Plastic Biodegradation by Microorganisms: Key Steps Explained

Scaling microbial processes involves four stages: (1) Research and development - isolation, purification, and identification of the microorganism, (2) Process research and development - testing different media and conditions in small reactors (100-500 mL), (3) Process optimization - scaling to larger reactors (1-20 L) and optimizing conditions, (4) Production - full-scale industrial production. Each stage builds on the previous one, with increasing complexity and scale. The first stage focuses on isolating, purifying, and identifying the microorganism of interest, including testing capabilities (e.g., phosphate solubilization in Pseudomonas). The second stage involves developing the optimal growth medium through various tests. The third stage involves scaling up from small reactors to larger ones, considering availability of medium components at industrial scale, sterilization challenges, maximizing substrate yield, and minimizing contamination risks.

Los microorganismos seleccionados y mejorados en la universidad funcionan como sistemas de purificación de aire, consumiendo carbono contaminante y transformándolo en oxígeno vital. El sistema tiene dos etapas: la primera donde la comunidad microbiana consume contaminantes y se reproduce, y la segunda de biorreactor donde la biomasa se convierte en biofertilizantes, revalorizando subproductos. La protección de propiedad intelectual (20 años) es un diferencial importante que permite manejar escalabilidad desde pequeñas oficinas hasta grandes centros comerciales, haciendo viable la inversión de empresas y aceleradores.

Monod kinetics models microorganism growth using substrate saturation constants, showing linear growth at low substrate concentrations transitioning to inhibition at higher levels. Maximum growth becomes independent of substrate concentration and depends solely on cell density. Airlift reactors offer alternatives to stirred tanks for shear-sensitive cells by eliminating mechanical mixing through gas-driven circulation, though they face height limitations due to oxygen deprivation periods. Scale-up always alters physical parameters, requiring adjustments like impeller speed optimization to maintain oxygen transfer rates. Increasing agitation improves oxygenation but raises power consumption and cooling demands. These trade-offs drive consideration of scale-out strategies using multiple smaller reactors instead of single large units.

Bioreactor scale-up requires achieving both geometrical similarity (where all linear dimensions are related by a constant scale factor) and dynamic similarity (where dimensionless numbers like Reynolds number, Power number, and Froude number are maintained). However, achieving complete dynamic similarity is challenging because satisfying all dimensionless parameters simultaneously is often impossible; therefore, engineers must select the most critical dimensionless parameter to keep constant based on the specific application and operating conditions.

The Micro-Val project, led by researchers at the University of Milan-Bicocca, is developing a biological process using specialized bacteria to digest polyethylene-based plastic residues (such as labels on fruit peels) that contaminate organic waste streams, offering a sustainable solution to plastic pollution in composting and waste management systems.

PHA (Polyhydroxyalkanoate) is a bio-based biodegradable plastic produced by microorganisms that accumulate this polymer when growth conditions are limited; it can be completely decomposed in soil, freshwater, and seawater environments, and its physical properties can be customized by adjusting the monomer composition and ratios, making it suitable for various applications including PLA modification additives and paper coating materials.

Biodegradable plastics (polyhydroxyalkanoates, PHAs) are produced through microbial fermentation, with polyhydroxybutyrate (PHB) being most important. PHB biosynthesis involves three enzymes: acetoacetyl-CoA reductase, PHA synthase, and PHA depolymerase. Initial cytoplasmic production in Arabidopsis yielded low quantities. Chloroplast targeting of all three genes achieved 14% dry weight production without adverse effects. Cotton fibers, containing beta-ketothiolase, were also engineered for PHB production. PHA copolymers are less crystalline and more flexible than PHB.

Polyhydroxyalkanoates (PHA) and polylactic acid (PLA) are biodegradable bioplastics produced through different biological and chemical processes; PHA is synthesized directly within bacterial cells under nitrogen-limited conditions, making it highly versatile with varying mechanical properties suitable for medical applications like sutures, while PLA is chemically polymerized from fermented lactic acid derived from renewable resources like corn starch, offering better thermal stability (melting point ~180°C vs PHB's ~175°C) and primarily degrading under industrial composting conditions rather than natural environments.

This extensive section provides a comprehensive introduction to bioplastics and PHA (Polyhydroxyalkanoates). The term 'bioplastics' is highly confusing because it can refer to two completely different concepts: bio-based materials (origin from renewable sources) or biodegradable materials (ability to degrade at end of life). Bio-based materials derive from renewable sources like vegetable cultivation rather than petroleum, resulting in carbon neutrality where CO2 absorbed during plant growth compensates for CO2 released during production. Biodegradable materials are defined by their end-of-life behavior, not their origin, and can come from any source including petroleum. The industry has created a classification system: conventional plastics (petroleum-based, non-degradable), bio-based plastics (renewable origin, non-degradable), biodegradable plastics (any origin, degradable), and bio-based biodegradable plastics (both characteristics). Bio-based polymers must derive from renewable sources but may or may not be biodegradable. Examples include bio-polyamides, bio-polyethylene, and bio-PET. Cellulose derivatives are inherently biodegradable, but acetate of cellulose is not. Bio-based content ranges from 10% to 100%, measured using ASTM D6866 standard through radiocarbon dating (carbon-14). Bio-based polyethylene is produced from sugarcane-derived ethanol, creating 'green ethylene' with identical properties to fossil ethylene. However, bio-based materials face significant challenges: producing one football field-sized area yields only 3 tons of bio-polyethylene, meaning a typical 250,000-300,000 ton facility would require 80,000-100,000 football fields. Biodegradable polymers are alternatives to traditional plastics that can degrade at end of life. Biodegradation occurs through five main mechanisms: UV degradation, oxidation, thermal degradation, mechanical degradation, and biological degradation. Only biological degradation leads to complete mineralization into simple inorganic compounds. Complete biodegradation requires natural microorganisms (bacteria, fungi, worms) to recognize and consume the material as food. PHA (Polyhydroxyalkanoates) is a natural polymer produced by microorganisms, fundamentally different from synthetic plastics because it is created by nature without chemical modification. PHA has been found in nature for millions of years within microorganisms. PHA was first discovered in 1925 by French scientist Le Guen in Bacillus megaterium bacteria. Natural PHA has low molecular weight and cannot be used industrially. Modern industrialization requires fermentation processes to increase molecular weight while maintaining natural properties. The EU Single-Use Plastics Directive (effective January 2021) bans single-use items made from plastic, including bio-plastics, but allows unmodified natural polymers. Only materials like PHA, cellulose, chitin, and natural fibers can be used for single-use applications. PHA is produced through fermentation in bioreactors where microorganisms consume nutrients (sugars, oils) and accumulate PHA as intracellular storage. The process requires simple conditions, normal temperatures, and low environmental impact. Different microorganisms produce different PHA types with varying properties. PHA polymers are classified by their chemical structure: PHB (with methyl group), PHBV (with valeric acid), PHB3HB (with 3-hydroxybutyrate), and others. Over 150 different PHA types have been identified. PHA can be compounded with various additives to modify properties: fillers (mineral, vegetable fibers like flax, hemp, jute), natural additives (orange peels, coffee residues), and biodegradable additives. This allows customization of flexibility, rigidity, and other properties while maintaining biodegradability.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by microorganisms as intracellular carbon and energy storage granules. Class 2 microorganisms consume fatty acids to produce medium-chain PHAs (6-14 carbons), while Class 3 utilize both sugars and fatty acids. The synthesis is controlled by genetic systems including the universal phc gene encoding PHA synthase. PHAs are rapidly biodegradable through depolymerases (pHz gene) that break them into monomers, with complete degradation occurring in less than 50 days. Applications include biodegradable plastics, biofuels, hygiene products, and medical implants. However, production costs remain higher than petroleum plastics, limiting market competitiveness. Extraction methods include mechanical (centrifugation) and chemical approaches.

Classical ecotoxicology measures apical endpoints (mortality, reproduction, behavior) to derive Predicted No Effect Concentrations (PNECs) for environmental quality standards. Advanced approaches use metabolomics to measure complete metabolite sets, providing mechanistic information on chemical interactions. Target analysis provides accurate quantitative data using reference standards, while suspect screening offers wider chemical coverage using high-resolution mass spectrometry without reference standards, revealing additional risks but potentially missing low-concentration compounds. Comparison showed semi-quantitative suspect screening values generally below one order of magnitude of target concentrations. Experimental design requires ensuring microplastic availability through equilibration periods and monitoring throughout experiments. Biofilm-microplastic interactions showed 10-17% microplastic incorporation, with microplastics surrounded by biofilm components, though microplastics did not significantly affect contaminant biotransformation or bioaccumulation in biofilms.

Some microorganisms lack enzymes for certain degradation steps, leading to incomplete degradation. For example, if a microorganism lacks the enzyme for the first step of PCE degradation, it cannot initiate the process. Additionally, incomplete degradation can produce toxic intermediates that may be more hazardous than the original compound.

The pharmaceutical and chemical industries, especially big oil and plastic industries, produce more chemicals than can be imagined. Toxic evaluation of these chemicals is impossible - it is beyond human capability. All these chemicals go into the environment. Additionally, macro plastics break down into micro and nano plastics (sub-micron particles that cannot be seen), which carry toxic chemicals and are dispersed in soil, water, and air.

Partial biodegradation creates serious environmental problems. When plastics break down into small fragments but aren't completely assimilated by microorganisms, these microplastics accumulate in ecosystems. Research shows plastic pellets absorb toxins from the environment, concentrating pollutants that then enter the food chain when organisms ingest them. Birds, fish, and ultimately humans consume these contaminated plastics, leading to bioaccumulation of toxins. Complete biodegradation ensures all carbon is converted to harmless CO2 and water, while partial degradation leaves persistent harmful residues that accumulate in nature.

When micropollutants are degraded by bacteria, they produce metabolites (degradation products). Some metabolites can be more toxic than the original compound. This complicates environmental monitoring because it requires tracking both the original micropollutants and their potentially more dangerous degradation products.

Scientists have discovered that microorganisms worldwide have evolved to digest plastic as a result of human plastic production. Analysis of over 200 million genes from environmental DNA samples revealed 30,000 different enzymes capable of breaking down 10 different types of plastic. This discovery suggests that nature has already developed solutions to plastic pollution, and humans can potentially harness these microorganisms to help address the plastic waste crisis.

Researchers discovered a unique microbial ecosystem called the 'plastisphere' on ocean plastic. This self-sustaining world includes predators, prey, parasites, and symbionts. Vibrio bacteria (up to 25% of communities) can cause disease and attach to fish intestines. Some bacteria accelerate plastic breakdown through enzymatic processes. This finding reveals how human pollution creates entirely new habitats that may alter marine biodiversity and ecosystem dynamics.

The plastisphere represents a newly emerging ecosystem where marine microbes colonize plastic surfaces, evolving over a century of plastic accumulation. Deep-sea environments, being nutrient-limited, may select organisms utilizing plastics as novel carbon sources. Researchers collected samples from 10m to 8,000m depths using submersibles and autonomous profilers, testing four plastic types through controlled incubations. Metagenomic analysis revealed distinct plastic-associated communities with reduced biodiversity compared to surrounding water, enriched for Proteobacteria capable of particle attachment. FTIR spectroscopy detected chemical changes indicating degradation. This research demonstrates how human-made materials are being incorporated into natural ecological systems, with microbes potentially adapting to utilize plastics as substrates.

The plastisphere refers to the unique microbial community that colonizes microplastic surfaces, forming a distinct ecological niche; researchers employ three main methodologies—field sampling, field culture, and laboratory simulation—to study these communities, with next-generation sequencing revealing that bacterial compositions vary significantly across environments (freshwater, marine, and terrestrial), primarily comprising proteobacteria and actinobacteria, while the plastisphere plays crucial roles in microplastic degradation, elemental cycling, and potentially serving as vectors for antibiotic resistance genes and pathogens.

Bacteria in landfills have evolved to break down complex polymer chains of modern plastics and use the carbon content as fuel. Laboratory researchers are enhancing and turbocharging this natural process for large-scale application. The Mix Up consortium (Mixed Plastics Biodegradation and Upcycling using Microbial Communities) brings together 14 international partners from Europe and China to create a truly circular economy for all types of plastic, aiming for commercially viable production of upcycled bioplastics by the end of the decade using engineered enzyme mixtures and mixed microbial communities.