Part B | 12.8
372 Part B Tools and Methods in Marine Biotechnology
media can be prepared and the system assembled in
an anaerobic chamber and transferred (via an anaerobic bag) to the presterilized and gas-flushed chamber body. The chamber also contains eight detachable
cells (up to 15 mL in volume) that allows microbial
growth in liquid medium within the chamber and also
transfer in and out of the isolation chamber. Enrichments for isolation were transferred into an individual
growth cell within the isolation chamber. A motorized
robotic arm in the isolation chamber enabled a sterile inoculation loop (electrically heated in situ) to be
dipped into the growth cell and its contents to be
streaked onto an agar plate. After incubation, individual colonies could then be picked off and transferred to
other cells containing sterile media for further growth.
Transfer of these purified cultures out of the isolation
chamber into high-pressure incubation chambers permitted further subculture and physiological tests to be
conducted on isolates. All manipulations in the isolation chamber were viewed through a sapphire window
via an endoscope attached to a digital camera and
monitor.
Pressure vessels (100 cm
3 total volume, 80 cm
3
medium) made of titanium, used for enrichment and
cultivation have a body and a lid, attached together with
a screw thread. They are sealed by a male cone in the
lid and a complementary female cone in the body, plus
O-ring seals. The low-pressure enrichment vessels have
a small-bore ball valve (4 mm) on the lid for gas input
and liquid transfer, as well as a large-bore ball valve
(32 mm) for sediment slice transfer. The high-pressure,
incubation vessels have just two small-bore ball valves.
Preprepared sterile anaerobic media were transferred
into sterile pressure vessels within an anaerobic chamber. For high-pressure liquid transfer between systems,
a small pressure differential ( 2 MPa) between vessels
was maintained. Ball valves were used to prevent shear
stresses and possible cell rupture during culture transfer by pressure differential. For routine high-pressure
sampling of pressure vessels and subculture into vessels
with fresh medium, a bored-out T-piece and a series
of valves were used, that enabled transfers without
depressurization.
The study accomplished the following, viz.:
a) The direct enrichment of subseafloor prokaryotes
under elevated pressure.
b) Enrichment of sedimentary, anaerobic prokaryotes
without depressurization.
c) High-pressure cultivation from subsurface gas hydrate sediments.
Results showed that many subseafloor prokaryotes
from gas hydrate containing sites could grow (anaerobically) up to at least 80 MPa in various media which
is a much higher pressure than that previously recorded
for subseafloor prokaryotes. As 70% of the ocean is at
a pressure of 38 MPa or higher and the average depth
of sediments is 500 m, growth at 80 MPa would enable the majority of the subseafloor environment to
be populated by prokaryotes. This study was claimed
as the first report on the isolation of Carnobacterium,
Marinilactobacillus, Acetobacterium, Clostridium, and
Bacteroidetes species from deep, subseafloor, gas hydrate deposits.
Miller et al. [12.73] developed a novel HPHTBR
to investigate pressure–temperature relationships (up
to 260
ı C and 350 bar) in the growth and productivity of the extremely thermophilic methanogenic archaea Methanococcus jannaschii isolated from a deep
sea hydrothermal vent. The corrosion-resistant reactor
system permits direct sampling (of both liquids and
gases) from a transparent culture vessel without affecting the internal growth conditions. It was found
that increasing the pressure from 7:8 to 100 bar accelerated the production of methane and unicellular
protein at 90
ı C; raised the maximum temperature for
growth from 90 to 92
ı C but further increase in pressure
did not affect growth. The reactor vessel or pressure
cell (internal volume 43 mL) is a transparent synthetic single crystal sapphire tube (I.D. 0.7
00 , O.D.
1.25
00 , length 6
00 ) which allows direct visual observation of the growth medium. It may be sealed by two
different configurations – one for working at 100 bar
consisting of a spring-loaded Teflon gasket held onto
the tube ends by flanges which, again, are held in
place by six spacer rods. In the other sealing technique, the distance between the flanges is reduced, thus
compressing the Teflon rings which, on application of
pressure, are further compressed thereby maintaining
the seal.
The pressure cell is connected to a magnetic pump
to recirculate vapor through the liquid, which serves
two purposes, viz. (1) agitation of the liquid and
(2) to promote vapor–liquid equilibrium. The pump
(I.D. 0:375 in, internal length 2 in) is actuated by a stack
of ceramic ring magnets located around the pump body
that are oscillated up and down through a motor driven
crank and cable. A piston with a plug travels the length
of the pump. On the upstroke, gas is pushed out of the
pump into the pressure cell, on the down stroke, gas
fills the pump for the next stroke. A crank valve at each
end of the pump prevents flow in the opposite direction.
372 Part B Tools and Methods in Marine Biotechnology
media can be prepared and the system assembled in
an anaerobic chamber and transferred (via an anaerobic bag) to the presterilized and gas-flushed chamber body. The chamber also contains eight detachable
cells (up to 15 mL in volume) that allows microbial
growth in liquid medium within the chamber and also
transfer in and out of the isolation chamber. Enrichments for isolation were transferred into an individual
growth cell within the isolation chamber. A motorized
robotic arm in the isolation chamber enabled a sterile inoculation loop (electrically heated in situ) to be
dipped into the growth cell and its contents to be
streaked onto an agar plate. After incubation, individual colonies could then be picked off and transferred to
other cells containing sterile media for further growth.
Transfer of these purified cultures out of the isolation
chamber into high-pressure incubation chambers permitted further subculture and physiological tests to be
conducted on isolates. All manipulations in the isolation chamber were viewed through a sapphire window
via an endoscope attached to a digital camera and
monitor.
Pressure vessels (100 cm
3 total volume, 80 cm
3
medium) made of titanium, used for enrichment and
cultivation have a body and a lid, attached together with
a screw thread. They are sealed by a male cone in the
lid and a complementary female cone in the body, plus
O-ring seals. The low-pressure enrichment vessels have
a small-bore ball valve (4 mm) on the lid for gas input
and liquid transfer, as well as a large-bore ball valve
(32 mm) for sediment slice transfer. The high-pressure,
incubation vessels have just two small-bore ball valves.
Preprepared sterile anaerobic media were transferred
into sterile pressure vessels within an anaerobic chamber. For high-pressure liquid transfer between systems,
a small pressure differential ( 2 MPa) between vessels
was maintained. Ball valves were used to prevent shear
stresses and possible cell rupture during culture transfer by pressure differential. For routine high-pressure
sampling of pressure vessels and subculture into vessels
with fresh medium, a bored-out T-piece and a series
of valves were used, that enabled transfers without
depressurization.
The study accomplished the following, viz.:
a) The direct enrichment of subseafloor prokaryotes
under elevated pressure.
b) Enrichment of sedimentary, anaerobic prokaryotes
without depressurization.
c) High-pressure cultivation from subsurface gas hydrate sediments.
Results showed that many subseafloor prokaryotes
from gas hydrate containing sites could grow (anaerobically) up to at least 80 MPa in various media which
is a much higher pressure than that previously recorded
for subseafloor prokaryotes. As 70% of the ocean is at
a pressure of 38 MPa or higher and the average depth
of sediments is 500 m, growth at 80 MPa would enable the majority of the subseafloor environment to
be populated by prokaryotes. This study was claimed
as the first report on the isolation of Carnobacterium,
Marinilactobacillus, Acetobacterium, Clostridium, and
Bacteroidetes species from deep, subseafloor, gas hydrate deposits.
Miller et al. [12.73] developed a novel HPHTBR
to investigate pressure–temperature relationships (up
to 260
ı C and 350 bar) in the growth and productivity of the extremely thermophilic methanogenic archaea Methanococcus jannaschii isolated from a deep
sea hydrothermal vent. The corrosion-resistant reactor
system permits direct sampling (of both liquids and
gases) from a transparent culture vessel without affecting the internal growth conditions. It was found
that increasing the pressure from 7:8 to 100 bar accelerated the production of methane and unicellular
protein at 90
ı C; raised the maximum temperature for
growth from 90 to 92
ı C but further increase in pressure
did not affect growth. The reactor vessel or pressure
cell (internal volume 43 mL) is a transparent synthetic single crystal sapphire tube (I.D. 0.7
00 , O.D.
1.25
00 , length 6
00 ) which allows direct visual observation of the growth medium. It may be sealed by two
different configurations – one for working at 100 bar
consisting of a spring-loaded Teflon gasket held onto
the tube ends by flanges which, again, are held in
place by six spacer rods. In the other sealing technique, the distance between the flanges is reduced, thus
compressing the Teflon rings which, on application of
pressure, are further compressed thereby maintaining
the seal.
The pressure cell is connected to a magnetic pump
to recirculate vapor through the liquid, which serves
two purposes, viz. (1) agitation of the liquid and
(2) to promote vapor–liquid equilibrium. The pump
(I.D. 0:375 in, internal length 2 in) is actuated by a stack
of ceramic ring magnets located around the pump body
that are oscillated up and down through a motor driven
crank and cable. A piston with a plug travels the length
of the pump. On the upstroke, gas is pushed out of the
pump into the pressure cell, on the down stroke, gas
fills the pump for the next stroke. A crank valve at each
end of the pump prevents flow in the opposite direction.