Experiment 2

This is the first 13C tracer experiment using glucose as the tracer.  In approximately 2 weeks the glucose in the defined medium will be replaced with 100% 13C-glucose in a step-up pulse, but the feed medium will otherwise be unchanged.  As bacteria consume the heavy isotope, as well as predators (such as protists, viruses and other bacteria) that consume the enriched bacteria, their RNA will become enriched with 13C and detectable using stable isotope probing techniques (also see Fortunato and Huber 2016).   Experimental data will be used to determine the extent of interconnectivity within microbial communities.

Experimental clock time started at 00:00 5 Feb 2019; however, chemostats where inoculated with Siders Pond water at 18:30 with medium feed rate turned off to allow the Siders Pond community time to adjust to warm conditions (25 °C).

On-line variables associated with MC1 (last 36 points):


Gas concentrations in outputDissolve oxygen and pH in Algal bioreactor
Plot of O2, CO2 over all time.  Plot of pH and DO over all time.  Interactive graphics are also available under the JavaScript Graphics menu item above.  Note, the analog output for the DO probe on MC1 is intermittent; however, the Modbus digital output is working fine, so see the text files below or JavaScript Graphics to check DO.

On-line variables associated with MC2 (Last 36 points):

Concentration of off gases in MC2DO and pH associated with MC2
Plot of O2, CO2 all time.  Plot of pH and DO over all time.  Interactive graphics are also available under the JavaScript Graphics menu item above.

Instrument Drift (between analyzer calibrations), Last 36 points

Instrument Drift
Instrument Drift over all time.

Experimental Events:

  1. At approximately 14:30 on 5-Feb-2019, a 20 L water sample was collected from the surface of Siders Pond, Falmouth MA (41.551115°N, -70.619438°E) and passed through a 335 µm Nytex mesh to remove large organisms and particles.
  2. Three liters of Siders Pond water was added to duplicate chemostats at approximately 16:30 and placed in a dark growth chamber at 25°C.
  3. Medium flow turned on at 14:39 6-Feb-2019 (t = 1.61 d) at a flow rate of 3.0 L d-1 or a dilution rate of 1.0 d-1.  Also, placement of DO probe in MC1 was corrected.
  4. Biofilm growing on DO probe in MC1, which is the cause of the low DO readings.  Biofilm was removed and also removed from chemostat wall at 9:20 11-Feb-2019 (t = 6.40 d).
  5. New Nafion gas dryer from Perma Pure installed (MD-070-24P-2) at 18:10 14-Feb-2019 (t = 9.76 d).
  6. Numerous modifications made: 1) Infrared heater added to gas handling equipment to minimize condensation in gas streams (see Photos), 2) medium feed to MC 1 interrupted for 12-24 hrs, 3) headspace exposed to atmosphere to install new medium feeding approach, 4) removed "cigarette butt" from MC1 (also see Photos), 5) ~3 m of Norprene tubing between SS drum (containing inorganic nutrient medium) and growth chamber was replaced with 1/4" SS tubing to minimize light input, 6) 1 L from each MC was cross inoculated to the other  to insure similar community composition after perturbations (15-Feb-2019, t = 10.77 d).
  7. Analog channel of DO probe in MC1 magically working again (12:56 17-Feb-2019, t = 12.93 d).
  8. Biofilm developing on DO probe in MC1 was removed (10:00 19-Feb-2019, t = 14.42 d).
  9. Microcosms sampled 10:38 20-Feb-2019 (t = 15.44 d).
  10. Nutrient medium not flowing properly, but clearer at 12:00 25-Feb-2019 (t = 20.5 d).
  11. Microbial respiration probably limited by N and/or P, so started test with modified nutrient medium, raising HNO3 from 15 to 136 µM and H3PO4 from 2 to 21.2 µM at 18:30 25-Feb-2019 (t = 20.77 d).
  12. Feed to chemostats has stopped at around 18:30 28-Feb-2019 (t = 23.77 d) as we spin down Exp. 2.  As the last 3 days have shown, the nutrient medium was deficient in N and P.  We will be repeating this experiment as Exp. 3, but with elevated HNO3 and H3PO4 concentrations.

 Data Files:

Primary gas and probe data: Exp2.dat
External variable values: Exp2.oc
Experimental info file: Exp2.info
Compiled high resolution data from DO and pH probes: Exp2_DOpH.csv

Higher resolution (10 min) probe data from Hamilton's RS485 ModBus (files updated here every 1 hr):
(Note, these data are now plotted on the JS Graphics page)
MC1 pH: Trace_2019-02-05_17h17_pH_243632-2353-1178.txt
MC1 DO: Trace_2019-02-05_17h18_DO_242453-02-210052.txt
MC2 pH: Trace_2019-02-05_17h18_pH_243632-2353-1176.txt
MC2 DO: Trace_2019-02-05_17h18_DO_242453-02-210053.txt

Experimental Equipment:

ChemostatsInstruement Rack
Image (left) shows Postdoc Ashely Bulseco-McKim setting up  the  Bellco Glass 3 L  microcosms (MCs) on 6-Feb-2019 just prior to start of Exp2. Instrument rack and environmental chamber housing the MCs (image on right) is equipped with MKS mass flow controllers, Oxigraf O2 and CO2 laser diode absorption spectrometer, Nafion drier and a computer for monitoring and experimental control via Valco selector valve, ASCO solenoid valves, Weeder Technologies WTDOT and WTAIN digital IO boards and Comtrol RocketPort serial card.  Monitoring and control software is written in Intel Fortran (gigidy) and all com is via rs232.

Reactor dissolved oxygen (DO) and pH are monitored with Hamilton VisiFerm DO Arc 335 and EasyFerm Plus Arc 325 probes, respectively.  Probes are controlled and calibrated via Hamilton Device Manager software via a USB-ModBus RS 485 Converter.  Data from probes are logged from 4-20 mA signal outputs that are converted to 0-10 V with Wayjun Signal Isolators and digitized with a Weeder Tech WTAIN A2D board (data is also logged via ModBus at higher frequency). 

The microcosms are sparged with air in a Conviron PGR15 environmental chamber that is currently operated at 25 ºC.  A closed gas sampling loop regulated at a flow rate of 270 mL/min by a Hargraves mini pump (B.1F15E4.A12VDC) delivers reactor headspace gas to analyzer.Sample loop has an approximate volume of 20 mL. A Masterflex L/S Digital drive connected to Easy-Load 3 peristaltic pump heads provides exchange flow between reactors.

Software

Program for monitoring instruments and control. Note, software makes use of Intel Visual Fortran's serial IO library:
Graphics are generated by Tecplot and HighCharts

Photos

Biofilm on MC1 DO probe and wall at 9:20 11-Feb-2019 (t = 6.40 d-1).
Biofilm on MC1 DO probe

"Fixed" condensation in gas handling system components and lines:
diner light

"Cigarette butt" in MC1.  Biomass formation/wad (formed in just ~10 days) within 1/4" SS tubing feeding chemostat right after carbon and nutrient media mix, thereby plugging line.  Returned to previous approach (Exp. 1) where each were added to chemostat via separate tubes.
"cigarette butt"