Surface water samples for these experiments were collected from West Bay of the Neuse River Estuary, North Carolina USA (34°55'32.42" N, 76°21'54.25" W). The microcosms were 60 liters. The experiments were conducted in biological duplicates under three light treatment incubations: 12-hour light-dark cycle of photosynthetically active radiation (PAR), 12-hour light-dark cycle of UV-B radiation, or darkness. Samples were collected from the microcosms in duplicate every few days for over one month to examine how light and the resulting microbial activity altered the DOC pool over time.

For each 60-liter (L) microcosm, water was sampled daily to weekly at the University of North Carolina over the month-long (September 2021) experiment or two-month-long experiment (April 2022). Water was sampled from the microcosms using a 50-milliliter (mL) serological pipette and auto pipettor. The pointed end of the pipette was snapped off to provide a wide, sterile pipette mouth. The pipette was twice rinsed with Milli-Q water prior to sampling to limit potential carbon contamination and attached to MasterFlex tubing. A vacuum pump with a trap was used under gentle pressure to fill 500 mL of sample water into a 1-L borosilicate bottle.

To size fractionate the sample water, a filter tower (47-millimeter (mm), 5-micrometer (um) polycarbonate filter) was twice rinsed with c.a. 50 mL of sample water and discarded. Then, 250 mL of sample was gravity filtered through the tower to generate a less 5 um fraction. For the respiration incubation assay, biological oxygen demand bottles (BODs) were rinsed three times with c.a. 20 mL of sample water (unfiltered or less 5 um), then filled to the brim. Air bubbles were removed from the BOD by taping the sides of the bottle with the stopper for 1 minute, then quickly inserting the glass stopper. The filtering and filling process was done in a temperature-controlled room to reduce changes in sample and bottle temperature during handling. All bottles, filter towers, and tubing in contact with sample water were acid washed with 10% HCl and triple rinsed with Milli-Q water before each sampling event and rinsed with Milli-Q, then sample water between treatment replicates.

The BODs have a PreSens foil-membrane optode mounted on the inside of the bottle and were fitted with a holder to align an external fiber optic cable with the sensor spot (detailed in Cohn, et al. 2023). The BODs and one abiotic control BOD of Milli-Q were then placed in a water bath in the temperature-controlled room and covered to prevent light from entering the bath. The BODs were incubated for an average of two days, recording an oxygen concentration every 30 seconds, then exported from the PreSens Measurement 2 Studio for analysis (note, the incubation time can be greatly shortened, however drawdown rates were linear and the long incubation was allowed due to logistical constraints).

Instruments and Materials:

Hydrochloric acid (36% w/w, ThermoFisher) was diluted to 10% (v/v) with Milli-Q water for acid washing procedures.

Serological pipette, 50 mL, sterile, cotton plug removed (VWR)

1/4 inch Masterflex L/S Platinum-Cured Silicone Tubing

GAST vacuum pump (Model: DOA-P704-AA) used at <-5 in Hg

1-L borosilicate media bottle (VWR)

500-mL amber bottle-top filter holder (Nalgene)

5 micron polycarbonate filter (Millipore Sigma, 47 mm)

60-mL borosilicate biological oxygen demand bottles with glass stopper (VWR)

PreSens oxygen optode sensor spot (SP-PSt3-NAU)

PreSens polymer optical fiber (POF)

PreSens temperature/pressure sensor (Pt-100)

PreSens sensor meter (Oxy-4 SMA [G2] for all sample bottles, Oxy-1 SMA for the abiotic control BOD)

water bath (60-L igloo cooler) filled with DI water, covered in black bags to block light, an aquarium pump for water circulation, all placed in an environmental controlled room at in situ temperature