strain. For *Alteromonas infernus*, differences between low-P and excess-P conditions were less pronounced than for *Pseudomonas denitrificans*. Occasionally, the average ratio of ELFA spots to DAPI counts was >1. Because ELFA spots and DAPI counts were made on 2 separate slides, it is difficult to conclude that percentages higher than 100% were really due to the formation of ELFA spots outside bacterial cells or to heterogeneity in the distribution of bacteria on the slide when abundances reached $10^7$ cells ml⁻¹.

## Seawater samples

Sampling was carried out during the spring phytoplankton bloom (Bourguet et al. in press) when heterotrophic bacteria ($1.5 \times 10^6$ ml⁻¹) and *Synechococcus*-like cells ($1 \times 10^5$ ml⁻¹) were abundant (Fig. 3b). SRP was below 20 nM or undetectable in surface layers down to 20 m depth (Fig. 3a). MUF-based AP activity was constant in the first 20 m of the profile and decreased significantly at 30 m depth (Fig. 3c). The Michaelis-Menten constant ($K_m$) was stable along the profile, with values ranging from 83 to 116 nM (results not presented). At most, we counted $2 \times 10^3$ ELFA spots ml⁻¹ at any particular location along the profile. Being aware of the difficulty of counting rare events on filters, the vast majority of these spots were identified as unassociated spots (34 to 75% over the depth range), in contrast to spots associated with detritus (yellow DAPI particles without chlorophyll fluorescence, 21 to 55%), and only a few spots were associated with identified orange fluorescent cell (cyanobacteria, 0 to 8%)

or red fluorescent cells (phototrophic nanoflagellates, 0 to 8%). The highest ratio of total ELFA spots to bacterial abundance reached was 0.01%.

A log-log plot of the distribution of ELFA spot abundance versus MUF-P based AP activity using the entire data set is shown in Fig. 4. Below a threshold of 5 to 10 nmol MUF-P hydrolyzed l⁻¹ h⁻¹, a range that is frequently found *in situ*, there was no significant relationship due to the high variability. MUF-P activity was linearly related to ELFA spots over a range of 5 to 200 nmol l⁻¹ h⁻¹. At MUF-P hydrolysis rates higher than 200 nmol l⁻¹ h⁻¹, ELFA spot abundance leveled off.

## CONCLUSIONS

Culture populations and field communities of heterotrophic bacteria exhibited different phosphatase activities as seen from the specific activities found with MUF-P substrate and the varying percentages of ELF labeling. A minimum threshold of extracellular phosphatase activity (measured by spectrofluorometry) of $10 \text{ fmol cell}^{-1} \text{ h}^{-1}$ for phytoplankton (Nedoma et al. 2003) and $0.17 \text{ fmol cell}^{-1} \text{ h}^{-1}$ for bacteria in acidified mountain lakes (Nedoma & Vrba 2006) is necessary to observe significant ELFA spot formation and to allow quantitative per cell estimates. Such levels were reached only in our cultures. In contrast, we were unable to detect ELFA labeling of bacteria from marine samples with the protocol developed in this study, even though SRP concentrations were undetectable in surface waters and MUF-P hydrolysis rates were relatively high. Heterotrophic bacteria were P-stressed, as shown by the stimulation of bacterial production after

<figure><img src="image_5.png" /><figcaption>Fig. 4. Log-log plot of abundance of ELFA spots versus MUF-P alkaline phosphatase activities using the whole data set; culture data presented in Table 1, profile of March 2003 (Fig. 3), and other measurements made using the same protocol within surface waters at the DYFAMED station in October 2004 (results not presented in text). Solid symbols represent *in situ* data, open symbols are pure cultures</figcaption></figure>
P amendments (results not presented). It is likely that either the concentration of ELF-P we added was different from the affinity of bacteria for this molecule, or that assay conditions were not optimal, e.g. only the most active bacteria were labeled. This problem has also been encountered with natural *Synechococcus* cells in the North Atlantic Gyre (Lomas et al. 2004). Detection of extracellular activity among small coccioids in an acidified lake was scarce, and when detectable, activity was lower than that of filaments and curved rods that systematically revealed higher activities (Nedoma & Vrba 2006). Future research should concentrate on more sensitive detection techniques such as flow cytometry, which could allow the detection of a greater number of events and the simultaneous quantification of ELF intensity per cell with a faster procedure than quantification by image analysis. This work is currently in progress.

*Acknowledgements.* We thank P. Catala for providing us with some marine strains from the AIRWIN collection; P. Raimbault, N. Garcia, and T. Moutin for providing us PP, SRP, and DOP data; and M. Goutx as one of the PIs of the PECHE project. Valuable comments and English improvement from 3 anonymous reviewers and J. Edmonds are appreciated. This study was supported by INSU-CNRS through the French program PROOF-PECHE, and by the PAI Barrande project 2005-06-009-01. This work is dedicated to V. Andersen, PI of the PECHE project, who passed away in March 2007.

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