Scalable Step-by-Step Approach of Sustainable Bioplastic Production from Food Waste

Summary

This protocol presents a scalable method for converting food waste into biodegradable polyhydroxyalkanoate (PHA) bioplastics. It utilizes arrested anaerobic digestion for food waste pretreatment, halophilic microbial fermentation for PHA biosynthesis, and a chemical-free downstream process for PHA recovery.

Abstract

The global microplastic crisis, coupled with the growing challenges of food waste disposal, necessitates innovative solutions to address these environmental issues together. Polyhydroxyalkanoates (PHAs) are unique bioplastics that are fully biodegradable in all environments, including marine ecosystems, offering a sustainable alternative to petroleum-based plastics. At the same time, utilizing food waste as a feedstock for PHA production provides an effective strategy for mitigating the challenges of food waste disposal while producing high-value biodegradable plastics. This study provides a step-by-step protocol for producing PHA from food waste, emphasizing the critical care required to ensure high cellular PHA content and quality. The process begins with arrested anaerobic digestion, which converts food waste into microbially assimilable volatile fatty acids (VFA) by maintaining an anaerobic environment and optimizing parameters, such as solid retention time, to maximize VFA production, a key precursor for PHA synthesis. The VFA-rich digestate is then used to cultivate Haloflex mediterranei, a halophilic microorganism capable of accumulating PHA up to 66% ± 5% of its dry cell weight. The high salinity cultivation environment of H. mediterranei prevented culture contamination, ensuring optimal PHA production. Cell growth is monitored by measuring optical density to determine the ideal time for PHA harvesting. Cells are lysed using a chemical-free, water-based method leveraging an osmotic pressure gradient, achieving 93% ± 3% PHA recovery, followed by solvent-based PHA purification to obtain a PHA purity of 96% ± 2%. Each step is vital to ensure the production of high-quality, biodegradable plastics. This paper provides detailed methods for arrested anaerobic digestion, pure culture fermentation, chemical-free cell lysis, and solvent-based PHA purification, offering a scalable and sustainable approach for converting food waste into biodegradable bioplastics suitable for pilot- and full-scale applications.

Introduction

Plastic pollution and food waste disposal are growing environmental concerns, highlighting the need for innovative approaches that address both challenges through sustainable waste management and material development¹. Conventional petroleum-based plastics are major contributors to environmental pollution and microplastic contamination, persisting in landfills and aquatic ecosystems for centuries². Polyhydroxyalkanoates (PHAs), a class of microbial bioplastics, offer a fully biodegradable alternative with mechanical properties comparable to conventional plastics while ensuring complete degradation in natural environments....

Protocol

1. Food waste collection and preparation

1. Collect food waste from local restaurants, as shown in Figure 1A. Assess the waste obtained visually. Observations revealed that the waste primarily consisted of vegetable peels, discarded raw meat, and carbohydrate-rich components such as rice and noodles. The carbon-to-nitrogen (C: N) ratio may need to be measured as detailed in steps 5.1 and 5.2, and adjusted if the food waste is predominantly carbon-rich, with an optimal C: N ratio near 20:1²⁸.
2. Put 10 kg of food waste into a 5-gallon bucket and add 2.5 L of water. Connect the blende....

Results

Food waste characteristics and VFA production from aAD
An example of TS, VS, and C:N ratio in prepared food waste slurry is shown in Table 1. Due to the inherent variability in food waste composition across different sampling sources and time, fluctuations in TS, VS, and C:N ratio values are expected. While the variability is acceptable, regular TS, VS, and C:N ratio measurements are essential for process monitoring, microbial balance, and overall aAD system optimization

Discussion

VFA production from aAD
VFA plays a crucial role as key precursor for H. mediterranei in converting food waste into PHA. Therefore, maintaining a high VFA concentration during aAD is essential. Several strategies can be employed to enhance VFA production in aAD²⁵. One effective approach is SRT optimization, with an ideal duration of 8 days or less, which promotes the washout of methanogens while maximizing hydrolysis and acidogenesis, thereby preventing excessive VF.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Air	Airgas	AI UZ300	Used for GC operation
Air pump	Cloudray	Hailea ACO-009D
Ammonium chloride	Acros Organics	A0396560
Autoclave	STERIS	AMSCO Lab 250	Used in ATCC medium preparation
Balance	METTLER TOLEDO	ME204TE/00
Blender	VEVOR	IB750LV+BLD300+WB-1
Bucket	Lowe's	Model 50640	5-Gallon BPA-Free Plastic General Bucket
Calcium chloride dihydrate	Acros Organics	A0362932
Chloroform	Millipore Sigma	C2432
Desiccator			Any type that can be used in TS, VS, VSS measurement
Digestion tubes, screw cap with PTFE liner	Millipore Sigma	CLS982613
Digital Reactor Block	Hach Company	DRB200	Used in Hach VFA quantification methods
Digital stirrer	SCILOGEX	SCI550-Pro
Disc centrifuge	Alfa Laval Inc.	Clara 20
Erlenmeyer flasks	Amazon	B07V3JPQMM
Ethanol	Amazon	B09DGVL4ML
Freeze dryer	Labconco	10030133, 1027319, and 0109621	FreeZone 2.5 Liter Freeze Dry Systems
Fume hood	Kewaunee Scientific Corporation	Supreme Air LV
Furnace	Thermo Scientific	Model F30428C
Gas chromatography (GC)	Agilent	Model 8890
Haloferax mediterranei	ATCC	33500
Hydrogen	Airgas	HY UHP300	Used for GC operation
Knife			Any type used for cutting food waste
Lubricant oil	Fisher Scientific Company	NC9844626
Magnesium chloride hexahydrate	Fisher Scientific Company	BP214-500
Magnesium sulfate heptahydrate	Millipore Sigma	M1880
Methonal	Millipore Sigma	34860
Methyl benzoate	Millipore Sigma	93583	Used as internal standard in PHA quantification
Microplate reader	BioTek Synergy	Synergy H1
Nitrogen	Airgas	NI UHP300	Used for GC operation
Oven	Thermo Scientific	OMH750
Peristaltic pump	Simply Pumps Inc.	PM6000 AC	High-flow, self-priming pump with excellent dry-running capability, capable of handling high-particulate slurries and designed for 1/8-inch wall tubing.
pH meter	Fisher Scientific Company	AE150
PHA standard	Millipore Sigma	403105
Pilot aAD digester	Quasar energy group, Independence, Ohio, USA	Stainless-steel fermenter with a total volume of 100 L and a working volume of 80 L
Pilot PHA fermenter	manukatree	SF-50l ISO 9001 CE	Double Layer Jacketed Glass Reactor
Potassium Chloride	G-BIOSCIENCES	162006
Pump tubing			Any brand of silicone tubing with a 1/8'' ID and a 3/8'' OD
Sodium Bicarbonate	Fisher Chemical	241787
Sodium Bromide	Carolina	21139-45
Sodium chloride	Amazon	B0787CFHF3
Sodium hydroxide, Pellets	Fisher Bioreagents	236088
Sodium hypochlorite	Millipore Sigma	425044
Sorvall LYNX 4000 Superspeed Centrifuge	Thermo Scientific	75006580	Used for PHA granule collection in downstream PHA recovery
Spectrophotometer	Hach Company	DR3900	Used in Hach VFA quantification methods
Sulfuric acid	Millipore Sigma	258105
Syringe filter	Amazon	B06X6GTKVY
TOC analyzer	Shimadzu	Model TOC-LCSN
Volatile Acids TNTplus Vial Test	Hach Company	TNT 872	Capable to measure VFA in the range of 50 - 2,500 mg/L Acetic Acid.
Water heater	Bosch Inc.	Tronic 3000 T 7-Gallon (ES8)
Yeast extract	Fisher Scientific Company	AAJ6028736