Bioplastic “Alive” with Bacterial Spores Can Compost Itself

Researchers led by a team at the University of California San Diego have developed a biodegradable form of thermoplastic polyurethane (TPU) that could help reduce the plastic industry’s environmental footprint. TPU is a soft yet durable commercial plastic used in footwear, floor mats, cushions and memory foam. The new biodegradable TPU bioplastic is filled with bacterial spores from a strain of Bacillus subtilis that has the ability to break down plastic polymer materials. When exposed to nutrients present in compost, the spores in the biodegradable TPU germinate and break down the material at the end of its life cycle.

“It’s an inherent property of these bacteria,” said Jon Pokorski, PhD, a nanoengineering professor at the UC San Diego Jacobs School of Engineering and co-lead of the university’s Materials Research Science and Engineering Center (MRSEC). “We took a few strains and evaluated their ability to use TPUs as a sole carbon source, then picked the one that grew the best.”

Pokorski and colleagues reported on their development in Nature Communications, in a paper entitled “Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration,” in which they concluded, “this work presents a scalable method for the fabrication of biocomposite materials with improved mechanical properties and programmed biological functionalities.”

The field of hybrid engineered living materials (ELMs) is a “burgeoning field” that seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function, “… since living systems can provide highly-programmable and complex behavior,” the authors explained. “The promise of incorporating living matter into biocomposites has generated materials that are capable of responding to stimuli (i.e. light, nutrients, inducers, etc), and consequently morphing their shapes and/or properties, which have been utilized for living biosensors, wearable bioelectronics,  drug delivery systems, wound healing patches and self-regenerating skin.”

Introducing live cells into polymer composites could significantly improve both their material properties and ecological footprint, the team continued. Live cells have ideal features as smart polymer additives. They are self replicating, self regulating, and can be programmed to respond to specific stimuli. Cells can also be genetically engineered to produce small and large molecules and to display desired functionalities. “Successfully harnessing living cells has limitless potential to develop polymer composites with enhanced properties such as improved mechanical performance and other performance characteristics, such as programmed/accelerated disintegration.”

There are challenges associated with working with live cells, however, the team continued. Cells are fragile and may need specific hydration, temperatures and pHs, for example. “Polymer processing into commercial parts typically requires heat, shear stress and/or solvents, all of which are detrimental to cell viability,” the researchers pointed out. These challenges together mean that incorporating live cells into polymers has only been demonstrated for a limited type of polymers that can be produced using ‘mild’ manufacturing conditions, such as low melting temperatures, aqueous conditions, and low shear stress.

For their study, the researchers instead turned to bacterial spores—a dormant form of bacteria—due to their resistance to harsh environmental conditions. Unlike fungal spores, which serve a reproductive role, bacterial spores have a protective protein shield that enables bacteria to survive while in a vegetative state. “Spores can preserve their viability against high temperatures, pressure, toxic chemicals (i.e. acids, bases, oxidants, and organic solvents), and radiation,” the researchers further explained.

The researchers were interested in Bacillus subtilis, a well known spore-forming bacteria, which features attributes that make them “promising additives in developing biocomposite polymers with facilitated biodegradation,” they noted. However, one challenge to using B. subtilis spores in polymer processing is their lack of heat tolerance. “Spores of several B. subtilis strains are known to be resistant at ~100 °C for several minutes, but most industrial thermoplastic processing requires higher temperatures above 130 °C,” they explained.

To negotiate this hurdle, the team used adaptive laboratory evolution (ALE), an evolutionary engineering approach that has been used in the past to generate the Bacillus species with enhanced tolerances to specific conditions. For their reported work the B. subtilis bacterial spores were evolutionary engineered to create a strain that would survive the high extrusion temperatures necessary for TPU production. The process involves growing the spores, subjecting them to extreme temperatures for escalating periods of time, and allowing them to naturally mutate. The strains that survive this process are then isolated and put through the cycle again. “We demonstrated that heat-shock tolerized B. subtilis spores retained ~100% viability in TPUs after hot melt extrusion (HME),” they commented. “We continually evolved the cells over and over again until we arrived at a strain that is optimized to tolerate the heat,” added study co-senior author Adam Feist, PhD, a bioengineering research scientist at the UC San Diego Jacobs School of Engineering. “It’s amazing how well this process of bacterial evolution and selection worked for this purpose.”

To make the biodegradable plastic, the researchers fed the heat-shock tolerized (HST)  Bacillus subtilis spores and TPU pellets into a plastic extruder. The ingredients were mixed and melted at 135°C, then extruded as thin strips of plastic. Interestingly, the authors found, the spores also serve as a strengthening filler, similar to how rebar reinforces concrete. The result is a TPU variant with enhanced mechanical properties, requiring more force to break, and exhibiting greater stretchability. “The incorporation of heat-shock tolerized spores as living fillers into TPUs resulted in an overall increase in the tensile properties of TPUs owing to the strong interfacial interactions between spores and polymer matrix.” This improvement in tensile properties of TPU by spore addition offers what they suggest is a promising solution to “overcome the trade off barrier between tensile stress and elongation at break in commercial TPUs.”

“Both of these properties are greatly improved just by adding the spores,” said Pokorski. “This is great because the addition of spores pushes the mechanical properties beyond known limitations where there was previously a trade off between tensile strength and stretchability.”

To assess the material’s biodegradability, the strips were placed in both microbially active and sterile compost environments. The compost setups were maintained at 37°C with a relative humidity ranging from 44 to 55 percent. Water and other nutrients in the compost triggered germination of the spores within the plastic strips, which reached 90 percent degradation within five months.

“This result depicted that nutrient and moisture content were sufficient for facilitating biodegradation. In other words, spores embedded in the TPU matrix triggered and facilitated the biodegradation of TPU with minimal intervention,” the team wrote. The experimental findings, they noted, “confirmed that the HST spores can successfully germinate with biological activity in compost after fabrication and they indicated that the disintegration process is significantly accelerated in environments lacking sufficient quantity of degrader strains.”

Added Pokorski, “What’s remarkable is that our material breaks down even without the presence of additional microbes. Chances are, most of these plastics will likely not end up in microbially rich composting facilities. So this ability to self-degrade in a microbe-free environment makes our technology more versatile.”

In addition, the authors were able to engineer biological function into the biocomposite by genetically engineering B. subtilis to express a model green fluorescent protein (GFP). After HME fluorescence could be detected once the spores were germinated. “… we showcased the potential of genetic engineering by incorporating a GFP-expressing plasmid into the strains,” they reported.

Although the researchers still need to study what gets left behind after the material degrades, they note that any lingering bacterial spores are likely harmless. Bacillus subtilis is a strain used in probiotics and is generally regarded as safe to humans and animals—it can even be beneficial to plant health.

“In conclusion, the incorporation of bacterial spores presents exciting opportunities for the introduction of living cells as renewable polymer fillers in industrial processes,” the researchers stated. “This innovative approach combines evolutionary and genetic engineering methodologies and shows potential for diverse applications in the advancement of biocomposite materials.”

While the current study focused on producing smaller lab-scale quantities to understand feasibility, the researchers are working on optimizing the approach for use at an industrial scale. Ongoing efforts include scaling up production to kilogram quantities, evolving the bacteria to break down plastic materials faster, and exploring other types of plastics beyond TPU.

“There are many different kinds of commercial plastics that end up in the environment—TPU is just one of them,” said Feist. “One of our next steps is to broaden the scope of biodegradable materials we can make with this technology.”