PHAsing Out microplastics

We ingest up to 5 grams of microplastics every week1. A large part of the microplastics we consume comes from eating food contaminated by plastics used in agriculture2,3. Microplastics are small plastic particles derived from non-degradable plastics that mechanically break down into smaller parts, which causes microplastic pollution4. This breakdown into microplastics, as well as even smaller nanoplastics, allows for the uptake of these particles into cells5,6. In soil, microplastics also act as a vector for organic pollutants like pesticides and industrial chemicals, as well as for heavy metals such as cadmium, chromium, and copper. These compounds adsorb to the surface of the microplastics and in turn are taken up by plants (Figure 1). When these plants are eaten, this leads to the accumulation of microplastics, heavy metals, and toxic compounds in the human body. To date, microplastic accumulation has been found in essentially all human tissues, in the bloodstream, and in fetuses7. Recent studies have already found a range of adverse effects on the respiratory tract, cardiac health, neurological health, mitochondrial health, and on oxidative stress8,9.


In light of these facts, it is essential for our current global health and for the generations to come that we eliminate plastics from our food. However, due to its advantageous material properties, the use of plastic is integral to many of the innovations and developments in the agricultural sector that increase productivity and efficiency10. Plastic mulch films and plastic-coated controlled release fertilizers (CRF) are major sources of microplastic pollution in agriculture11. Mulch films are sheets laid over soil that help modify soil temperature, prevent contamination of crops, and limit weed growth, which leads to improved crop yields12. CRFs are plastic coated fertilizers used to gradually release fertilizers at the plant roots, thus limiting nitrogen volatilization and allowing for the fertilizer to stay in the correct place for longer. Use of CRFs has helped us make tremendous steps towards preventing eutrophication of groundwater and to mitigate harmful impacts on the climate13. These beneficial qualities make it necessary to develop sustainable alternatives to the current plastics, instead of avoiding the use of CRFs and mulch films. This is where PHAse Out comes in!


To PHAse Out fossil-based non-degradable plastics in agriculture, the iGEM Leiden 2023 team has been working on producing sustainable and cost-effective biobased biodegradable polymers from the family of polyhydroxyalkanoates (PHA)14.


PHA is a microbial biopolymer that is already available on the market, however, current production is expensive and relies on feedstocks like sugar that compete with our food security15,16. This makes current PHA production unsustainable and more costly than the fossil-based plastics available. To change this, we focused on developing a process using an alternative carbon source as feedstock, and landed on green methanol . Methanol can be made sustainably, is cheaper than sugar, and can be integrated into most existing production and transport infrastructures17. Using green methanol would allow for addressing the two core issues of PHA production, price and sustainability, which makes it a great potential alternative feedstock18.


To harness the characteristics of green methanol, we chose to work with Methylobacterium extorquens AM1. M. extorquens is a promising non-model organism for PHA production, as it is able to grow on methanol as its sole carbon source and endogenously accumulates polyhydroxyalkanoates (PHA)19,20. With the combination of green methanol and M. extorquens we look forward to PHAsing Out fossil-based plastics together!

something.
Fig. 1 | Visualization of heavy metals in soil adsorbing to microplastics, which then get taken up by plants.



Learn more about plastic in agriculture

    With a growing world population and increasing demand for food, we continue to need plastics in agriculture to safeguard our crop yield and food supply. However, we need to replace non-degradable plastics in agriculture to remove microplastics from entering our food chain.


    Microplastics in agriculture largely originate from the breakdown of non-degradable mulch films and CRF pellets21. Mulch films support plant growth and reduce the use of water and pesticides. They also limit volatilization and unwanted ground water eutrophication. Addition of mulch film to croplands has been shown to improve crop yield by up to 39.5%22. CRF pellets allow for fertilizer to be applied with better spatial and temporal precision, which in turn allows for a decrease in the amount of fertilizer needed. Due to the slow-release characteristics of CRFs, they are favorable in comparison to other fertilizers, making them a valuable asset in agriculture23.


    The current plastics used in agriculture are predominantly polyethylene, polypropylene, and ethylene-vinyl acetate11. These plastics are overwhelmingly non-degradable, fossil-based, and are environmentally unsustainable. Conversely, the ideal sustainable plastic needs to be biobased, i.e. made from organic material and from renewable sources, and biodegradable in all ambient conditions. This market need is currently unmet, as the few available alternatives to fossil-based plastics only partly meet these requirements11. These alternatives to fossil-based plastics, such as polylactic acid (PLA) or starch-based plastics, can be made from renewable or organic material and some biodegrade under certain conditions and/or can be composted under certain conditions. However, they are not both biobased and biodegradable24,25. PHA is both biobased and biodegradable, and has similar material properties to fossil-based plastics, and as such holds great potential for use in agriculture if the cost can be reduced26.

Learn more about PHA

    PHAs are a family of polyesters that are produced by many micro-organisms, including M. extorquens, as a carbon and energy reserve compound. As it is an energy reserve, this means PHA can be broken down by the bacteria in circumstances of carbon deficiency. PHA is broken down by the bacteria, releasing the stored energy for use in essential metabolic processes27.

    PHAs can be classified as short-chain-length (SCL) or medium-chain-length (MCL), depending on the amount of carbon monomers in the chain. Depending on the length of carbon chains in their monomers, PHA has different physical characteristics. SCL PHAs are brittle and rigid, and are due to this commonly used for stiff applications such as disposable cutlery. MCL PHAs have a good balance between flexibility and toughness, which makes it suitable for applications like packaging and agricultural films28.

    Examples of different types of short-chain-length PHAs are poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxyvalerate) (P3HV). In addition, co-polymers also exist, such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)28.

    Fig. 2 | a. General molecular formula of PHA. b. Some commonly synthesized PHA monomers.
    3HB: 3-hydroxybutyrate,
    3HV: 3-hydroxyvalerate,
    3HHx: 3-hydroxyhexanoate,
    3HO: 3-hydroxyoctonaote,
    3HD: 3-hydroxydecanoate,
    3HDD: hydroxydodecanoate.
    Adapted from Mukherjee A, Koller M. (2023)28


    In our project, we focus on producing P3HB, which is a part of the PHA family of polymers and is a good potential alternative to the conventional plastics in agricultural applications due to its properties. Similar to polypropylene, It is rigid and strong, making it suitable for applications that require structural integrity, such as CRFs29,30. Its thermal properties, including a moderate melting point and low glass transition temperature, make it suitable in various environmental conditions31. P3HB's ability to degrade at ambient temperatures also makes it suitable for agricultural purposes. However, as it is a SCL polymer it is brittle, and to endow P3HB with the flexibility needed for some applications copolymerization may be needed. Co-polymerizing P3HB with other PHA polymers would allow for customization of the material properties to meet specific agricultural needs, offering an environmentally friendly and versatile choice for replacing traditional plastics14,20. A possible polymer for aiding in flexibility is P3HV. M. extorquens can produce both P3HB and P3HV, either separately or as copolymers. In a copolymer, both P3HB and P3HV exist in the same chain. In most cases, including in our application as mulch film, a copolymer is more desired as the material tends to be more homogeneous and thus also material properties are more consistent leading to better predictability in the application.

Green methanol as microbial feedstock

In the face of global food shortage and the need for increasing the sustainability of biotechnological processes, we chose to use M. extorquens as our PHA producing microbial cell factory. Using a methylotrophic bacteria allows for the utilization of methanol instead of sugar, which is the current standard feedstock for bioproduction15. The use of sugar as a feedstock for industrial bioproduction competes with food production, as it uses arable land. With our growing world population and the insufficient availability of arable land, food insecurity and global hunger are a persistent issue affecting over 700 million people worldwide16. To combat this, exploring the use of alternative feedstocks is crucial. Methanol is a promising alternative feedstock, as it can be produced from carbon dioxide and hydrogen using renewable energy or from biomass waste streams, called e-methanol and biomethanol respectively. Methanol production is independent from oil prices, cheaper than sugar, does not depend on arable land for production, and contrary to sugar the price is expected to remain stable over time32, 33. Storage and transportation methods for methanol also suit the current fossil-fuel based infrastructure, which makes it an attractive alternative for industry17. Increased industry adoption would also have the benefit of allowing for even further infrastructure improvements and would push e-methanol prices lower.

Sustainable

Using M. extorquens for biodegradable plastic production allows us to leverage the major sustainability and cost benefits of methanol as an alternative feedstock. You can find more information on the advantages of green methanol on our Sustainable Development page .

Future-proof

PHAse Out’s focus on producing biodegradable plastics for agriculture addresses a pressing concern for the future – the pollution of microplastics in food and the environment. PHAse Out actively participates in the transition to a circular carbon economy as our project ensures that carbon resources are continuously reused, reducing the reliance on fossil fuels and aligning with global efforts to mitigate climate change. This is in line with what circular plastics experts told us about the circularity of our project. You can read more about these interviews on the Integrated Human Practices page .

Cost-effective

Using methanol as a feedstock for PHA production significantly reduces raw material expenses and makes PHA production cost-effective (Table 1). This makes PHA an attractive choice for industries seeking sustainable alternatives to conventional plastics, driving market adoption and long-term profitability.

To underscore the economic viability of methanol as a carbon source for PHA-production, we calculated the comparative feedstock costs of methanol and sugar for 1kg of PHA (Table 1). This gives an overview of the current and future projected feedstock costs, showing that methanol is a more economically viable alternative for the future.

Table 1 | Comparison of feedstock costs.
Feedstock Yield (kg substrate / kg PHA) Price (US$ / kg) Feedstock costs ($ / kg PHA)
Sugar (2023) 0.434 $0.5336 $1.33
Green methanol (2023)

0.5935 $0.7037 $1.19
Green methanol (2050) 0.5935 $0.338 $0.51

The project

M. extorquens naturally produces PHA, as it is a carbon and energy reserve within the bacteria, similar to fat in humans27. However, the bacteria do not produce PHA as efficiently as is needed for large-scale industrial biobased biodegradable plastic production39. To make M. extorquens suitable for efficient production of PHA we worked on multiple aspects to increase the yield and improve the ease and safety of extraction (Figure 3).

lab-lines
Fig. 3 | Overview of the the lab lines.



To improve the PHA production yield, we genetically engineered M. extorquens in several ways: through overexpressing the phaC and groE genes and by knocking out genes from the carotenoid pathway. To increase the ease and safety of extraction, we engineered an inducible autolysis system with the aim of allowing for PHA extraction without chemical or mechanical cell disruption. Our project combines genetic engineering and synthetic biology principles to address pressing sustainability and environmental challenges. Make sure to also read our Integrated Human Practices page to learn more about the experts we interviewed, what role they played in our project, and how their expertise influenced the project.

Promoter characterization

As M. extorquens is a non-model organism, the genetic engineering tools available for this are not yet on par with those available for model organisms, such as E. coli40. Additionally, as M. extorquens is classified as an alphaproteobacteria, many established promoters from E. coli do not work as they are intended for use in gammaproteobacteria. However, our application necessitates various promoters for the different applications. This led us to dedicate our first lab line towards characterizing various inducible and constitutive promoters. We incorporated the results from this in the other aspects of our lab work to optimize either overexpression of genes or the inducible expression of genes. You can read more about the specific promoters characterized on the Parts page .

Improved PHA yield

Overexpression of PhaC and GroE

The phaC gene codes for the enzyme polyhydroxybutyrate (PHB) synthase. It is a key enzyme in the PHA biosynthesis pathway where it plays a role in the polymerization of monomers into polymers41. Literature shows that overexpressing PhaC in bacteria considerably increases PHA accumulation, indicating that phaC is a promising gene target42.


Based on this, we created multiple constructs to overexpress phaC in M. extorquens. We used the characterized constitutive promoters to test whether the differing promoter strengths affected how well phaC overexpression increased PHA production.


In addition, we evaluated the added benefit of co-expressing PhaC with the GroEL/GroES chaperone system. This system aids in the folding of proteins, ensuring a high soluble concentration of active protein43. We hypothesized that co-overexpression of phaC and groEL/groES may improve the total activity of the overexpressed PhaC enzyme, increasing PHA yield. As no scientific literature exists detailing the effects of this coexpression in our non-model organism, we wanted to experimentally validate this system.


To measure the efficacy of our modifications in vivo, we adapted a quantifiable fluorescence-based method from published methods44,45. The development of this method allowed for PHA quantification without extraction.




Carotenoid knock-out

To identify promising metabolic engineering strategies for PHA production and to increase the success rate of our modifications we used a metabolic network model of M. extorquens46. We specifically looked further into the carotenoid biosynthesis pathway, as production of carotenoid compounds takes up a lot of energy and carbon, which could otherwise be used in the PHA production pathway47. Based on our modeling results, we created a carotenoid knockout strain for increased PHA production.

Improved PHA extraction

Inducible autolysis system

A major aspect of what makes current PHA production costly and unsustainable is the extraction. Currently, extraction relies on either physical or chemical disruption of the cell39. However, these methods can be both costly and harmful to the environment as well as human health48. Depending on the extraction technique used, this may also mean that the end product is not food safe, which severely limits the number of possible applications. Some estimates have also found that the extraction process can account for as much as 50% of the total production costs39. To improve the sustainability, safety, and cost of PHA extraction, we developed an inducible autolysis system. Our aim to create a cell-integrated system that allows for inducible lysis without the requirement for mechanical disruption or toxic chemicals. We developed this inducible system by creating constructs using various cell lysis genes from different bacteriophages50. As bacteriophages rely on lysis to propagate infection, we hypothesized that using these lysis genes would allow us to disrupt the cell wall, removing the need for external input51. To optimize this system, we incorporated the results from our characterization of inducible promoters in this construct.

Safer extraction

Safety is of high priority, and with the specific goal of minimizing the need and use of harmful chemicals in PHA extraction, we discussed the risks with experts and considered various alternatives. You can find more information on the evaluation of safety risks of hazardous chemicals on our Safety page .

Conclusion

PHAse Out has contributed to the development of sustainable and cost-effective production of PHA. Overall, we characterized the strength of constitutive and inducible promoters, created strains for PHA overexpression, created a carotenoid knockout strain, developed and optimized an inducible autolysis-system, and looked into improving the ease and safety of PHA extraction. Our goal was to create a sustainable PHA production process and extraction, with the goal of using PHA in agricultural films and fertilizer coatings to remove microplastics from our food. Our experimental work shows promising results for cost-effective and sustainable PHA production using M. extorquens and green methanol. We are one step closer to PHAsing Out microplastics from our diet! Visit our Results page for an in-depth look into our experimental outcomes or visit our Engineering page to see how different aspects of our lab are intertwined. Check out our Entrepreneurship page to have a look at our project development plans and future business perspectives.

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