rates of growth and metabolism of natural microbial
populations collected from surface waters as well as from the
deep sea” (Jannasch and Wirsen 1973). Contrary to this early
conclusion, virtually all subsequently collected data from the
water column under in situ conditions have shown that the
situation is the reverse, namely, those microorganisms autochthonous to depth are adapted to both the high pressure and low
temperature of their environment (Tamburini et al. 2013).
17.2.3.3 High-Pressure Retaining Deep-Seawater
Samplers
A limited number of high-pressure vessels have been
constructed during the past 50 years to measure microbial activity in the cold deep ocean and evaluate the effects of hydrostatic
pressure, as well as decompression, on deep-sea microbial activity. Sterilizable pressure-retaining samplers for retrieving and
subsampling undecompressed deep-seawater samples have been
developed independently by three laboratories including
Jannasch/Wirsen at the Woods Hole Oceanographic Institution
(USA), Colwell/Tabor/Deming at the University of Maryland
(USA), and Bianchi at the Aix-Marseille University (Marseille,
France) (Jannasch and Wirsen 1973; Jannasch et al. 1973; Tabor
and Colwell 1976; Jannasch and Wirsen 1977; Deming et al.
1980; Bianchi and Garcin 1993; Bianchi et al. 1999; Tholosan
et al. 1999; Tamburini et al. 2003). Extensive sampling equipment for cold deep-sea high-pressure work has also been developed by Horikoshi and his team (Jamstec, Japan) exclusively
devoted to recovering new piezophilic microorganisms and to
study the effect of pressure on those isolates, as described in the
Extremophiles Handbook (Horikoshi 2011). At least two other
groups are developing pressure-retaining samplers, the Royal
Netherlands Institute for Sea Research (NIOZ) and the National
University of Ireland (Galway), but the designs or initial results
have not yet been published.
Relatively few interactions occurred between these laboratories; then only the high-pressure serial sampler (HPSS),
always used today, is briefly presented in Fig. 17.11. More
details can be found in Bianchi et al. (1999) and in Tamburini
et al. (2003). The HPSS is based on a commercially available
multi-sampling device that includes a CTD (Sea-Bird Carousel)
and equipped with 500-ml high-pressure bottles (HPBs) fitted on
polypropylene boards, totally adaptable to Sea-Bird Rosette 12
or 24 bottles (Fig. 17.11a). The HPSS allows collection of
several ambient-pressure water samples at different depths during the same hydrocast down to a depth of 3,500 m.
HPBs are 500-ml APX4 stainless steel cylinders (75-mm OD,
58-mm ID, and 505-mm total length) with a 4-mm thick
polyetheretherketone (PEEK®) coating. The PEEK® floating
piston (56-mm total length) is fitted with two O-rings. The
screw-top endcap is covered with a sheet of PEEK to avoid
contact between the sample and the stainless steel. Viton® Orings are used to ensure that the system is pressure-tight; Viton®
O-rings are chosen instead of nitrile O-rings to eliminate possible
carbon contamination of the sample. The screwed bottom
endcap is connected, via a stainless steel tube, to the piloted
pressure generator. HPBs are autoclaved to ensure sterility of
sampling. Functioning of the HPBs is depicted in Fig. 17.11b;
when the filling valve is opened at depth by the operator via the
deck unit connected to the electric wire, the natural hydrostatic
pressure moves the floating piston downward and seawater
enters the upper chambers of two HPBs. The distilled water
flushes from the lower chambers to the exhaust tanks through a
nozzle that acts as a hydraulic brake avoiding decompression
during filling. Samples are collected into two identical highpressure bottles filled at the same time through the same twoway valve (Fig. 17.11b). One of these bottles retains the in situ
pressure, while the other which lacks a check valve is progressively decompressed when the sampler is climbed back up to the
surface. The decompressed bottle, like a classical retrieving
bottle (e.g., Niskin bottle), is used in comparative fashion to
estimate the effect of decompression on measurements of
deep-sea microbial activity. Decompressed and high-pressure
(ambient) samples are treated in exactly the same way. Highpressure sampling, subsampling, and transfer are performed
using hydrostatic pressure (instead of gas pressure) thanks to a
piloted pressure generator (described in Tamburini et al. 2009) in
order to diminish risk during manipulation. In contrast to classical high-pressure pumps based on alternating movement of a
small piston associated with inlet and exhaust valves, the piloted
pressure generator is based on a step motor-driven syringe. A
step motor with a high starting torque is connected to a highprecision worm screw operating the high-pressure syringe.
Adjustment of the movement of the syringe is controlled by a
digital computer fitted with a high-power processor. Subsampling is performed by the use of counterpressure (a maximum of 0.05 MPa) from the bottom of the HP bottle; opening the
sampling valve allows the counterpressure to move the floating
piston upward. The primary sample within the HPB is
maintained under in situ pressure while the secondary sample
(decompressed) is analyzed directly or else fixed (by adding a
preservative solution like formaldehyde), stored, and analyzed
later.
To evaluate the state of the field of piezomicrobiology,
Tamburini et al. (2013) have compiled data from published
studies of deep samples where prokaryotic activities were
measured under conditions of in situ pressure and the results
compared with those obtained using incubation at atmospheric
pressure after decompression. The pressure effect (Pe) has been
calculated from this compiled data set (n ¼ 52 pairs of samples
maintained under in situ pressure condition versus decompressed
and incubated at atmospheric pressure condition). Pe is defined
as the ratio between activity obtained under HP and that obtained
under DEC conditions (Pe ¼ HP/DEC), where a ratio >1
indicates piezophily (adaptation to high pressure) and a ratio
<1, piezosensitivity. Calculation of Pe values has proven to be a
useful diagnostic tool for evaluating the effect of decompression
770
F. Joux et al.
populations collected from surface waters as well as from the
deep sea” (Jannasch and Wirsen 1973). Contrary to this early
conclusion, virtually all subsequently collected data from the
water column under in situ conditions have shown that the
situation is the reverse, namely, those microorganisms autochthonous to depth are adapted to both the high pressure and low
temperature of their environment (Tamburini et al. 2013).
17.2.3.3 High-Pressure Retaining Deep-Seawater
Samplers
A limited number of high-pressure vessels have been
constructed during the past 50 years to measure microbial activity in the cold deep ocean and evaluate the effects of hydrostatic
pressure, as well as decompression, on deep-sea microbial activity. Sterilizable pressure-retaining samplers for retrieving and
subsampling undecompressed deep-seawater samples have been
developed independently by three laboratories including
Jannasch/Wirsen at the Woods Hole Oceanographic Institution
(USA), Colwell/Tabor/Deming at the University of Maryland
(USA), and Bianchi at the Aix-Marseille University (Marseille,
France) (Jannasch and Wirsen 1973; Jannasch et al. 1973; Tabor
and Colwell 1976; Jannasch and Wirsen 1977; Deming et al.
1980; Bianchi and Garcin 1993; Bianchi et al. 1999; Tholosan
et al. 1999; Tamburini et al. 2003). Extensive sampling equipment for cold deep-sea high-pressure work has also been developed by Horikoshi and his team (Jamstec, Japan) exclusively
devoted to recovering new piezophilic microorganisms and to
study the effect of pressure on those isolates, as described in the
Extremophiles Handbook (Horikoshi 2011). At least two other
groups are developing pressure-retaining samplers, the Royal
Netherlands Institute for Sea Research (NIOZ) and the National
University of Ireland (Galway), but the designs or initial results
have not yet been published.
Relatively few interactions occurred between these laboratories; then only the high-pressure serial sampler (HPSS),
always used today, is briefly presented in Fig. 17.11. More
details can be found in Bianchi et al. (1999) and in Tamburini
et al. (2003). The HPSS is based on a commercially available
multi-sampling device that includes a CTD (Sea-Bird Carousel)
and equipped with 500-ml high-pressure bottles (HPBs) fitted on
polypropylene boards, totally adaptable to Sea-Bird Rosette 12
or 24 bottles (Fig. 17.11a). The HPSS allows collection of
several ambient-pressure water samples at different depths during the same hydrocast down to a depth of 3,500 m.
HPBs are 500-ml APX4 stainless steel cylinders (75-mm OD,
58-mm ID, and 505-mm total length) with a 4-mm thick
polyetheretherketone (PEEK®) coating. The PEEK® floating
piston (56-mm total length) is fitted with two O-rings. The
screw-top endcap is covered with a sheet of PEEK to avoid
contact between the sample and the stainless steel. Viton® Orings are used to ensure that the system is pressure-tight; Viton®
O-rings are chosen instead of nitrile O-rings to eliminate possible
carbon contamination of the sample. The screwed bottom
endcap is connected, via a stainless steel tube, to the piloted
pressure generator. HPBs are autoclaved to ensure sterility of
sampling. Functioning of the HPBs is depicted in Fig. 17.11b;
when the filling valve is opened at depth by the operator via the
deck unit connected to the electric wire, the natural hydrostatic
pressure moves the floating piston downward and seawater
enters the upper chambers of two HPBs. The distilled water
flushes from the lower chambers to the exhaust tanks through a
nozzle that acts as a hydraulic brake avoiding decompression
during filling. Samples are collected into two identical highpressure bottles filled at the same time through the same twoway valve (Fig. 17.11b). One of these bottles retains the in situ
pressure, while the other which lacks a check valve is progressively decompressed when the sampler is climbed back up to the
surface. The decompressed bottle, like a classical retrieving
bottle (e.g., Niskin bottle), is used in comparative fashion to
estimate the effect of decompression on measurements of
deep-sea microbial activity. Decompressed and high-pressure
(ambient) samples are treated in exactly the same way. Highpressure sampling, subsampling, and transfer are performed
using hydrostatic pressure (instead of gas pressure) thanks to a
piloted pressure generator (described in Tamburini et al. 2009) in
order to diminish risk during manipulation. In contrast to classical high-pressure pumps based on alternating movement of a
small piston associated with inlet and exhaust valves, the piloted
pressure generator is based on a step motor-driven syringe. A
step motor with a high starting torque is connected to a highprecision worm screw operating the high-pressure syringe.
Adjustment of the movement of the syringe is controlled by a
digital computer fitted with a high-power processor. Subsampling is performed by the use of counterpressure (a maximum of 0.05 MPa) from the bottom of the HP bottle; opening the
sampling valve allows the counterpressure to move the floating
piston upward. The primary sample within the HPB is
maintained under in situ pressure while the secondary sample
(decompressed) is analyzed directly or else fixed (by adding a
preservative solution like formaldehyde), stored, and analyzed
later.
To evaluate the state of the field of piezomicrobiology,
Tamburini et al. (2013) have compiled data from published
studies of deep samples where prokaryotic activities were
measured under conditions of in situ pressure and the results
compared with those obtained using incubation at atmospheric
pressure after decompression. The pressure effect (Pe) has been
calculated from this compiled data set (n ¼ 52 pairs of samples
maintained under in situ pressure condition versus decompressed
and incubated at atmospheric pressure condition). Pe is defined
as the ratio between activity obtained under HP and that obtained
under DEC conditions (Pe ¼ HP/DEC), where a ratio >1
indicates piezophily (adaptation to high pressure) and a ratio
<1, piezosensitivity. Calculation of Pe values has proven to be a
useful diagnostic tool for evaluating the effect of decompression
770
F. Joux et al.