On-Line Data from Experiment 1

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

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

Instrument Drift (between analyzer calibrations), Last 36 points

Experimental Events:

1. Experiment begun.  Filter sterilized medium added to reactors and monitoring program and analyzers started (19:13 21 May 2015)
2. Algal bioreactor inoculated with 1 L of Chlamydomonas reinhartii CW15 mutant lacking cell wall (12:30 22 May 2013).  Note, 18 L digestor has not been inoculated and flow between reactors has been stopped to allow algae to grow.
3. Gas flow rate of air increased from 20 to 50 mL/min due to decreasing DO concentration in algal bioreactor (21:35 24 May 2015)
4. Gas flow rate of air increased from 50 to 100 mL/min due to decreasing DO concentration in algal bioreactor (9:00 25 May 2015).  Neither increase in flow help situation very much.  
5. Removed shade screen from algal culture. This reversed the declining DO and net heterotrophy (12:00 25 May 2015)
6. Returned air flow to algal reactor to 20 mL/min (10:20 26 May 2015).
7. Decreased sample interval from every 5 hr to every 2 hr. (22:40 26 May 2015, t = 5.94 d)
8. Time on growth chamber was ~ 4 hrs slow.  It has been updated to correct EDT (14:10 27 May 2015, t = 6.59 d)
9. Impeller speed for algal reactor increased to 60 RPM (12:41 28 May 2015, t = 7.53 d)
10. Decrease air flow to algal reactor to 5 mL/min (18:10 28 May 2015, t = 7.76 d)
11. Currently, algal reactor is becoming CO2 limited very quickly after lights turn on.  Gas diffusers will be installed soon to improve gas-liquid transfer characteristics.
12. Reduce N₂ flow rate to digestor to 5 mL/min (16:10 10 Jun 2015, t = 20.67 d)
13. Installed gas diffusers on algal and methanogenic reactors (2 µm) and increased gas flow in algal reactor to 20 mL/min (11:07 20 Jun 2015, t = 40.46 d).
14. Decreased gas flow in algal reactor to 5 mL/min (17:41 8 Jul 2015, t = 48.37 d)
15. Increase light level in growth chamber to 2 metal halides plus 2 HP sodium lamps (17:13 10 Jul 2015, t = 50.72 d)
16. Increase gas flow in algal reactor to 20 mL/min (14:05 14 Jul 2015, t = 54.59 d)
17. Added CO₂ to air stream to bring CO₂ partial pressure to 2% from ambient air (~0.03%) (11:40 16 Jul 2015, t = 56.58 d).
18. External calibration of gas analyzers (13:15 17 Jul 2015, t = 57.55 d)
19. CO₂ in air to algal culture has been turned off due to pH crash (9:12 19 Jul 2015, t = 59.39 d)
20. Approximately 10 mL of a 4 M K₂HPO₄ solution was added to algal reactor increasing the PO₄ concentration by 10 mM. pH increased rapidly to ~7.45 (13:15 20 Jul 2015, t = 60.55 d)
21. CO₂ enrichment (2%) restored to algal reactor.  pH (currently at 7.45) will be monitored and adjusted with K₂HPO₄ as needed. (15:07 20 Jul 2015, t = 60.63 d)
22. Potassium phosphate buffer (10 mM) adjusted to pH 7.5 was added to the digestor (17:49 22 Jul 2015, t = 62.74 d).
23. Coupled chemostat flow between reactors was turned on at an effective dilution rate of 0.1 d^-1 based on algal reactor volume (17:50 22 Jul 2015, t = 62.74 d).
24. Increased exchange flow between reactors to 1.0 d^-1, or 4.032 L d^-1.  Also increased N₂ gas flow to digestor from 5 to 20 mL min^-1 (9:17 23 Jul 2015, t = 63.39 d)
25. Methanogenic reactor (aka digestor) was inoculated with ~500 mL of an anaerobic groundwater sample collected from a 6 m deep well located near the inlet of Little Pond, Falmouth (well LP7: 41.558512º Lat, -70.591887º Long).  Sample measurements: DO, 0 mg/L; pH 5.75; Salinity 0.2 ppt; Temp, 13.9ºC (19:11 30 Jul 2015, t = 70.80 d).
26. Monitoring computer locked up for ~ 9 hr. (starting at 10:42  to 19:42 1 Aug 2015, t = 72.40 - 72.82 d).
27. External calibration of gas analyzers conducted (11:31 4 Aug 2015, t = 75.48 d)
28. Flow between bioreactors stopped to facilitate assessment of respiration in digestor (12:30 10 Aug 2015, t = 81.54 d)
29. Added 10 mL of 4 M K₂HPO₄ solution to algal reactor only (11:38 14 Aug 2015, t = 85.48 d)
30. Approximately 0.4 mL of anaerobic sediments collected from station P22 along the Parker River was added to digestor to augment inoculum (15:31 21 Aug 2015, t = 92.65 d)
31. Around 10:20 on 25 Aug 2015 (t = 96.4 d), the CO₂ gas cylinder went empty, so CO₂ algal feed concentration returned to atmospheric levels (~0.03%).
32. Enrichment of CO₂ to algal feed gas returned to 2% (10:52 28 Aug 2015, t = 99.45 d).
33. Approximately 0.5 L of medium from digestor was transfered to algal reactor to makeup for samples that have been withdrawn for algal reactor (15:30 28 Aug 2015, t = 99.65 d).
34. Increased N₂ flow rate to digestor from 20 to 30 mL min^-1 (14:48 1 Sep 2015, t = 103.62 d).
35. Approximately 100 mL of suspended sediments (i.e., microbial aggregations or flocs) from the sequence batch reactor at the Falmouth Wastewater treatment plant was added to the digestor to augment inoculum (13:13 3 Sep 2015, t = 105.55 d)
36. Added 2.45 g of sodium acetate trihydrate to digestor to stimulate methanogens.  Concentration increased by approximately 1 mM (16:04 9 Sep 2015, t = 111.67 d).
37. Added 3.24 g of D-glucose to digestor.  Concentration increased by approximately 1 mM (9:34 15 Sep 2015, t = 117.40 d).
38. Upgraded MonitorA2M program and hardware for active control and monitoring of CO₂ enrichment of air feed to algal reactor (17:39 22 Sep 2015, t = 124.74 d)
39. Returned to coupling algal and methanogenic reactors at a dilution rate of 1 d^-1 with respect to algal reactor volume (exchange flow at 4 L d^-1) (10:11 23 Sep 2015, t = 125.42 d)
40. Increased air+CO₂ gas flow to algal reactor to 30 mL min^-1 (16:20 23-Sep-2015, t = 125.68 d)
41. Increased air+CO₂ gas flow to algal reactor to 100 mL min^-1. DO in algal reactor near zero, likely because of carry over of glucose and acetate from digestor that was previously added (notes 36 and 37 above). (21:55 25-Sep-2015, t = 127.91 d)
42. Exchange flow between algal and methanogenic reactors temporarily stopped (14:33 26-Sep-2015, t = 128.62 d)
43. Returned algal reactor gas flow rate to 30 mL min^-1 (9:46 30-Sep-2015, t = 132.41 d)
44. Returning to 100 mL min^-1 in algal reactor due to continued high respiration rate (12:19 30-Sep-2015, t = 132.51 d)
45. Returned algal reactor gas flow rate to 30 mL min^-1 since system is nearing net autotrophy (9:30 6-Oct-2015, t = 138.40 d)
46. Added hardware and modified MonitorA2M program for computer control of exchange pump. Also added two point calibration for CO₂ analyzer to improve CO₂ readings in digestor. This resulting in  format change for the Exp1.dat file (new column) (17:20 6-Oct-2015, t = 138.72 d)
47. Impeller direction reversed in Algal reactor, but speed still at 60 RPM (9:20 9-Oct-2015, t = 141.39 d)
48. Calculations indicate that all of the added glucose and acetate have been consumed (see Mathematica file below), and there has been a turn down in CH₄ production.  Consequently, the algal and methanogenic reactors have been recoupled at a flow rate of 0.4 L d^-1, or a dilution rate of 0.1 d^-1 (10:20 18-Oct-2015, t = 150.43 d).
49. Reduced exchange flow rate to 0.05 d^-1 (0.20 L d^-1) to reduce low DO in algal reactor (9:39 23-Oct-2015, t = 155.40 d).
50. Reduced exchange flow rate to 0.01 d^-1 (0.04 L d^-1) to reduce low DO in algal reactor (21:32 27-Oct-2015, t = 159.90 d).
51. Increased air+CO₂ gas flow to algal reactor to 100 mL min^-1. DO in algal reactor near zero (10:25 29-Oct-2015, t = 161.43 d).
52. Flow between reactors stopped (21:38 7-Nov-2015, t = 170.90 d).
53. Several changes made (11:35 10-Nov-2015, t = 173.45 d)
1. Decreased Air+CO₂ gas flow to algal reactor to 30 mL min^-1
2. Changed direction of agitation in algal reactor
3. Added 10 mL of 4 M K₂HPO₄ to increase pH to 7.0
54. Returned Air+CO₂ gas flow to algal reactor to 100 mL min^-1 (21:35 10-Nov-2015, t = 173.90 d)
55. Reduced Air+CO₂ gas flow to algal reactor to 30 mL min^-1 due to low tank pressure (11:09 12-Nov-2015, t = 175.46 d)
56. Returned Air+CO₂ gas flow to algal reactor to 100 mL min^-1 with new air tank (10:51 13-Nov-2015, t = 176.45 d)
57. Reduced Air+CO₂ gas flow to algal reactor to 30 mL min^-1 due to lower metabolic rates (16:00 4-Dec-2015, t = 197.67 d)
58. 500 mL of TAP media was added to both reactors (12:30 9-Dec-2015, t = 202.52 d)
59. 4.5 g of FeCl₃ as added to digestor to simulate methanogenesis (10:58 11-Dec-2015, t = 204.46 d)
60. Reduced N₂ gas flow to digestor from 30 to 5 mL min^-1 to see if CH₄ production can be detected (16:51 15-Dec-2015, t = 208.70 d)
61. Changed A2M monitoring program to increase input gas flow to reactors during gas sampling to prevent intrusion of air if nominal flow rate is low (below 30 mL/min) (15:27 23-Dec-2015, t = 208.70 d)
62. New air tank on line and gas analyzers calibrated (17:22 15-Jan-2016, t = 239.7 d)
63. Exchange flow between digestor and algal reactor reestablished at a dilution rate of 0.05 d^-1 or 0.2 L d^-1 (10:44 19-Jan-2016, t = 243.45 d)
64. Flow between reactors turned off (12:17 29-Jan-2016, t = 253.51)
65. In-line UV sterilizers installed on both flows connecting the reactors, flow between the reactors returned to 0.05 d^-1 or 0.2 L d^-1 and approximately 600 mL of fresh medium was added to the system (18:41 22-Feb-2016, t = 277.78 d)
66. Reactor exchange flow rate increased to 0.4 L d^-1, or 0.1 d^-1 (10:23 3-Mar-2016, t = 287.43 d)
67. New air tank on-line and calibrated gas analyzers (10:33 5-Apr-206, t = 320.44 d)
68. Increase exchange flow between reactors to to 0.8 L d^-1, or 0.2 d^-1 (11:45 5-Apr-2016, t = 320.49 d)
69. Several modifications were made (11:52 29 Apr 2016, t = 344.49 d)
• Added 15 mL of 4 M K₂HPO₄ to increase pH in Algal reactor
• Added 60 mL of water from freshwater pond to increase biodiversity
• Installed a closed loop circulating pump in flow from Algal to digestor to facilitate cell disruption (picture here).
• Discovered flow between reactors had stopped because of algae biomass plugging tubing due to low flow in UV irradiators.  Have removed UV irradiators from system for now.
70. Increased exchange flow between reactors to 1.0 L d^-1 or 0.25 d^-1 (10:31 3-May-2016, t = 348.44 d)
71. Increased exchange flow between reactors to 2.0 L d^-1 or 0.5 d^-1 (15:21 6-May-2016, t = 351.64 d)
72. Increased exchange flow between reactors to 4.0 L d^-1 or 1.0 d^-1 (10:50 9-May-2016, t = 354.45 d)
73. Added 7 mL of 4 M K₂HPO₄ to increase pH in Algal reactor (10:11 12-May-2016, t = 357.42 d)
74. Added 30 mL of 4 M K₂HPO₄ to increase pH in Algal reactor (18:25 18-May-2016, t = 363.77 d)
75. Increased aggitation in digestor from 10 to 30 RPM (9:44 19-May-2016, t = 364.41 d)
76. Added 90 mL of 4 M K₂HPO₄ to increase pH in Algal reactor (9:38 31-May-2016, t = 376.40 d)
77. At approximatly 2:00 3-Jun-2016 (379.1 d), the pressure in the N₂ gas cylinder went to 0 psig.  This was not corrected until 9:52 3-Jun-2016 (379.41 d).  The lack of N₂ gas flow allowed some air to enter the digestor and corrupted the methane analyzer high calibration. 
78. Nitrogen tanked emptied approximately 22:48 26-Jun-2016 (t = 402.94 d) and was not corrected until 10:20 27-Jun-2016 (t = 403.43 d).
79. New air tank on-line and gas instruments calibrated (11:15 28-Jun-2016, t = 404.47 d)
80. Two UV sterilizers were placed within cell disruptor loop feeding algae to digestor (see here).  Loop volume approximately 600 mL (18:00 30-Jun-2016, t = 406.75 d)
81. Increased exchange flow between reactors to 6.0 L d^-1 or 1.5 d^-1 (14:57 15-Jul-2016, t = 421.62 d)
82. Increased exchange flow between reactors to 8.0 L d^-1 or 2.0 d^-1 (9:51 28-Jul-2016, t = 434.41 d)
83. Returned flow between reactors to 6.0 L d^-1 or 1.5 d^-1.  Dilution rate of digestor at 8 L d^-1 is 0.44 d^-1, which is around maximum growth rate of methanogenes (see here), so washout may be occuring (14:57 30-Jul-2016, t = 436.38 d).
84. Reduced flow between reactors to 4.0 L d^-1 or 1.0 d^-1 (14:08 18-Aug-2016, t = 455.59 d).  
85. Replaced air tank and calibrated gas analyzers (13:43 16-Sep-2016, t = 484.57 d).
86. Reduced flow between reactors to 2.0 L d^-1 or 0.5 d^-1 (16:54 19-Sep-2016, t = 487.70 d).
87. Failure of UV circulation loop resulted in a loss of approximately 4 L of medium from the algal reactor some time around 21-Sep-2016, which was not noticed until 8:19 22-Sep-2016, t = 490.35 d.  UV loop was repaired and 4 L of fresh TAP media was added to the system and exchange pump returned to  previous flow rate of 2.0 L d^-1 or 0.5 d^-1 (17:16 22-Sep-2016, t = 490.72 d).
88. An additional 500 mL of TAP medium was added to algal reactor (9:50 23-Sep-2016, t = 491.41 d).
89. Increase flow between reactors to 6.0 L d^-1 or 1.5 d^-1(15:49 21-Oct-2016, t = 519.66 d)
90. Gas flow to reactors turned off for ~1.5 hr while reactors were moved in growth chamber (15:00 15-Nov-2016, t = 544.62 d)
91. Flow between reactors was blocked for about two days (starting 13:00 15-Nov-2016) until now. Flow has been reestablished (10:30 17-Nov-2016, t =  546.44 d)
92. Modifications to this experiment are still occuring, but are now documented in the file Exp1.info
93. Monitoring program temporarily stopped while the methane analyzer is sent out for repairs. (16:30 9-May-2017, t = 719.69 d)

Data Files:

Experimental Equipment:

Software

• Version 1.10: MonitorA2M_V1.10.f90
• Version 1.12: MonitorA2M_V1.12.f90: Adds computer control of exchange pump and two point CO₂ calibration.
• Version 1.20: MonitorA2M_V1.20.f90: Gas flow to reactors increased during sampling if minimum flow is not met.

• CO2integration.nb (PDF version) Notebook to look at methane cumulative production.

Photos