Aerobic Biodegradation Testing of Materials Using a Natural Marine Seawater Inoculum and Closed Loop Respirometer

Our research quantifies the aerobic biodegradation rate and degree of natural materials in the marine environment using natural seawater inoculum for data comparison. Our current experimental challenges include limited testing capacity and a lack of infrastructure to meet growing demand for biodegradation testing, while ensuring uniform and transparent methods across laboratories. To begin, collect 10 to 20 liters of sea water into a 20-liter acid-leached carboy using a large Niskin bottle.

Transport the filled carboy back to the laboratory. Transfer one liter of whole sea water from the carboy into an acid-leached, one-liter amber HDPE bottle for particulate carbon and nitrogen analysis. Filter it through a 0.2-micrometer, low-nitrogen, cellulose acetate membrane.

Now, fill a one, two, or four-liter volumetric flask halfway with seawater. If the seawater has high-particulate organic matter, sieve it through a 20-micrometer mesh to remove large heterogeneous particles. Then collect 60 milliliters of filtrate into an acid-leached polyethylene bottle for dissolved inorganic and organic nitrogen and phosphorus analysis.

Loosely cover the opening of the carboy to allow aerobic conditions. Store in the dark at 30 degrees Celsius for up to seven days. Next, place 10 to 15 grams of the experimental substrate in a 50-milliliter, stainless-steel milling jar.

With a 20-millimeter steel ball. Submerge the milling jar in liquid nitrogen for 15 minutes to embrittle it. Secure the milling jar in the ball mill attachment.

Then set the instrument to 30 hertz and two minutes, 30 seconds, and press start. Perform milling four times with 30 seconds of cooling in liquid nitrogen between each cycle. Mill the substrate to a uniform particle size between 0.10 and 0.25 millimeters prior to the start of the test.

Using a precision balance with 0.001-gram sensitivity, weigh out 0.5 grams per liter of ammonium chloride and 0.1 grams per liter of monobasic potassium phosphate. Add the inorganic nutrients to the flask. Bring the volume to mark using seawater.

Then drop in a stir bar and stir on a stir plate until all salts dissolve. On an analytical balance, weigh out 20 milligrams of the experimental or control substrate. Record the full readout weight and set the vial aside.

To set up the reactor vessels, use an acid-leached, 75-milliliter volumetric pipette with a motorized pipette controller and aspirate the sea water to dispense 75 milliliters of the nutrient-supplemented sea water into each reactor vessel. Remove approximately three milliliters of sea water from each vessel. Then place it on a clean surface next to the vessel to rinse the milled substrate from the teared vial.

Now transfer the milled substrate into the corresponding reactor vessel from the teared vial. Place an O-ring on the reactor vessel rim. Then place a two-port lid over the O-ring and secure with a screw cap.

Connect each reactor vessel to its matching gas or condensation lines. Firmly insert the gas lines into the appropriate inlet and outlet ports on the lid. Turn on the shaker platform to 0.05G in continuous mode.

Visually check that all vessels are secure, then close the incubator door. On the instrument software, navigate to Experiment and click Setup. Enter the start and end channel numbers from the dropdown menus corresponding to occupied channels.

Set the refresh threshold to 0.50. Refresh interval to NA and refresh window to Auto. Then check the box for purge sensors under Miscellaneous Setup.

Ensure that the Auto Volume Measurement, Oxygen Consumption Positive and Enable Open Flow Mode are not selected. Set the sample interval to 8.00. Then set the gas and time units as required.

If using a primary temperature probe for incubation temperature correction, ensure Manually Enter Chamber Temps is not selected. Check the box for Venting Mode and ensure Drain Mode is not selected. Now, select Chamber Setup and enter the descriptive channel labels.

If a leak is indicated, then ensure the O-ring is clean, seated and free of particles or fibers. Tighten the screw cap. Inspect the tubing connections at inlet and outlet.

Trim worn or deformed tubing ends with a sharp blade to produce clean cuts. From the menu, select Tools, then click on Service Menu, and confirm that the primary temperature probe measures 30 degrees Celsius with two degree variations and is stable. Click Run to begin the experiment.

Save the experiment file under a descriptive name, including the date, and record the file name on the data sheet. Cumulative carbon dioxide production over 84 days confirmed expected performance. Over 84 days, cellulose showed rapid carbon dioxide production after a short lag, and reached about 700 micromoles.

PHA showed a slower initial rate over three days. And the sea water control remained low at 150 micromoles. Individual negative control replicates showed low variability, with all curves remaining close together.

Cellulose positive control replicates showed consistently low variability, confirming stable performance across all four replicates. In the negative result dataset, replicate five exhibited an anomalous spike in carbon dioxide production rate, likely due to labile organic matter, but its cumulative production began to plateau at 100 micromoles. Replicates one in six had lower cumulative carbon dioxide production compared to others, suggesting potential technical issues.

The standard error of average cumulative carbon dioxide production in the negative control was 2.5%indicating low variability among replicates. The standard error for the cellulose positive control was 0.83%further confirming high reproducibility of the biodegradation assay. PHA has high mineralization close to the positive control with standard error comparable to the control.

This protocol addresses the gap in standardized accessible methods for screening the biodegradability of materials in the marine environment for industry, academia and regulatory bodies. The protocol provides a faster standardized way to directly measure this biodegradation, with shorter test duration in simultaneous sample evaluation. The protocol enables high throughput standardized testing for material and laboratory comparability for machine learning and predictive modeling of material performance.