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

Summary

The protocol describes standard operating procedure for testing potentially biodegradable materials both synthetic and natural under aerobic conditions using a natural seawater inoculum and automated closed loop respirometry system.

Abstract

Proper assessment of biodegradable materials depends on the wide adoption of standard test methods. Although such methods exist, testing capacity remains limited, constrained by cost and the start-up time to establish new laboratories. This modified screening protocol is based on the ASTM International D6691-24a, Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in the Marine Environment by a Defined Microbial Consortium or Natural Sea Water Inoculum. It assesses the biodegradation of materials in a marine environment, comparing results to positive and negative controls, and uses a natural seawater inoculum supplemented with ammonium and phosphate to prevent nutrient limitation. Materials are exposed to this nutrient-enriched seawater and incubated at 30 °C. A Micro-Oxymax respirometer measures the production of biogas (carbon dioxide, CO₂) over time. The degree of mineralization (biodegradation) is determined by calculating the proportion of material-derived carbon converted to biogas-carbon. The percent CO₂ production, expressed as a fraction of the measured or theoretical carbon content, is reported as a function of time. The closed-loop respirometry system accommodates a range of reactor vessels, prepared in triplicate for each material, a negative control (seawater inoculum only), and a positive control (thin-layer chromatography cellulose). This method is important for evaluating materials in the marine environment and addresses global pollution prevention.

Introduction

Plastic waste poses a threat to marine ecosystems and human health¹. The growing concern over plastic pollution, along with marine debris from any materials in the ocean, underscores the need for sustainable solutions, such as improved end-of-life waste management and biodegradable materials that readily biodegrade in the marine environment. Unlike traditional synthetic plastics, which persist for decades to centuries and generate microplastics that accumulate in food webs^2,3,4, biodegradable materials, organic components of carbon, may be metabolized to CO₂ and methane (CH₄), or assimilated into microbial biomass⁵. Understanding biodegradation of materials in seawater is therefore key for quantifying and mitigating the environmental impacts of marine debris.

Biodegradation in the marine environment is controlled by the interaction of spatiotemporal variables, including light and nutrient availability, pressure, temperature, microbial species composition, and physicochemical nature and structure of the polymer, which all determine the rate, degree, and mechanism of degradation⁶. These variables present a significant challenge for laboratory-based biodegradation studies simulating marine environments. This standard operating procedure (SOP) uses natural seawater as the inoculum, capturing the in situ microbial community present, and records relevant variables including seawater dissolved nutrients, particulate carbon and nitrogen, chlorophyll, salinity, pH, and temperature. This controlled laboratory-based approach optimizes conditions for promoting mineralization and allows for direct biodegradation measurements that are not feasible in situ. The ASTM International D6691-24a, Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in the Marine Environment by a Defined Microbial Consortium or Natural Sea Water Inoculum (hereafter ASTM D6691), and this SOP were developed as a rapid, reliable screening tool for assessing the biodegradability of materials^7,8,9,10.

Aligned with ASTM D6691, a screening test for inherent polymer biodegradability, this protocol evaluates biodegradation of materials under optimal conditions, 30 °C, maximum surface area from milled sample, and excess inorganic nutrients (nitrogen, 130 mg/L; phosphorus, 23 mg/L)⁹, to promote mineralization and prevent nutrient limitations. While screening tests offer valuable initial insights, further testing, such as coastal and deep-sea moorings, which measure weight loss as a function of time, might be necessary for a comprehensive evaluation under natural marine conditions. Since these coastal and deep-sea mooring tests estimate biodegradation based on weight loss as a function of time^10,11, researchers should first perform this screening test, which directly measures microbial metabolism, before going to an in situ coastal and deep-sea mooring experiment, which is also more expensive.

The ASTM D6691 standard test method outlines procedures for evaluating the aerobic biodegradation of non-floating plastic materials in marine environments. This standard, along with specification standards, such as ASTM D7081 (Standard Specification for Non-Floating Biodegradable Plastics in the Marine Environment; focusing on biodegradability in an aqueous medium, currently withdrawn from ASTM and being reballoted), provides a framework for certifying experimental polymers as marine biodegradable^9,12. In general, a specification is a consensus standard from ASTM International that sets requirements that a material, product, or system must meet. It ensures that materials designed to biodegrade in marine environments do so within acceptable timeframes and do not release potentially toxic additives, such as dyes or plasticizers.

Building on ASTM D6691, this protocol quantifies the rate and degree of aerobic biodegradation of materials (experimental substrate) in natural seawater and expands on positive material controls (i.e., cellulose, Kraft paper, chitin/chitosan), historically shown to be biodegradable in the marine environment^7,9,10. The goal of this method is to screen materials under optimized conditions to see if the materials biodegrade in the marine environment. The inoculum leverages the natural microbial community present in the seawater, collected following specific guidelines outlined in ASTM. These guidelines emphasize using seawater with a salinity of approximately 32, collected from a location free from pollutants like sewage, chemical dumping, or oil spills. The test uses an automated closed-loop respirometer with 250 mL reactor vessels maintained at 30 °C, each containing 75 mL of seawater and ~20 mg of the experimental or control substrate⁸. Mineralization (biodegradation) is expressed as the percentage of net carbon biogas (CO₂-C) produced relative to the initial carbon mass added. This method does not account for carbon assimilation into microbial biomass.

Protocol

1. Collection and characterization of seawater

1. Identify a seawater collection site unaffected by wastewater, chemicals, oil slicks, or strong freshwater influence.
2. At the collection site, record source (location, latitude and longitude, collection depth, total depth, and temperature), date of sample collection, and observed water conditions.
3. Collect and combine seawater samples from multiple depths, site locations, and sampling events to promote a diverse microbial community composition.
4. In a 20 L acid leached carboy, collect 10 L to 20 L of seawater using a large Niskin bottle, and transport back to the laboratory.
5. From the carboy, transfer 1 L of whole seawater subsample into an acid-leached 1 L amber high-density polyethylene (HDPE) bottle for laboratory analysis of particulate carbon and nitrogen for initial seawater conditions. From the 1 L sample, filter (0.2 µm; low nitrogen cellulose acetate) and collect a 60 mL subsample in an acid-leached polyethylene bottle for analysis of dissolved inorganic and organic nitrogen (N) and phosphorus (P) constituents.
6. Label subsamples with project name, sample ID, and date. Measure NH₄⁺ concentrations using the indophenol method (4500-NH3)^13,14 and PO₄^3- levels using the single solution method (4500-P E)^13,15. Quantify particulate nitrogen and carbon using the elemental analysis method¹⁶. Measure chlorophyll-a concentrations following the protocols outlined by Parsons et al.¹⁷(10150 A, 10150 C)¹³.
   NOTE: Nutrient-related water quality analyses are optional. This protocol assumes analyses are conducted by an outside laboratory.
7. Loosely cover the carboy opening to maintain aerobic conditions and store seawater at 30 °C in the dark for up to 7 days.
   NOTE: Seawater may also be aerated using an air pump and bubbler system.

2. Preparation of experimental substrate

1. Add 10-15 g of experimental substrate (i.e., natural polysaccharides, wood, fiberboard, polymers) that may be in the form of pellets, fragments, and formed pieces to a 50 mL stainless steel milling jar with a 20 mm steel ball. Mill the experimental substrate to a uniform particle size (0.10-0.25 mm) prior to the start of the test.
2. Submerge the milling jar in liquid nitrogen and embrittle for 15 min until the liquid nitrogen stops boiling.
   CAUTION: Liquid nitrogen is a cryogenic liquid that may cause burns, injury, or frostbite. Read and follow all Safety Data Sheets before use. Wear appropriate personal protective equipment.
3. Secure the milling jar in the ball mill attachment and set the instrument parameters to 30 Hz and 2:30 min, and press Start. Repeat 4x with 30 s cooling in liquid nitrogen in between trials. If the material is a polymer with a low glass transition temperature, perform additional milling cycles as necessary to produce a uniform particle size distribution.
4. Verify size uniformity and characterize particle size distribution by sieve analysis as described in ASTM D1921-96¹⁸. Assemble standard sieves spanning the expected particle size range, with the smallest mesh size on the bottom. Place the milled sample on the top sieve and secure the sieve stack in a mechanical shaker. Agitate until particles pass through the mesh. Collect and weigh the retained material from each sieve to calculate particle size distribution. Use the same set of sieves across treatments to maintain comparability. Record the distribution on the data sheet.
5. Determine carbon content per subsample dry weight of the milled experimental substrate by elemental analysis¹⁶.
   NOTE: This protocol assumes elemental analysis is conducted by an outside laboratory.

3. Preparation of the additional nutrients for seawater

1. Fill a 2 L or 4 L volumetric flask halfway with seawater. The total volume needed depends on the number of reactor vessels to be used in the experiment. Each reactor vessel will contain 75 mL of seawater. If the seawater source contains high concentrations of particulate organic matter, sieve the seawater through a 20 µm mesh to eliminate heterogeneous contamination by large particulates.
2. Using a precision balance with sensitivity to 0.001 g, tare a weigh pan and weigh out 0.5 g/L ammonium chloride (NH₄Cl) and then 0.1 g/L potassium phosphate monobasic (KH₂PO₄) based on 2 L or 4 L of seawater.
   CAUTION: Ammonium chloride can cause eye damage or irritation. Read and follow all Safety Data Sheets before use and wear appropriate personal protective equipment.
   NOTE: Dispose of ammonium chloride and potassium phosphate monobasic waste in dedicated hazardous waste containers per institutional procedures and local regulations. Small volumes of dilute (<1%) NH₄Cl and KH₂PO₄ solutions may be eligible for drain disposal after dilution with copious water, if permitted by the institution and local wastewater authority.
3. Add the inorganic nutrients to the 2 L or 4 L volumetric flask, bring to volume with seawater, add a stir bar, and stir on a stir plate until all salts dissolves. The solution should be clear with no visible particulates.
4. Subsample 20-30 mL of the nutrient-supplemented seawater for pH and salinity measurement. Filter an additional 60 mL through a 0.2 µm low-nitrogen cellulose acetate filter into an acid-leached polyethylene bottle for dissolved inorganic N and P analysis following the standard methods in step 1.5.
5. Measure pH and salinity on a benchtop meter following the manufacturer's instructions. A pH decrease of approximately one unit is expected for full-strength seawater due to the acidity of the nutrient supplement.

4. Preparation for reactor vessel incubation

1. Acid leach all glassware, including 250 mL reactor vessels for the samples and 75 mL volumetric pipette in 10% hydrochloric acid (HCl) for 24 h and rinse 3x with deionized water.
   CAUTION: HCl may cause severe burns and eye damage. Read and follow all Safety Data Sheets before use. Wear appropriate personal protective equipment.
   NOTE: 10% HCl waste can be neutralized with sodium bicarbonate to form a neutral solution prior to disposal. Perform neutralization in accordance with institutional hazardous waste procedures and applicable local regulations.
2. Autoclave the acid-leached 250 mL empty reactor vessels, lids with ports, silicone seal O-rings, and screw caps in an autoclave at 121 °C for 15 min prior to the day of the experiment.
3. Label sample vials with the channel number/ID corresponding to vessels receiving sample or control materials.
4. Using the analytical balance, tare a vial (with cap) and weigh out 20 mg (± 0.1 mg) of the experimental or control, and record the full readout weight, and set aside. Repeat for all experimental and control replicates.
   NOTE: Be sure weights are accurate. It is recommended to weigh all substrates on the same day and by the same person.
5. Turn on the incubator the day prior to adding vessels and set the temperature to 30 °C. Turn on the respirometer at least 2 h prior to running system diagnostics and sensor calibration based on manufacturer instructions.
   NOTE: Reactor vessel preparation can proceed once the incubator temperature reaches and maintains 30 ± 2 °C.
6. Prepare the data sheet by recording sample IDs/replicates and the corresponding channel number. Organize the reactor vessels and cap assemblies (lid with ports/O-ring) in order of channel number/ID (make sure each vessel is clearly labeled). Check the O-rings and lids/ports to ensure they are clean, dry, and intact.

5. Setting up the reactor vessels

1. Use an acid leached 75 mL volumetric pipet and motorized pipet controller to dispense 75 mL of nutrient supplemented seawater into each reactor vessel.
2. Remove one disposable pipette worth of seawater (~3 mL) from each vessel and set it down on a clean surface next to it. This seawater will be used to rinse the milled experimental and control substrates from the tared vial (Figure 1).
3. Transfer the substrates into appropriate vessels.
4. Place an O-ring on the reactor vessel rim, then place a two-port lid directly on top of the O-ring. Secure the O-ring and lid in place with a screw cap.
5. Match the reactor vessel to its corresponding gas/condensation lines. Firmly insert the gas lines into the appropriate inlet/outlet ports in the vessel lid.
6. Turn on the shaker platform to 0.05 x g (continuous mode). Conduct a visual check to ensure all vessels are secure, then close the incubator door.
7. Record incubator temperature (readout and thermistor probe) and atmospheric pressure using a barometer.

Figure 1: Vessel setup and preparation. (A) Adding seawater, (B) adding the sample, and (C) adding the O-ring, metal lid, and screw cap. Please click here to view a larger version of this figure.

6. Starting the respirometry system

1. Launch the software on the connected computer. Verify that the system detects the hardware through the RS-232 connection. If the hardware is not found, a notification will indicate that the device is not connected, and the program must be restarted.
2. In the software main menu, select Tools > Diagnostics > Basic Operations. Click Save log file. Click Valves and Sensors. The software will indicate PASS or FAIL for each test.Proceed to two-point calibration of the CO₂ sensor only if both tests pass.

7. Calibrating the CO₂ sensor

1. Connect a soda lime column to the nitrogen port on the sample pump back panel to remove ambient CO₂ for zero calibration. Ensure soda lime appears chalky white and not discolored (e.g., pink or purple).
2. Select Tools > Calibration. Click Start Calibration in the lower right corner. When prompted, adjust the sample flow to 0.5 L/min using the front panel sample pump control. Click OK.
3. When the timer reaches zero, press the Up and Down offset buttons on the CO₂ sensor to adjust the reading on the screen to 0.000 (or as close as possible). Values will turn green when within the acceptable range. Click Next Gas.
4. When prompted, connect the calibration gas cylinder to the calibration port on the sample pump back panel. Adjust the calibration gas outlet pressure to 5 PSIG. Ensure excess gas flows through the T fitting whereby the user can hear and feel the gas near the fitting, before clicking OK.
5. When the timer reaches zero, press the Up and Down span buttons on the CO₂ sensor to adjust the reading on the screen to 2.700 (or as close as possible). Values will turn green when within the acceptable range. Click Next Gas to complete the calibration.
   NOTE: Span gas concentrations are determined by the certified primary standard gas mixture, as documented in the supplier's certificate of analysis.

8. Experiment setup in software

1. Select Experiment > Setup. Enter start and end channel numbers from the drop-down menus based on occupied channels.
   1. Set Refresh Threshold (%) to 0.50, Refresh interval to N.A., and Refresh Window (sec) to Auto.
   2. Under Misc Setup, check the box for Purge Sensors; ensure Auto Volume Measurement, O₂ Consumption Positive, and Enable Open Flow Mode are not selected.
   3. Set Sample Interval (Hours) to 8.00 and Experiment Duration (Hours) to N.A. Set gas and time units as needed.
   4. If using the 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.
   5. Ensure Anaerobic Venting Mode is not selected.
2. Select Chamber Setup. Enter descriptive channel labels (e.g., NegCtrl_1, PosCtrl_2, Test_PHA_3).
3. Click Utilities. Click Cells in the EN Column for occupied channels only. Click Restriction. The software will indicate PASS or FAIL for each channel. Proceed only if all restriction tests pass.
4. Click Volume to measure headspace volumes.
   NOTE: Headspace volume equals the volume of the air within the sample reactor vessel and closed-loop tubing. Confirm volumes are reasonable for vessel size and tubing length.
5. Click Leakage. The software will indicate PASS or FAIL for each channel based on the headspace volume. If a channel fails, perform the following steps.
   1. Ensure the O-ring is clean and free from particles and fibers and properly seated between the vessel rim and the metal lid.
   2. Tighten screw cap. Check the connection and tubing at the IN/OUT ports and fittings. Trim worn or deformed tubing ends with a sharp blade to ensure a clean, straight cut.
   3. If the channel continues to fail, replace the O-ring with a new, compatible one specified by the manufacturer.
6. Click Volume to remeasure headspace volumes. Click Quit. Ensure the Volume column of the Chamber Setup tab is populated for all occupied channels.
7. In the main menu, select Tools > Service Menu. Confirm that the primary temperature probe, measuring the incubator temperature, is at 30 °C ± 2 °C and stable.
8. Click Run. Save the file using a descriptive name that includes the date (e.g., 030325PositiveControlValidation). Record the filename on the data sheet.
9. Select Experiment > View > Graphing to view real-time plots. Biogas production appears as positive CO₂ production rates. Negative controls may fluctuate about zero; however, their average rate should be zero or positive.
10. If the system includes condensing air driers, maintain a daily log of visible condensation in tubing. Gently agitate tubing to disrupt blockages and allow drainage, if necessary.
11. Update data files (ASCII Text Files) at the end of each sampling interval. Data, including temperature, gas % (gas composition), rate, and accumulation, can be downloaded to various other software packages during the experiment or at the end.
    1. To open .dat files in a spreadsheet, launch Excel and select File > Open. Navigate to the folder containing the .dat file. In the file type drop-down menu, select All Files and choose the desired file.
    2. When prompted by the Text Import Wizard, select Delimited as the file type and click Next.
    3. Under Delimiters, check the box for Comma. Ensure no other delimiters are selected. The preview window should display data separated into columns. Click Finish to load the data into the spreadsheet.
    4. Save the file as an .xlsx or .csv format for further analysis and archiving.
12. Periodically, analyze data to ensure treatment replicates are consistent. Standard error of treatment replicates should be less than 5% of the mean cumulative CO₂ production. Repeat tests failing this threshold.
13. Click Stop when incubation is complete.
    1. Incubations can last 10-90 days. If test materials are highly biodegradable, terminate the incubation when cumulative CO₂ production plateaus or extend it if no degradation is observed after 90 days.
    2. Conclude experiments when no net gas production is observed for at least 5 days in both control and test vessels. No net production is defined as less than 1% change in percent biodegradation over 5 days.
14. Shut down the system or prepare for the next experiment.

9. Breaking down the experiment

1. Record incubator temperature (thermistor probe and incubator readout). Disconnect the tubing from the reactor vessels from the respirometer and weigh each sample reactor vessel using a precision balance. Record weights to determine if any weight/water loss occurred over the incubation period.
2. Subsample 20-30 mL of seawater from each reactor vessel to measure pH and salinity and compare these values to those recorded in step 3.4 to verify stability during incubation. Filter an additional 60 mL through a 0.2 µm low nitrogen cellulose acetate filter into an acid-leached polyethylene bottle for dissolved inorganic N and P analysis following standard methods in Step 1.5.
   NOTE: Residual seawater samples can be filtered to remove remaining plastics, diluted to reduce nutrient concentrations, and disposed of by drain discharge in accordance with institutional and local regulations.
3. Rinse vessels, O-rings, metal lids, and screw caps with tap water. Place glassware in a 10% HCl acid bath for at least 24 h. Autoclave and store covered in aluminum foil for the next incubation

10. Calculating percent biodegradation

1. Calculate the total carbon in the experimental/control substrate as follows:
   
   where % C (carbon content) is determined through elemental analysis.
   NOTE: Total carbon in the experimental and control substrates can undergo the following biochemical transformation during the aerobic incubation:
   C + O₂ → CO₂
   Each mmole (12 mg) of organic carbon from the experimental/control substrate can be converted into 1 mmol of gaseous CO₂. One mmole of gas produced occupies 22.4 mL at standard temperature and pressure (STP).
2. Calculate the percent biodegradation as follows:
   
   where C_g = amount of gaseous carbon produced from the experimental/control substrate and negative control (seawater inoculum only), mmoles, and C_i = amount of carbon in experimental substrate added, mmoles.
3. Calculate the standard error, s_e, of the percentage of biodegradation as follows:
   
   where n1 and n2 = number of replicate experimental/control substrate and negative control reactor vessels, respectively, and s = standard deviation of the total gaseous carbon produced.
4. Calculate the 95% confidence limits (CL) as follows:
   
   where t = t-distribution value for 95% probability with (n1 + n2 − 2) degrees of freedom, thus n = 3 + 3 − 2 = 4.

11. End of protocol

1. After completing calculations and data analysis, compile results into the final report or enter them into a shared database in accordance with project or institutional data management plans. Ensure all materials are disposed of according to institutional hazardous waste guidelines, and all equipment is cleaned and stored.

Results

The results from marine biodegradation tests are presented. Biodegradation was assessed for each sample by measuring the proportion of carbon converted to carbon dioxide. Cumulative, rate, and percent biodegradation plots typically exhibit a lag phase, followed by a growth phase, and eventually a plateau. The timing and magnitude of these phases depend on the sample material, experimental conditions, and the composition of the seawater inoculum.

Seawater collected year-round from a temperate e...

Discussion

Positive-control performance determines test validity. A minimum of 70% biodegradation of a readily marine biodegradable positive control substrate (e.g., analytical grade cellulose, Kraft paper, etc.), based on its theoretical maximum gas yield, is required. This benchmark verifies favorable conditions and sufficient microbial activity in the natural seawater during the incubation. Failure to meet this threshold indicates a potential issue with replicate preparation, seawater inoculum handling (i.e., microbial community...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm, 47 mm cellulose acetate filter	Geotech Geofilter	83100056	Filter seawater
100 mL Volumetric pipet	Thermo Fisher Scientific, Fisherbrand	13-650-2U	Class A; acid leached and rinsed with deionized water
100-600 mL Glass beakers	Thermo Fisher Scientific, Fisherbrand	50-194-5690	Class A; acid leached and rinsed with deionized water
20 mm milling balls	Retsch	53680062	Stainless steel. Used with milling jar for cryogenic milling of polymer
50 mL milling jars	Retsch	14620216	Stainless steel. Used with Mixer Mill 400 and 20 mm milling balls for cryogenic milling of polymer
75 mL Volumetric pipet	Thermo Fisher Scientific, Fisherbrand	13-650-2T	Class A; acid leached and rinsed with deionized water
Air pump and bubbler system	Tetra	77846	Aerate seawater during pre-incubation equilibration
Aluminum foil	Fisherbrand	01-213-100	Cover for autoclaving and clean storage
Ammonium chloride (NH4Cl)	Thermo Fisher Scientific, Fisher Chemical	A661-500	Seawater nutrient spike
Analytical balance	Mettler	AT201	To weigh out experimental and control substrates
Autoclave	Tuttnauer	MFI-TTN-3870EA	To sterilize vessels and lid assemblies
Base Micro-Oxymax 120VAC Single Channel	Columbus Instruments	0135-8000	Used to store positive controls and polymer
Calibration gas	Airgas	X03NI92P3000001	Primary Standard Gas Mixtures; 2.7% Carbon dioxide, 4.5% Methane, balance Nitrogen
Computer Monitor	Dell	210-BMKK	Dell Pro 24 Plus USB-C Hub Monitor. Used with computer to run Micro-Oxymax software
Cryogenic Gloves	Thermo Fisher Scientific, Tempshield, Cryo Gloves	11-394-306	Used to handle liquid nitrogen
Dial Thermometer	Thermo Fisher Scientific, H-B Instrument Durac	13-201-433	Calibrated thermometer for seawater temperature at time of collection
Disposable pipettes	Thermo Fisher Scientific, Fisherbrand	13-711-7M	Rinse sides of vessel to free up material
ENC52 Temperature and Light Controlled Enclosure	Columbus Instruments	4740-4054	Maintain temperature and light conditions
Flask Tongs	Thermo Fisher Scientific, Fisherbrand	S07387	Used to handle milling jars in liquid nitrogen
Flasks, 250 mL	DWK Life Sciences	14395-250	Sample reactor vessels
Glass Shell Vials with Plug Style Closures	Thermo Fisher Scientific, Fisherbrand	03-339-26D	Sample storage
H22PX Handheld Depthfinder	West Marine, Norcross Marine	6849368	Determine seawater collection site depth
Handheld GPS 73	Garmin	10-01504-00	Used to determine seawater collection site location
HDPE Pan	Thermo Scientific, Nalgene	13-359-26	Used to acid leach glassware  in 10% HCl solution
Hydrochloric Acid (acidic water solution)	Thermo Fisher Scientific, Fisher Chemical	A144-212	Certified ACS Plus, 36.5 to 38.0%. CO2 sensor check and acid leaching glassware
Laminar flow hood	Air Science	FLOW-48-A	Minimize airborne contaminants during sample preperation
Large Niskin; Beta Plus Bottle Only - 8.2L Horizontal, PVC	Science First	3-1980-H65	For seawater collection from pier or boat
Liquid Nitrogen Pan	Thermo Fisher Scientific, Bel Art Magic Touch 2	11-999-063	Hold Liquid Nitrogen
Liquid Nitrogen Transfer Vessel, 10 L	Thermo Scientific	TY509X2	Store and dispense liquid nitrogen for cryomilling
Liquid Nitrogen, Industrial Grade, 22 PSI	Airgas	NI 180LT22	Used to embrittle and mill polymers
Mechanical Sieve Shaker	Humbolt, Grainger	5DPL6	Sieve analysis for particle size distribution
Micro-Oxymax	Columbus Instrumentts	7395-113-D64	Silicon sealed o-ring, metal port w/ 3 fittings, and GL45 screw cap
Micro-Oxymax 10 Channel Expansion Interface	Columbus Instruments	0135-8101	Respirometry System Component
Micro-Oxymax Respirometer (3% CO2 sensor)	Columbus Instruments	0135-8403	Non-dispersive infrared
Mixer Mill 400	Retsch	207450001	Mixer Mill 400. Used for cryogenic milling of polymer
Motorized pipette controller	Eppendorf	4430000018	Transfer seawater to sample reactor vessels
Narrow-Mouth Amber HDPE Lab Quality Bottle, 1 L	Thermo Scientific, Nalgene	02-923-5	Discrete wholewater seawater sample
Nylon Mesh 20 Micron	ELKO Filtering Co.	41718	Used to sieve raw seawater to remove larger heterogeneous particulates
Open Air Orbital Shaker	Thermo Fisher Scientific	SK4000	Maintain orbital motion of reactor vessels during incubation
OptiPlex 5090 Small Form Factor Computer	Dell	C0SL03	Desktom computer used to run Micro-Oxymax software
pH/Cond Meter	Mettler Toledo, SevenExcellence S470-Basic	30046252	Benchtop pH/conductivity meter to measure initial and final seawater inoculum
Polyethylene bottle, 60 mL	Qorpak	PLC-03564	Discrete filtered seawater sample
Potassium phosphate monobasic (KH2(PO4))	Thermo Fisher Scientific, Fisher BioReagents	BP362-500	Seawater nutrient spike
Precision balance	Sartorius	Practum2102-1S	To weigh out tare and whole weight of vessels
Round Carboy, 20 L	Thermo Fisher Scientific	02961A	Acid-leached for seawater storage
Safety Goggles	Thermo Fisher Scientific, Fisherbrand	19-181-513
Scoopula	Thermo Fisher Scientific, Fisherbrand, Scoopula	01-257-567	Stainless steel lab scoopula
Sieves	Advantech, Grainger	40PR18-40PR55	Stainless steel sieves. Number of sieves and sizes will depend on the material being tested.
Sigmacell Cellulose, Type 101	Sigma-Aldrich	435236-250G	Powder, positive control substrate
Sodium Bicarbonate	Thermo Fisher Scientific, Fisher Chemical	S233-500	CO2 sensor check
Sodium sulfite, anhydrous, 98%	Thermo Fisher Scientific, Thermo Scientific Chemicals	AAA1793336	CO2 sensor check
Thermometer/thermistor probe	Fisherbrand	15-079-700	Verify incubator temperature
Traceable Precision Dial Barometer	Fisherbrand	14-648-51	Record atmospheric pressure at start of experiment
Victor Medium Duty Hydrogen, Methane Two Stage Regulator, CGA - 350	AirGas	VIC0781-3559	Calibration gas regulator
Volumetric flasks, 2 L	Thermo Fisher Scientific, Fisherbrand	FB4002000	Class A; acid leached and rinsed with deionized water. Used to prepare nutrient spiked seawater.
Weigh pans	Thermo Fisher Scientific	02-202-101	Polyethylene