Part B | 12.8
380 Part B Tools and Methods in Marine Biotechnology
the hydrostatic pressure relative to the pressure in R1
from P1 to P2 and allows incubation at increased hydrostatic pressure compared to the methane partial pressure
in R1. The fluid flow in R2 is also automatically adjusted with pump P2 working in constant flow mode. R2
is also equipped with an internal vessel (made of PEEK
or POM) to prevent potential chemical contamination
of the biomass. Due to the mode of headspace-free incubation, the internal vessel is designed as a closed
vessel. For compensating pressure or volume changes
it is has a top and a bottom part and its volume is adjustable. The bottom part is equipped with an O-ring
sealing that permits axial movement of the two parts
relative to each other. The internal vessel is housed inside the pressure vessel surrounded by a hydraulic fluid
that can be pressurized independently. Under standard
operating conditions, the free volume for incubation
is 420 mL within R2. Medium transport upstream and
downstream of R2 is carried out through a tubing that is
directly connected to the internal vessel through flexible
lead-through systems. The internal vessel can be heatsterilized and inoculated. The culture broth inside R2
is agitated with a magnetic stirrer. Liquid samples were
taken from downstream section of both R1 and R2 at
the sampling ports, directly into glass vials closed with
butyl stoppers or into glass syringes after opening the
shut-off needle valves in the sampling lines.
The HP-MI system (R3), originally developed
(Kallmeyer et al. [12.71]) for high-pressure/hightemperature (HPHT) batch incubation, was modified
and adapted in this study to be operated for manifold fed-batch incubation. Gas enrichment of liquid
medium and delivery is attained in a similar method as
in the HP-CI system. Each pressure vessel is equipped
with shut-off valves at the inlet and outlet, respectively,
and can be manipulated independently. Gas-enriched
medium is transferred separately to each of the highpressure vessels and each vessel is filled in upstream
mode. Like R2, pressure vessels in R3 are operated
without free gas and are equipped with internal vessels
(free volume for incubation, 7 mL) (made of PEEK or
POM), which are constructed as open vessels that seal
against the steel by a polytetrafluoroethylene (PTFE)
plate that presses into the cap of the steel vessel. While
the medium is delivered into an internal vessel via leadthrough tubings (similar to R2 in the HP-CI system),
the upper outlet of an internal vessel has a built-in glass
filter plate which allows permeation by both gas and liquid medium. Before sampling the biomass was allowed
to settle to the bottom of the internal vessel; subsequently, an aliquot of the supernatant was collected
through the bottom capillary while the sampled liquid
volume was replaced with gaseous methane through the
top capillary at the pressure corresponding to the saturation methane partial pressure applied during incubation.
On commencing the fed-batch mode of operation, the
design of the internal vessels ensured that the gas introduced during sampling was completely removed during
filling of the vessels under high-pressure conditions.
This study demonstrated that biomass-specific activity increases substantially as a function of methane
partial pressure in both continuous and fed-batch incubation. The systems could be used to control incubation
parameters and to simulate environmental conditions
with regard to constant substrate supply and product removal in continuous incubation. Stability with respect
to sulfide production in continuous operation and reproducibility of results from manifold fed-batch incubation
indicate that both the system components and the experimental procedure could be successfully applied for the
study and incubation of AOM-performing marine microbial consortia.
Zhang et al. [12.83] developed a novel, continuous
high-pressure bioreactor (HPBR) system to mimic a diverse methane-rich deep-sea ecological niche, to stimulate in vitro sulfate reduction (SR)-coupled- anaerobic
oxidation of methane (AOM) activity; and to examine
the kinetics of the SR-AOM process. The HPBR system (Fig. 12.45) comprises three main parts: (a) the
conditioning vessel, (b) the high-pressure pump, and
(c) the incubation vessel. The conditioning vessel (volume 1 L) made of S.S. 316 is directly connected to two
pipettes – a liquid pipette (to charge liquid medium at
high-pressure) and a gas pipette (to charge methane gas
at high-pressure) both of which are pressurized by the
methane gas bottle. Artificial seawater medium is saturated with methane at a determined pressure (up to
8 MPa) in the conditioning vessel and transferred to
the incubation vessel by a high-pressure pump. In the
conditioning vessel, built-up gas pressure determines
the amount of methane saturated into the medium and
available as substrate. A motor-mixer (40 rpm) is used
to achieve a homogeneous dissolved methane concentration in the whole vessel. The high-pressure pump
is piston based with a separated chamber to rinse the
piston while it is moving – this self-flushing mechanism allows feed of saline water without damage to
the piston and its seals. The incubation vessel (volume
0:6 L), made of titanium to prevent potential microbial
corrosion, is placed inside a thermal regulated incubator (555
ı C) to control the incubation temperature.
The pressure inside this incubation vessel could be in-
380 Part B Tools and Methods in Marine Biotechnology
the hydrostatic pressure relative to the pressure in R1
from P1 to P2 and allows incubation at increased hydrostatic pressure compared to the methane partial pressure
in R1. The fluid flow in R2 is also automatically adjusted with pump P2 working in constant flow mode. R2
is also equipped with an internal vessel (made of PEEK
or POM) to prevent potential chemical contamination
of the biomass. Due to the mode of headspace-free incubation, the internal vessel is designed as a closed
vessel. For compensating pressure or volume changes
it is has a top and a bottom part and its volume is adjustable. The bottom part is equipped with an O-ring
sealing that permits axial movement of the two parts
relative to each other. The internal vessel is housed inside the pressure vessel surrounded by a hydraulic fluid
that can be pressurized independently. Under standard
operating conditions, the free volume for incubation
is 420 mL within R2. Medium transport upstream and
downstream of R2 is carried out through a tubing that is
directly connected to the internal vessel through flexible
lead-through systems. The internal vessel can be heatsterilized and inoculated. The culture broth inside R2
is agitated with a magnetic stirrer. Liquid samples were
taken from downstream section of both R1 and R2 at
the sampling ports, directly into glass vials closed with
butyl stoppers or into glass syringes after opening the
shut-off needle valves in the sampling lines.
The HP-MI system (R3), originally developed
(Kallmeyer et al. [12.71]) for high-pressure/hightemperature (HPHT) batch incubation, was modified
and adapted in this study to be operated for manifold fed-batch incubation. Gas enrichment of liquid
medium and delivery is attained in a similar method as
in the HP-CI system. Each pressure vessel is equipped
with shut-off valves at the inlet and outlet, respectively,
and can be manipulated independently. Gas-enriched
medium is transferred separately to each of the highpressure vessels and each vessel is filled in upstream
mode. Like R2, pressure vessels in R3 are operated
without free gas and are equipped with internal vessels
(free volume for incubation, 7 mL) (made of PEEK or
POM), which are constructed as open vessels that seal
against the steel by a polytetrafluoroethylene (PTFE)
plate that presses into the cap of the steel vessel. While
the medium is delivered into an internal vessel via leadthrough tubings (similar to R2 in the HP-CI system),
the upper outlet of an internal vessel has a built-in glass
filter plate which allows permeation by both gas and liquid medium. Before sampling the biomass was allowed
to settle to the bottom of the internal vessel; subsequently, an aliquot of the supernatant was collected
through the bottom capillary while the sampled liquid
volume was replaced with gaseous methane through the
top capillary at the pressure corresponding to the saturation methane partial pressure applied during incubation.
On commencing the fed-batch mode of operation, the
design of the internal vessels ensured that the gas introduced during sampling was completely removed during
filling of the vessels under high-pressure conditions.
This study demonstrated that biomass-specific activity increases substantially as a function of methane
partial pressure in both continuous and fed-batch incubation. The systems could be used to control incubation
parameters and to simulate environmental conditions
with regard to constant substrate supply and product removal in continuous incubation. Stability with respect
to sulfide production in continuous operation and reproducibility of results from manifold fed-batch incubation
indicate that both the system components and the experimental procedure could be successfully applied for the
study and incubation of AOM-performing marine microbial consortia.
Zhang et al. [12.83] developed a novel, continuous
high-pressure bioreactor (HPBR) system to mimic a diverse methane-rich deep-sea ecological niche, to stimulate in vitro sulfate reduction (SR)-coupled- anaerobic
oxidation of methane (AOM) activity; and to examine
the kinetics of the SR-AOM process. The HPBR system (Fig. 12.45) comprises three main parts: (a) the
conditioning vessel, (b) the high-pressure pump, and
(c) the incubation vessel. The conditioning vessel (volume 1 L) made of S.S. 316 is directly connected to two
pipettes – a liquid pipette (to charge liquid medium at
high-pressure) and a gas pipette (to charge methane gas
at high-pressure) both of which are pressurized by the
methane gas bottle. Artificial seawater medium is saturated with methane at a determined pressure (up to
8 MPa) in the conditioning vessel and transferred to
the incubation vessel by a high-pressure pump. In the
conditioning vessel, built-up gas pressure determines
the amount of methane saturated into the medium and
available as substrate. A motor-mixer (40 rpm) is used
to achieve a homogeneous dissolved methane concentration in the whole vessel. The high-pressure pump
is piston based with a separated chamber to rinse the
piston while it is moving – this self-flushing mechanism allows feed of saline water without damage to
the piston and its seals. The incubation vessel (volume
0:6 L), made of titanium to prevent potential microbial
corrosion, is placed inside a thermal regulated incubator (555
ı C) to control the incubation temperature.
The pressure inside this incubation vessel could be in-