High-pressure systems for gas phase-free continuous incubation of enriched marine
microbial communities performing anaerobic oxidation of methane
Christian Deusner1,2, Volker Meyer1 and Timothy G. Ferdelman1
1
Max Planck Institute for Marine Microbiology, Bremen 28359, Germany
2
Current Address: Leibniz Institute for Marine Sciences (IFM GEOMAR), Kiel 24148,
Germany
Corresponding author: c.deusner@ifm-geomar.de, IFM GEOMAR, Wischhofstrasse 1-3, Kiel
24148, Germany
Running title: High-pressure continuous incubation systems
Keywords: AOM, methane, sulfate reduction, deep-sea, high-pressure, piezophilic
Supporting information
Components of the high-pressure incubation systems
The basic system components and the overall experimental setup are shown in
Supplementary Figure S1. The tubing system for transfer of liquid medium installed upstream
of biomass incubation was made from either PEEK, polyetheretherketone (1/8“, 1/16“) or
titanium (1/4“, 1/8“, 1/16“). All other connections including tubing for gas delivery were
made from PEEK or stainless steel (1/2“, ¼“, 1/8“, 1/16“). All fittings and connections were
made from PEEK, titanium or stainless steel.
The pressure vessel R1 (Büchi, Uster, Switzerland) is made from stainless steel and
has an internal volume of 1000 ml. It can be operated up to 20 MPa and 50°C. R1 is equipped
with multiple ports for medium delivery, online measurements and sampling. Tubings for
medium and electrical wiring for sensor connection are attached via lead-through with sealing
gland assemblies, respectively (Conax, Buffalo, NY, USA). The incubation vessel R2 (PARR
Instrument, Frankfurt, Germany) is made from titanium. It has an internal volume of 1000 ml
and can be operated up to 35 MPa and 50°C. It is equipped with multiple ports for medium
delivery, online measurements and sampling similar to R1.
The HPLC pumps (High-performance liquid chromatography, Sykam, Eresing,
Germany) P1, P2.1, P2.2 can be operated alternatively in constant flow mode (0.1-9.95 ml/min)
or constant pressure mode. The HPLC pumps were modified by the manufacturer with
stronger motors and gear units for a higher pressure range up to 60 MPa. The pump heads are
made from stainless steel. The two-piston operation principle of the pumps effectively
minimizes pulsation and pressure oszillations during operation.
The two-stage gas compressor station B1 (Haskel, Wesel, Germany) is driven by
compressed air. The pressure buildup is regulated proportional to the pressure of compressed
air and the booster can hence be operated in constant pressure mode. The compressor is
specifically designed and certified for flammable gases and it is integrated in a compressor
unit which is controlled by pressure switches and bursting disks.
Gas flow is regulated by shut-off valves, high-pressure regulating valves (V1, V2.1V2.3, V4.1-V4.3) and metering valves (V3.1-V3.3, all TESCOM Europe, Selmsdorf, Germany).
Liquid flow is adjusted with back-pressure regulation valves (V7.1-V7.2) in line with metering
valves (V6.1-V6.2, all TESCOM Europe). This combination of regulation units is chosen to
limit fluid pressure and flow velocity throughout the incubation system. It also acts to avoid
pressure oscillations and eliminates the possibility of rapid pressure changes during normal
operation. All valves and regulators are made from stainless steel. Parallel supply of
pressurized methane at different flow rates and pressure levels to a maximum of three
experimental reaction units is currently possible.
The overall HP-CI system and particularily R1 is connected to a specific exhaust gas
system which allows collection and discharge of exhaust gases to the exterior of the building.
To comply with health and safety requirements, the exhaust gas system is comprised of lines
dedicated to to the discharge of pressurized fluids in the case of a pressure deviation above the
maximum pressure limit. These lines are separately connected to each single reaction unit.
The HP-MI system R3 allows for manifold incubation of up to 45 pressure vessels.
The high-pressure vessels consist of stainless steel standard HPLC columns (ID 20 mm,
height 120 mm). The system can be applied up to 45 MPa and 220°C. The maximum
temperature gradient that can be achieved is 160°C.
The high-pressure system is equipped with several pressure sensors, manometers and
temperature sensors as also shown in Supplementary Figure S1. R1 is further equipped with a
custom-made conductivity sensor for liquid level monitoring. Several security valves (V8.1V8.4) and bursting discs are integrated in accordance to health and safety standards.
Monitoring and control of the high-pressure incubation systems is achieved with a custommade programmable controller connected to a PC and process software that was developed
inhouse. The controller unit is designed to be connected to the gas-safety-management-system
(Dräger Medical ANSY, Bremen, Germany) which continuously monitors deviations from
working pressure and flammable gas atmosphere.
Supplementary Figure S2 shows a combined illustration of the system flow scheme
with image representations of the central system components of the HP-CI system.
Supplementary Figure S3 shows an image of the basic setup of the HP-CI system.
Sampling ports and sampling devices
Liquid samples were taken either directly from the reservoir bottle and the pressure vessels R 1
and R2, or alternatively downstream from each of these components. The samples were taken
into closed glass vials or into glass syringes (see Sampling and analysis). The sampling ports
(SP) consist of capillary tubing (PEEK or stainless steel, 1/16”) equipped with needle valves.
A specific recirculation system for biomass sampling was installed in the incubation vessel R 2
since the settling characteristics of the biomass do not allow for the usual downstream
sampling. The recirculation system was implemented to minimize loss of biomass. Since
rapid decompression from gas-enriched media can have adverse effects on cellular structures
(Park and Clark, 2002) a specific pressure release device was integrated in the system for
biomass sampling (not shown). With that system the pressure within a given volume of liquid
sample can be steadily decreased by controlled volume expansion.
Process Monitoring and Control
The HP-CI system and HP-MI system are equipped with several pressure sensors and
manometers, as well as temperature sensors. R1 is equipped with a custom-made level sensor.
Pressure, temperature and level sensors are connected to the controller unit via a 4-20mA
interface. The level sensor is a potential-free conductivity sensor with three discrete levels for
detection of upper and lower level settings as well as sensor-failure. Detected failure on a
sensor is used to hardware-inhibit an actuator controlled by the sensor-signal. Monitoring and
control of the high-pressure incubation systems is achieved with a custom-made
programmable controller connected to a PC and process software which was developed
inhouse. The PC can be programmed to operate R1 pressure-controlled with secondary level
limits (current mode of operation) or level-controlled with secondary pressure limits. Both
modes are implemented by controlling P1. The controller unit is designed to be connected to
the gas-safety-management-system (see Health and Safety Requirements below). It can
process a management-failure-information from the gas-safety-management-system and send
a controlling output / error signal to the super-ordinated gas-safety-management-system.
Health and safety requirements
The setup and operation of the high-pressure incubation systems must conform to health and
safety requirements. The system design physically minimizes the possibility of any rapid
pressure build-up. All system components are continuously monitored for deviations from
working pressure and are further equipped with security valves and bursting discs. Pressure
increase above the allowable working pressure results in immediate system cutoff in safemode and a pressure release. In this case, all pressurized fluids are released and discharged
outside the building. Furthermore, the experimental area is continuously monitored for
flammable gases. Accumulation of methane above 40 % LEL causes a system cutoff in safemode. Monitoring and control for safety purposes is achieved using a stationary gas-safetymanagement-system (Dräger Medical ANSY, Bremen, Germany).
Sampling and analysis
Liquid samples during continuous operation of the HP-CI system were taken downstream of
both reaction phases R1 and R2, respectively. Liquid samples were taken into closed glass
vials with butyl stoppers (Supplementary Figure S4) or glass syringes (Supplementary Figure
S5) by opening the shut-off needle valves in the sampling lines. The samples were fixed in
either zinc acetate solution (5% w/v) or saturated sodium chloride solution. The concentration
of dissolved methane was measured either by expansion of the gas after depressurization in a
glas syringe as a volume ratio referring to the liquid volume. Alternatively, methane
concentration was measured from the headspace of the fixed samples and calculated referring
to sample pressure and volume. Sample pressure was measured with a custom-made pressure
sensor setup which was developed for pressure measurements in small gas volumes. This
system uses a small semiconductor differential pressure sensor (Motorola mpx 2010 dp).
References
Park CB, Clark DS. 2002. Rupture of the cell envelope by decompression of the deep-sea
methanogen Methanococcus jannaschii. Applied and Environmental Microbiology
68(3):1458-1463.