Experiment 3

This is basically Exp. 2 restarted with a modified nutrient medium.  Nitrate has been increased from 15 然 to 136 然 and phosphate has been increased from 2 然 to 21.2 然.  Also, a more defined artificial seawater mixture was used to bring salinity to 3 PSU.

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 6 Mar 2019; however, chemostats were filled with 3 L of Siders Pond water at 11:50, but with medium feed rate turned off to allow the Siders Pond community time to adjust to warm conditions (25 蚓).

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 10:30 on 6-Mar-2019, a 20 L water sample was collected from the surface of Siders Pond, Falmouth MA (41.551115衹, -70.619438蚩) and passed through a 335 痠 Nytex mesh to remove large organisms and particles.
  2. Three liters of Siders Pond water was added to duplicate chemostats at approximately 11:50 6-Mar-2019 and placed in a dark growth chamber at 25蚓 and sparged with air at a flow rate of 10 sccm. Initial medium flow rate was turned off.
  3. Medium flow turned on at 17:00 6-Mar-2019 (t = 0.71 d) at a flow rate of 0.3 L d-1 or a dilution rate of 0.1 d-1. Starting with low dilution rate for ~ 1 d to allow community spin-up.
  4. Increased dilution rate to normal operating value of 1.0 d-1 or 3.0 L d-1 at 10:30 7-Mar-2019 (t = 1.44 d).
  5. Some turbidity has been noticed in the nutrient feed to MC 2, which has caused some plugging of the Sterivex in-line filter and decrease in flow rate to MC 2 at times. First notice around 9-Mar-2019 (t = 4 d).
  6. MC's sampled at 15:00 13-Mar-2019 (t = 7.63 d).  Introduction of air into the MCs from sample withdrawal is evident by the temporary increase in DO and oxygen concentrations around this time.
  7. MC's sampled at 15:40 14-Mar-2019 (t = 8.65 d). 
  8. New nutrient medium prepared and placed on-line at 18:30 14-Mar-2019 (t = 8.77 d).
  9. Potassium phosphate injected into both MCs to increase PO4 concentration by 10 然 to test of P limitation at 15:21 15-Mar-2019 (t = 9.65 d)
  10. Another 10 然 increase in phosphate to both MCs occurred at 18:42 15-Mar-2019 (t = 9.78 d)
  11. Power failure occurred at 21:52 on 15-Mar-2019 (t = 9.91 d), which turned off the pump for the carbon medium.  Pump was not turned on until 13:18 16-Mar-2019 (t = 10.55 d).
  12. Three mL of trace elements (adjusted to pH 7) where added to both MCs to test for macro/micro element limitation at 10:52 18-Mar-2019 (t = 12.45 d).
  13. Several modifications on 19-Mar-2019: 1) 10:58 (t = 13.65 d) Calibrated pumps, so feed interrupted for ~30 min., 2) 16:08 (t = 13.76 d) Switch to newly prepared nutrient medium, but did not 0.2 痠 filter to avoid removal an any metal-phosphate precipitates, 3) 16:16 (t = 13.68 d) carbon pump malfunction due to software bug in Masterflex pump corrected. Pump running at higher rate than specified (10x?) for approximately 1.25 hr.
  14. Microcosms and feed sampled starting at 15:40 20-Mar-2019 (t = 14.65 d).
  15. Switched to a new batch of nutrient medium and calibrated pumps at 19:45 24-Mar-2019 (t = 18.82 d).
  16. MCs sampled then carbon medium replaced with carbon medium containing 99% 13C enriched glucose at 9:40 25-Mar-2019 (t = 19.40 d).  The main experiment has begun! Sampling will be frequent over the next several days.
  17. The last 13C samples were taken at 9:40 4-Apr-2019 (t = 29.40 d). The flow to the MCs will be stopped soon, then run in batch mode for a short time.
  18. Media flow to MCs stopped at 8:34 6-Apr-2019 (t = 31.36 d).

 Data Files:

Primary gas and probe data: Exp3.dat
External variable values: Exp3.oc
Experimental info file: Exp3.info
Compiled high resolution data from DO and pH probes: Exp3_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_2019-03-06_14h30_pH_243632-2353-1178.txt
MC1 DO: Trace_2019-03-06_14h30_DO_242453-02-210052.txt
MC2 pH: Trace_2019-03-06_14h31_pH_243632-2353-1176.txt
MC2 DO: Trace_2019-03-06_14h31_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 慢.  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

Ashley sampling recently collected inoculum in the "field"
Ashley sampling inoculum

Sadly, the stainless steel nutrient medium feed drum (left) was retired due to some observed growth and/or corrosion.  It has been replaced with two 22 L Belco glass vessels (right) that can be autoclaved; medium is filter sterilized now. The SS drum now collects chemostat output.
UPDATE: It appears the "growth" was actually FePO4 precipitate, which was being removed by filtration.  As of 16:08 19-Mar-2019 (t = 13.76 d), feed medium is no longer being 0.2 痠 filtered, but glass vessels are still being used but with constant mixing to insure suspension of micron size precipitate.
SS DrumBelco Media Vessels

Microcosms and feed on 17:46 22-Mar-2019 (t = 16.74 d)
MC and feed on 22-Mar-2019, t = 16.74 d