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 ....

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 labor....

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