MSc Chemistry
Science for Energy and Sustainability
Literature Thesis
____________________________________________________________
PET Biodegradation and PET Biorecycling
Economic Perspectives
By
Sabrina Maria Brandjes
10549005
May 2020
12 Credits
January 2020 - May 2020
Supervisor/Examiner
Prof. Dr. G.J.M (Gert-Jan) Gruter
Examiner:
Prof. Dr. J. H. (Jan) van Maarseveen
Industrial Sustainable Chemistry
Faculty of Science
2 Summary
Using plastics has been beneficial for improving efficiencies along many value chains, but it has come at a cost. Not only are we using plastics and burning them when we feel that it is too difficult to recycle them, there are also many unwanted losses of plastics to the environment. Polyethylene terephthalate (PET) and other plastics have been developed and are used for their durability and inertness. However, research into the biodegradation of PET has seen a spike in activity since the discovery of a PET degrading bacterium at a PET recycling facility in Japan. Unprecedented substrate specificity of the so-called PETase enzyme has aided the bioengineering of more heat stable PET hydrolyzing enzymes with the ability to hydrolyze crystalline PET into terephthalic acid (TA), mono-hydroxy ethylene glycol (MHET) and bis-hydroxy ethylene glycol (BHET). PET biorecycling poses a solution for currently unused undervalued PET waste that would otherwise be incinerated. According to the calculations in this report, PET biorecycling producing recycled TA, will need very specific conditions to become market competitive. This resource intensive method for PET recycling would have to be optimized in multiple different aspects to become profitable.
Index
Summary ... 2
Introduction into PET Waste and Pollution ... 3
Biodegradation and Biorecycling ... 4
Biodegradation ... 6
Biorecycling ... 8
Challenges for Economic Viability of PET Biorecycling ... 10
How far advanced is Biorecycling? ... 11
Discussion on the Biodegradation of PET ... 13
Discussion on the Biorecycling of PET ... 15
Conclusions ... 16
Introduction into PET Waste and Pollution Since its discovery, plastic in all its forms has been praised for its durability and versatility. However, the way we use the material has serious consequences. In all forms, plastics have penetrated all ‘corners’ of the globe and are even found at the bottom of our deepest ocean trenches (Peng, Bellerby, Zhang, Sun, & Li, 2020). As such, it is important to understand the consequences that come with polluting our environment with plastics.
If large plastic debris does not kill instantly when entangling animals, it is often mistaken for food, killing animals in the long term as plastic can build up in the intestines leading to starvation. But most importantly, plastic litter is most disruptive in marine ecosystems because microplastics tend to travel up the food chain. Hundreds of aquatic species have shown negative health effects as a result of exposure to both macro and microplastics. The species that ingest the most plastic are the procellariform seabirds (Kawai, Kawabata, & Oda, 2019; Kühn, 2020). These include albatrosses, petrels, shearwaters, fulmars and storm petrels. Research has shown that plastics are found in 63% of these birds. Figure 1 illustrates the large volume of plastics that these birds typically ingest (Van Franeker & Kühn, 2019).
Plastics can also act like a hydrophobic type of sponge for environmental pollutants, making plastic particles in the environment even more toxic. Multiple papers have highlighted the dangers of plastic particles acting as transportation vectors for persistent organic pollutants (POP’s) as polycyclic aromatic hydrocarbons (PAH’s), polychlorinated biphenols (PCB’s), and DDT and other organochlorine pesticides (LI, Tse, & Fok, 2016). If POP’s are concentrated in plastics as various articles refer to, they can easily harm any organism that mistakes the plastic particles for food. What is more disturbing is that research has hinted that plastic particles might actually smell like food for some organisms (Procter, Hopkins, Fileman, & Lindeque, 2019; Savoca, Wohlfeil, Ebeler, &
Nevitt, 2016; Savoca, Tyson, McGill, & Slager, 2017).
Furthermore, plastics have shown the ability to carry invasive bacterial species throughout the ocean. Marine ecosystems can be severely impacted by the introduction of foreign microbes (Chatterjee & Sharma, 2019). Plastic particles could transport bacteria, viruses and other microbes across the globe to infect whole communities that are crucial to their respective ecosystems.
Not only nature suffers from plastic pollution, but plastics are now entering our own bodies through the food chain and drinking water sources in the form of microplastics. This is a major concern as all types of plastics including microplastics can release harmful additives that affect our hormonal balances and those of other species. For example, plastic medical implants have shown harmful endocrine disrupting effects in humans (Gallo et al., 2018).
Microplastics (1-1000 µm in size) account for the largest share of plastic particles in marine environments. The majority of which come from losses of pellets along the plastic value chain before the pellets are processed into plastic products. The second largest source of microplastics comes from the laundering of synthetic textiles(Boucher & Friot, 2017). This means that synthetic fibers enter water bodies Figure 1: Plastic ingested by Northern Fulmars. Source: Van Franeker, J.A. & Kühn, S. 2019. Fulmar Litter
EcoQO monitoring in the Netherlands - Update 2018. Wageningen Marine Research Report C077/19 & RWS
Centrale Informatievoorziening BM 19.16. Den Helder, 60pp.
4 after passing the cleaning systems of wastewater
treatment plants if these are in place.
Surprisingly, another source of microplastic pollution is the production of organic fertilizer(Weithmann et al., 2018). It is produced from organic waste which should not contain any plastics. Unfortunately, consumer errors pollute the otherwise organic waste stream with different types of plastics. If this organic waste contains too much plastic, it is not digested to produce fertilizer, but it is burned with other household waste instead.
Furthermore, it seems that we have nearly no understanding of plastic nano particles which are a class of particles that is three orders of magnitude smaller than microparticles. As such, these could penetrate cell walls and tissues with more ease than micro particles. So far, only models have been made in the laboratory to predict the behaviour of plastic nano particles as no measuring techniques are available yet for the detection of plastic nanoparticles in environmental samples (Mitrano, 2019).
A publication from the Ellen MacArthur Foundation revealed that, in 2014, global plastic production had reached a staggering 311 million tonnes and that in a business-as-usual case this amount will at least have tripled by the year 2050. It was also noted that a quarter of plastic production is used for packaging of which only 14% is collected for recycling. Another 14% is incinerated for energy recovery, 40% is landfilled and a staggering 32% escaped the
collection system(Kawai et al., 2019; Neufeld, Stassen, Sheppard, & Gilman, 2016). This means that 72% of plastic packaging is polluting our environment, and 14% is incinerated while producing greenhouse gasses that accelerate unwanted climate change. In total, 86% of plastic packaging is disposed of unsustainable and only 14% is disposed of in a sustainable manner. Though, even here, the plastics are mostly
thermally or mechanically recycled into lower quality plastic products that could never again serve its initial purpose as, most often, food packaging material.
As presented in Figure 1, plastic production in 2018 has increased to 388 million tonnes. That is 77 million tonnes more plastics produced than in 2014.
Radical change has to be made to protect natural resources from plastic pollution and create a more sustainable economy where resources are not wasted due to legislative barriers or unnecessary cross contamination of waste streams. These natural resources include the great forests that produce oxygen that humanity desperately needs, marine ecosystems that humanity relies upon for a large share of food supplies, but also sources of drinking water as underground aquifers and agricultural land which are all currently being polluted with plastics or destroyed by carbon emission induced climate change. Figure 2 shows some evidence supporting these findings (O'Connor, 2018).
Biodegradation and Biorecycling
Two techniques that could reduce plastic pollution and the waste of valuable resources, are biodegradation and biorecycling respectively. Biodegradation refers to biological degradation using microbes that ‘feed’ on plastic, and biorecycling refers to the use of enzymes from the aforementioned microbes to cleave the plastic polymer chains into the monomers a Figure 2: A 2018 study found microfibers in the vast majority of sea salt,
beer, and tap water samples tested.
Source: Kosuth M, Mason SA, Wattenberg EV (2018) Anthropogenic contamination of tap water, beer, and sea salt.
5 polymer was originally formed from. The
monomers can then be retrieved to produce new high quality plastic products. As such, only biorecycling leads to recycling, biodegradation does not.
Although, plastics were initially made to be durable, some enzymes have the ability to cleave polymer chains. Research in this field has gained renewed interest since the discovery of a bacteria called Ideonella Sakaiensis. This species has probably evolved through natural selection with lateral gene transfer and specific mutations as a result of external conditions to degrade and assimilate on poly-(ethylene terephtalate) (PET) as its only carbon source(Yoshida et al., 2016). It was the first time that a bacteria was discovered that shows such a remarkable selectivity for PET.
In March 2016, the discovery of a bacterium that degrades and assimilates poly-ethylene terephthalate (PET) was presented (Yoshida et al., 2016). By sourcing and screening 250 different samples of PET contaminated environmental (water, soil, waste water and active sludge) samples from a PET waste recycling
site, the researchers observed that the consortium named ‘no. 46’ consisted of a mixture of bacteria that could degrade low-crystalline PET with a rate of 0.13mg cm-2 day-1 at 30 degrees Celsius. Hereafter, the species responsible for the degradation and assimilation of PET was successfully isolated and represented a new species of the genus Ideonella, hence its name Ideonella Sakaiensis 201-F6. After isolating the strain that was responsible for PET degradation, the isolated strain showed a PET degradation that was twice as fast as the original consortium ‘no. 46’ that it originated from.
These PET degrading properties seem to arise from a combination of genes coding for enzymes and a terephthalic acid (TPA) transporter (TPATP) that is unprecedented by any other known organism. The predicted pathway for PET degradation is presented in Figure 3. A combination of an enzyme that degrades mono-hydroxyethylene terephtalate (MHETase), a TPATP, a TPA deoxygenase (TPADO), 1,2-digydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase (DCDDH) and PCA
3,4-Figure 3: I. Sakaiensis PET Metabolism.
Source: Yoshida, S. et al. (2016). A bacterium that degrades and assimilates poly (ethylene terephthalate). Science, 351(6278), 1196-1199.
6 dioxygenase (Pca34) results in the efficient
degradation of PET into environmentally benign molecules.
The discovery of I. Sakaiensis has had several consequences for both the field of biodegradation and biorecycling. Bioengineering can aid both fields of research by inspiring the design of mutant microbes as well as mutant enzymes that have superior performance to degrade or hydrolyze PET respectively.
As such this paper will discuss if PET biodegradation and PET biorecycling can play a role in creating a more sustainable future, which role that could be and how to reach this stage. There are several niches in which PET biodegradation could be a useful and sustainable tool, examples are: (1) PET removal from organic waste, and (2) microplastic removal from wastewater. (3) PET Biorecycling could be used to recycle PET more sustainably.
Biodegradation
The enzyme from I. Sakaiensis 201-F6 responsible for hydrolyzing PET, shows a PET hydrolyzing activity (PHA) that is 5.5 to 20 times larger than other enzymes that show PHA. Due to the enzyme’s remarkable selectivity towards PET and its high activity, it is referred to as PETase and in some cases IsPETase to refer to the microbial species it is obtained from. There are few published examples of enzymes other than PETase that show PHA and they mainly belong to the cutinase, lipase and other esterase families(Dvorak & Fletcher, 2013; Ferrario, Pellis, Cespugli, Guebitz, & Gardossi, 2016). Cutinases naturally evolved in some fungi to breakdown a natural hydrophobic polymer called cutin that covers the outer layer of higher plants(Ferrario et al., 2016). This means that cutinases are the first and essential actors in plant matter degradation processes, removing the protective cutin layer from leaves that reach the forest floor. Lipases occur in many different organisms and catalyze the hydrolysis of fats and lipids.
In late 2017 another research group in the United Kingdom determined that PETase has
a typical α/β-hydrolase structure with an open active site cleft (Furukawa, Kawakami, Tomizawa, & Miyamoto, 2019). From this group of hydrolases PETase especially resembles cutinases and lipases. This same research group also hypothesized that a cutinase inspired double mutation could alter substrate-binding interactions which were confirmed with improved PHA by the PETase double mutant. Tweaking the enzyme resulted in a PETase double mutant that could degrade both PET and polyethylene-2,5-furandicarboxylate (PEF).
Although the wild type PETase is highly selective to PET and shows a relatively high PHA, it is very heat labile. In contrast to other known PET hydrolyzing enzymes (PHEs), PETase loses its PHA quite quickly when approaching the glass transition temperature of PET. Other known PHEs are more stable and perform better than PETase at higher temperatures.
A cutinase from Thermobifida fusca (TfCut2) has recently been mutated to resemble a PETase structure at the active site. With the help of cationic surfactants a degradation rate of 31±0.1 nmol min−1 cm−2 was achieved on a low crystalline PET film (200 μm). The paper published in 2019 noted that this was the fastest PET degradation rate thus far. After consulting the authors of that paper, it was clear that due to uncertainties in measurements, the degradation rate could not be expressed in g min-1 cm-2, but had to be expressed in the amount of moles of degradation products formed (TPA, MHET and BHET). The true value of PET loss by weight can not be derived precisely from the degradation products as there is a possibility that other degradation products have been formed as well. Long duration tests showed that 97±1.8% of the PET film was degraded within 30 hours (Furukawa et al., 2019).
For a different cutinase-like PHE from Saccharomonospora Viridis AHK190, improved PHA could be explained by later research as a result of conformational changes in the enzyme’s structure in the presence of calcium ions(Numoto et al., 2018). The calcium ions seemed to
7 facilitate a weaker product bonded conformation,
resulting in a quicker release of product. As such, the enzyme is more quickly accessible for new substrates.
Efforts resulting in the improved performance of a double mutant of PETase, and the fact that PET was only put into widespread use in the 1970s, demonstrates that microbes evolve relatively fast to feed on new abundant molecules and that PETase may still be optimized by evolution to improve its PET hydrolyzing capacity. If natural evolution of these bacteria will procreate rapid PET degraders, this might still take centuries, which is why it is important to invest in the bio-engineering of PET degrading bacteria and enzymes if not for degrading other plastics as well.
In addition to the intrinsic properties of a
PHE, other factors influence the hydrolyzation of a PET material as well. Factors influencing the hydrolyzability of PET are hydrophobicity, molecular size, crystallinity and surface topography of the PET material. These should
always be taken into consideration when assessing research performed on the biodegradation of PET and other synthetic polymers(Kawai et al., 2019).
The paper researching PETase presented results achieved from testing the degradation of low crystalline PET films with 1.9% crystallinity. Though, such a low crystallinity does not at all represent material properties of PET encountered in the industry, consumer products or the environment. For example, a soft drink bottle has a crystallinity of around 15.7%4 and PET fibers used in polyester clothing have a crystallinity of 14.1% (Włochowicz & Jeziorny, 1972).
Until recently, hydrolysis of highly crystalline PET was still very inefficient, making the realisation of tertiary bio-recycling of PET waste a difficult one. An extensive review was
published by Fusako Kawai et al. in 2019 that reviewed the current status of research into PHE’s (Kawai et al., 2019). Here, the authors proposed to categorize PHE’s as either PET hydrolases or as PET surface-modifying enzymes. Figure 4: Global Production of Plastics
Source: Ryberg, M., Laurent, A., & Hauschild, M. Z. (2018). Mapping of global plastic value chain and plastic losses to the environment: with a particular focus on marine environment.
8 Here, PET hydrolases are defined as enzymes that
can significantly degrade the inner block of PET materials up to at least 10% with visible change by SEM-observation, whereas PET surface-modifying enzymes can only hydrolyze easily accessible surface residues of polymer strands on a PET surface without any SEM observable change. Surprisingly, the aforementioned PETase did not meet all requirements to be called a PET hydrolase as its PET hydrolyzing activity was too weak to significantly degrade the inner block of (crystalline) PET materials.
To sketch a pathway towards the implementation of the biorecycling of PET (and other plastics), we need to look at how other PETase related enzymes are implemented and determine the level of performance that is needed from the ideal PHE. We choose to look into the industrial biocatalysis of enzymes as enzymes have been used and researched extensively to provide industry with more sustainable alternatives for various reaction processes. The use of enzymes as bio-catalysts generally offers milder (safer) reaction conditions, outstanding selectivity and lower physiologic and environmental toxicity (Choi, Han, & Kim, 2015). Hence, if economically viable, biocatalysis would certainly aid the transition towards a more sustainable future for the whole global population.
Biorecycling
As plastics have been crucial to the optimization of various value chains, it is difficult to eradicate it from our daily lives. In various ways it is also essential for sustainable value chains as the lightweight of plastic containers have resulted in less CO2 emissions from transport. Also, the durability of plastics have aided the preservation of fresh fruit and vegetables which prevents food waste, and durability and inertness has proven to be an important feature for medical tools. As such, it would be foolish to try to completely eliminate the use of plastics from society all together. Figure 4 shows the annual global production of
plastics in million tonnes (Ryberg, Laurent, & Hauschild, 2018). As can be concluded from the figure, PET materials make up about 10 percent of the total production . However, some single use plastics have shown to be expendible or replaceable with more sustainable alternatives and should therefore definitely be banned from our daily lives.
Currently, only a small part of plastics produced are recycled because recycling methods are still very expensive as they either require large amounts of energy or hazardous chemicals, but most importantly because the collection and sorting of different types of plastics is a logistical nightmare. Consumer behaviour results in the contamination of all waste streams imaginable including household separated plastic waste. Though, conventional recycling methods would already reduce greenhouse gasses compared to the production of plastics produced from virgin fossil resources (virgin plastics) with 79% (PlasticsToday Staff, 2017), many plastics can not be recycled to re fulfill the purpose for which the plastics were made originally due to legislative barriers or because many plastic materials are colored. To sustainably and efficiently dispose of plastics needed for efficient value chains, products need to be designed so that they are easily sorted by their type of plastic and don’t contain multiple types of plastic in one product. This will aid plastic recycling logistics and make recycling technologies less expensive. Multilayered materials containing multiple different layers of plastics can not be recycled and have already proven to be a headache to both consumers and recycling facilities.
Plastic recycling processes are divided into four categories: primary, secondary, tertiary and quaternary recycling(Ryberg et al., 2018). Primary recycling is only covers pre-consumer plastic waste. Cuttings, trimmings and other fall-out waste from within the factory re-enter the production process. However, secondary, tertiary and quaternary recycling are all applied to post-consumer plastic waste.
9 Secondary recycling is also called
mechanical recycling or plastic reprocessing and is the most widely applied method for plastic recycling. Here, plastic waste can be upcycled to re fullfil the function that the recycled plastic was originally made for. Though, most plastic waste can only be downcycled with secondary recycling
as the process often uses heat to re-extrude the plastics into new shapes. Heat could drastically lower the quality of the recycled product, but outdated legislation and difficult sorting of plastics and colors pose obstacles as well.
Tertiary recycling degrades plastic waste into its monomers allowing the production of high quality monomer resources that can then be used again to produce new high quality plastic products.
The least sustainable option for processing plastic waste is quaternary recycling. Here, plastic is incinerated together with other waste for the purpose of energy recovery after which the ashes are used to fill up roads. Though in my opinion, this is still landfilling waste.
Most plastic recycling efforts fall into secondary recycling and rely on physical properties. Thermal recycling, which heats plastic to its melting temperature to mold it into new products, can greatly reduce the quality of the
plastic product. This means that old plastic bottles can’t be formed into new plastic bottles with the same quality. Though, PET bottles have proven the only exception to this rule, the PET recycling industry knows large losses as a result of screening and filtering methods to improve the purity of the recovered PET flakes. Only, clear, colorless PET flakes from separate PET collection systems can be used to reproduce high quality food grade recycled plastics. Contaminants as PVC and additives to PET bottles can greatly reduce the quality of PET upon heating and remolding. To ensure high quality bottles for the food and beverage industry, recycled PET is so far still mixed up with virgin PET(Dvorak & Fletcher, 2013; Thoden van Velzen, E. U., Brouwer, & Molenveld, 2016).
As such, it could
prove beneficial to find a
technique/method/process that is able to separate PET from these microparticle contaminants that hinder upcycling into high quality PET materials.
Hence, chemical recycling, as a form of tertiary recycling, offers a solution by breaking the polymer down into the monomers that it was originally produced from. Figure 5 presents a recent analysis done by CE Delft showing that chemical recycling of PET produces more CO2
equivalent greenhouse gas emissions than mechanical recycling, but it still produces less CO2-eq emissions than incineration (quaternary
recycling). Nonetheless, this process is both energy and resource intensive, and ,therefore, expensive. As such, it is important to find more sustainable, and possibly more economically viable, alternatives for tertiary plastic recycling that can degrade plastics into its monomers and Figure 5: Climate impact of different PET processing strategies. CO2-equivalent
emissions per tonne PET input from using (from left to right): Waste incineration (quaternary, Storage, Chemical recycling (tertiary), Mechanical Recycling
(secondary).
10 thus allow for the production of high quality
recycled plastics.
By using enzymes, less hazardous chemicals are needed to hydrolyze PET into its monomers. This can make the process of PET recycling into high quality plastic products more sustainable by using fewer chemicals, as well as safer by using less hazardous chemicals. These are both sustainability goals that should be aimed for.
In addition, enzymes are highly selective, which means that even when the plastic feed is not 100%, we might obtain a 100% monomer product as the enzyme only cleaves one specific type of chemical structure. As a result, more PET waste can be tertiary recycled into high quality plastic materials instead of being downcycled or even incinerated for being too polluted with (other plastic) contaminants. This will lower the need for virgin plastics and in turn lower the emission of greenhouse gasses while creating a more circular plastic economy.
Challenges for Economic Viability of PET Biorecycling
Until now, PHE’s did not seem to be efficient enough to be implemented as a method for tertiary recycling. Several aspects of PHE needed drastic improvement before reaction conditions could be optimized for the biocatalysis of PET hydrolysis.
The main problem with the biodegradation of PET is that PET is crystalline, which hinders enzymes from getting in contact with the ester linkage. As such, it would be wise to elevate the reaction temperature to above the PET glass transition temperature (Tg). Wild type PETase is deactivated/denatured before reaching this temperature.
So a solution would be to bioengineer an enzyme with high PETase activity that is thermally stable around the Tg of PET. Research has already engineered a mutant PETase that showed improved thermal stability compared to wild type PETase, but this had to be improved to withstand higher temperatures of those around the PET Tg
as well. Though, the improvement in thermal stability was indeed promising for future research. Perhaps immobilization of enzymes could improve the thermal stability even further, creating a very appealing industrial process where the monomer containing liquid is easily separated from the catalyst.
Furthermore, the final biocatalyst needs to be of such an efficiency that the total production costs are similar to that of virgin PET monomers. Only then, does this technique have a promise of profitability, meaning the process can be up scaled. If PET can be easily separated from other types of plastic, logistics will also help reach economic viability of PET recycling with biocatalysis.
Note, that the final catalyst might not be a PETase mutant. It could be a different type of bioengineered hydrolase that has been inspired by PETase or its mutants or perhaps a new PET degrading bacteria. But when it comes to the bioengineering of catalysts, the field does run into some constraints. The main challenges that biocatalysis faces are the understanding of protein folding, the hydrophobic effect, the complex effects of organic solvents and the understanding of catalysis itself (Chapman, Ismail, & Dinu, 2018a).
From an economic perspective, the final goal would be for biorecycled PET material to be competitive with virgin PET. Recycled PET would have to be traded at an estimated 1000 euros per tonne to be competitive with virgin PET. Clean high quality recovered PET flakes are currently at about 960 euros per tonne (Dvorak & Fletcher, 2013). Therefore, biorecycled PET could never be competitive with virgin PET if produced from clean high quality recovered PET flakes.
Another argument in favor of PET recycling was proposed saying that the price of plastic waste is not as volatile as that of crude oil as society uses quite a fixed and growing amount of plastic continuously. This produces a continuous plastic waste stream (The Manufacturer, 2019). In turn, this can result in plastic products that are not prone to price
11 fluctuations as strong as those of virgin PET (The
Manufacturer, 2019). Secured prices and feed stocks can lead to a stronger market position for recycled PET.
However, what is more interesting, is to research the use of PET waste that is currently not recycled into high quality plastic. Being able to produce low valued plastic waste into high quality food-grade plastic makes for a much more interesting value chain from a financial point of view. Recovered colored PET bottles have a very low market value in the range of 50 euros per tonne (WRAP, 2019).
How far advanced is biorecycling?
One company called CARBIOS is actively researching and bioengineering enzymes for the purpose of biorecycling PET and other polymers. Newsflashes published at the CARBIOS website have shown that the company is very busy with improving plastic biorecycling efficiencies. Additionally, Carbios recently published an article in Nature presenting an enzymatic PET depolymerization of 90% within 10 hours (Tournier et al., 2020). This equals a depolymerization speed of 16.7 gTA/L/h or
200 g per hour per kg of PET suspension, using 3 mg of enzyme per gram of PET. Calculated per gram of enzyme, the total reaction rate was 27.9 gTA/L/h/genzyme. The mutant
responsible for these results is an engineered leaf-branch compost cutinase (LCC) of which the wild type outperformed all other tested wild-type enzymes for degradation of amorphous PET. Further
tests with LCC on crystalline PET showed that the largest barrier for efficient PET degradation was the thermostability of the enzyme. Subsequently, site directed mutagenesis allowed for the development of a more thermostable enzyme through several mutations that resulted in the aforementioned degradation rates. Though, the initially measured degradation rate was faster than the one reported as the final rate. The maximum yield was determined at 42.1 gTA/L/h or in relation to the amount of enzyme: 70.1 gTAeq/L/h/genzyme (where TAeq means terephtalic
acid equivalents).
In addition to labscale tests, the researchers from CARBIOS also presented the biorecycling potential with a 150L pilot-scale demonstration. Here, less enzyme was used to compensate for estimated enzyme costs, the depolymerization reaction was performed with 2mgenzyme/gPcW-PET (PcW-PET refers to
post-Figure 6: Estimated PET Biorecycling Costs
* Left: Considering that reactant costs represent 65% of total TA production costs. Right: Considering that that is 75% instead.
Reactants:
Costs for producing
1 tonne of TA
Reference
PcW-PET colored (LOW)
€ 173,81 (WRAP, 2019)
Enzyme ^^
€ 53,30
(Tournier et al.,
2020)
Solid NaOH
€ 195,77
Estimation
Water
€ 8,54
(Van der Zeijden
et al., 2009)
H2SO4
€ 171,45
Estimation
Extrusion (front-end)
€ 100,00
Estimate
Micronization (front end) € 100,00
Estimate
Filtration (back-end)
€ 100,00
Estimate
Discolouration (back-end) € 100,00
Estimate
Crystallization (back-end) € 100,00
Estimate
Total Reactant Cost*:
€ 1.102,88
Total Electricity Cost:
€ 93,51 (Statline, 2018)
Income from Na2SO4 Sales: € -100,00
Estimate
Additional Costs
(65%reactants,35%OTHER)
€ 500,35
Estimate
12 consumer coloured waste PET). Note that this
PcW-PET material is the under-valorized colored PET waste that is separated from clear PET flakes that are used for the mechanical recycling of PET. Only the clear PET flakes are used for mechanical recycling, the colored PET waste can not be mechanically recycled into new clear PET bottles and is burned or downcycled into low value plastic products.
CARBIOS might pose a solution for all the PcW-PET that can not be mechanically recycled into high value plastic (bottles/containers/etc.). Figure 6 presents a gross estimate of the economic viability of the biorecycling process used in the pilot scale trials presented by the researches from CARBIOS.
To calculate the operational costs several assumptions had to be made and several estimates were required to get to the final production cost of around 1597 euros per tonne TA. The input feed consists of undervalued colored PET waste. Although, one source mentioned colored PET waste bottle prices as low ass 50 euros per tonne, we decided to use an estimated 150 euros per tonne for colored PET waste based on prices promoted on google search results. Another of the assumptions made was to add 100 euros of operational costs for the processes of extrusion and micronization on the front-end of the recycling process, and for possible filtration, decolourization and crystallization at the back-end of the process. The publication in Nature by CARBIOS’ researchers mentioned decolourization and crystallization as back-end steps before producing plastic bottles, but do not mention any other back-end steps. As such we decided to add at least one filtration step.
To determine the energy needed for heating the reaction solution, several assumptions were made. If the factory was located in the Netherlands, the average outside temperature would be 11 degrees Celsius, resulting in an average heating of 54 degrees to reach the preferred temperature of 65 degrees Celsius. A processing time of 10 hours was
assumed as the actual reaction time for the pilot scale test was not mentioned in the publication. The final energy costs were 93,51 euros, but this considered a batch reactor made from a steel container with no insulation (Statline, 2018). Adding insulation may save some energy, but practicality all depends on the design of the reactor and heating systems.
For a hypothetical biorecycling facility producing 500.000 tonnes of TA annually, two batches per day of 6000m3 (rounded up) were considered appropriate if the factory operates 275 days a year. Furthermore, it was assumed that 65% of production costs are determined by reactant costs and the other 35% are determined by energy costs, labor costs, etc.. As such, the row ‘additional costs’ was formed as (reactant costs) divided by 65, multiplied by 35 minus (heating costs). Furthermore, income from Na2SO4 was estimated to reduce operating costs
by 100 euros per tonne of TA produced. However, this does not seem to result a very viable recycling process.
TA prices seem to vary greatly between 450 and 1500 euros per tonne. Reasons for this could be purity or geographical differences between manufacturers. If the quality of the TA produced using biorecycling is of sufficient purity and geographical aspects are stimulative, the recycled TA may be sold for 1500 euros per tonne, resulting an unprofitable biorecycling process.
But, if process optimization could lead to reactants making up 75% of operational costs instead of 65% as estimated (75% is the case for many established industrial processes), production costs can be reduced by about 200 euros per tonne. An example of process optimization could be the addition of insulation to reduce heating costs. In addition, the calculated operational costs could be overestimated as the recycled EG is not considered as a valuable by product. This could hypothetically account for a cost reduction of around 72 euros per tonne TA considering that EG has an estimated market value of 460 euros
13 per tonne. Note that the EG value will probably
increase significantly with the world recovering from the COVID-19 crisis. Furthermore, it is unfortunate that reaction times were not mentioned by CARBIOS as this is a major factor in the viability of the process. If the biorecycling process takes too long, operational costs increase drastically due to higher labor costs, maintenance, higher energy bills, etc.. From the lacking information on the processing time we must conclude that the biorecycling process is not efficient enough yet to result a profitable process. No explanation has been given in the paper as to how exactly the process deals with waste and by products as EG and Na2SO4
(Tournier et al., 2020). But looking at the jumps that CARBIOS has made regarding biorecycling, they may also be just a few steps away from optimizing the process enough to start making profitable business cases.
However, looking at the large amount of chemicals needed for this recycling process, it does not incorporate any other sustainability goal than to recycle waste. Although it does recycle waste that would otherwise be burned or downcycled, this process uses 2 equivalents of NaOH instead of a catalytic amount of 0.01 equivalents that is used in other chemical recycling processes.
From Figure 6, we can conclude that the viability of this process is greatly dependent on both the cost of the recovered PET material as well as the reaction time, the amount of operational steps in the recycling process and the resource intensity of the steps.
Upon contacting the company Plastic Recycling Amsterdam, we were informed that highly pure (99.5 %) recovered colored PET flakes in the Netherlands can be sold for 500-800 euros. Unfortunately, this price would render the biorecycling process unprofitable.
Compared to other waste separators, Plastic Recycling Amsterdam produces a very pure waste stream. Despite the high purity of the PET waste stream from Plastic Recycling Amsterdam, legislation does not allow the
remolding of new PET bottles from any of their PET waste streams as it has been in contact with a lot of other types of waste. Outdated legislation has been a large obstacle for many different waste recycling companies to find proper buyers for their recycled products. As such, care should be taken to properly communicate the needs of new and emerging recycling strategies with authorities, including legislational needs for biorecycling.
It would be interesting for future research on biorecycling to find out what the best way is to obtain a cheap (colored) PET waste streams to recycle. To name an idea, perhaps combined technologies could facilitate the recovery of mixed plastics from landfills. Next to facilitating society with recycled plastics, this would reduce leaching of hazardous additives in plastics from the landfill to groundwater and other parts in the environment, and it would lower biodegradation of plastics in the environment where it would only emit more CO2
gasses.
Discussion on the Biodegradation of PET
To determine the faith of biodegradation of PET, performance criteria, production criteria, compatibility with current recycling and production processes and additional pro’s and con’s must be determined.
To determine the rate of biodegradation of PET, the application is of great importance. For example, if digesting organic waste that is contaminated with PET for the production of organic fertilizer, then the PHE should have a Kcat of about 10 s-1 and a Km of about 1. This is based on the knowledge that the biodegradation of natural compounds by for example cellulases exhibits a similar catalytic rate and substrate affinity. Research should aim at a hypothetical enzymatic activity that hydrolyzes 1 mol of PET every 14 seconds per 1 mole of catalyst in an industrial digester. However, due to the high crystallinity of PET, bioengineering research is nowhere near the biodegradation of PET waste in a ‘dry’ environment (dirt, solid organic waste).
14 There are two drivers that may push
research towards the development of methods to biodegrade plastics (Chapman et al., 2018a; Choi et al., 2015). Initially, biodegradation can prevent the incineration of organic waste contaminated with plastic. So, instead of burning the contaminated waste, it can be composted still and be used as fertilizer after digestion. This solution would help close a loop for organic waste and prevent but it would not prevent CO2 emissions as the plastics are degraded by bacteria that use the plastics for their metabolism causing them to emit CO2. As such it seems that avoiding
plastic contamination in organic waste is the most sustainable and efficient way to keep agricultural soils free from plastics as much as possible.
Secondly, biodegradation could pose a solution to the unintended losses of plastics to the environment through waste water. For example, when washing laundry, fibers are released from all types of fabrics including synthetic fibers as polyesters which persist in the environment. These fibers are microplastics and enter our food chain when they accumulate in seafood and drinking water reservoirs. This problem will become even bigger as more and more plastics are being used by society in the coming decades.
Regarding the lack of research into the biodegradation of microplastics for the use in waste water treatment, it seems that this application is nowhere near efficient or other treatments are more promising. Researchers in Finland determined that conventional methods in WWTPs already remove around 80% of all microplastics in sewage water, but 20% still reaches surface waters if no new filtration technologies are added (Talvitie, 2018). Research into the removal of microplastics from waste water through biodegradation could add a new perspective on the removal of microplastic particles as microbes that remove plastics could be added to the active. In this way, no extra operational units would have to be added to the WWTPs. Additionally, intentional losses of
microplastics through self care products should be avoided at all costs with the help of authorities.
From the plastic producer’s point of view, it is understandable that the release of PET degrading bacteria might seem like a threat to the production and use of PET. However, as mentioned in the previous paragraph, all biodegradable plastics need specific conditions and microbes in an industrial environment to efficiently degrade. As an example, the recently developed PEF material is also biodegradable and shows superior performance over PET. As such, PET biodegradation could potentially save the future of PET use and production which already has an established value chain.
Before the discovery of I. Sakaiensis, other organisms were found with the ability to degrade plastics, but none of them were specialized to prioritize any type of plastic. Those organisms as well as I. Sakaiensis need very specific conditions to degrade the plastic. Either specific temperature ranges, moisture levels, additional nutrients or a high enough concentration of bacteria are needed to reach high level of efficiency. These properties may create misconceptions about biodegradable materials in general (Neufeld et al., 2016). Products that are labeled as ‘biodegradable’ or ‘compostable’ hardly degrade at all in most environments or in the average compost pile, because they need specific bacteria or conditions to be fully degraded. This is a problem and will become a bigger problem in the future as the terms biodegradable plastics and bio-based plastics are already creating confusion among uninformed consumers.
Biodegradation of plastic does not pose a solution to prevent or remove litter with beneficial consequences. Biodegradation could only degrade plastics, while releasing harmful toxicants that are embedded in the plastic structures. In addition, it would create CO2
emissions that the global community is now trying to combat. In addition, the promotion of biodegradation of plastics in marketing strategies
15 should be done cautiously as biodegradation
would not prevent the use and production of these plastics and might even promote littering and mismanagement of waste streams. It should be communicated that all forms of biodegradation of plastics need a specific environment (industrial digesters) and that littering is, therefore, inherently harmful for flora and fauna (Rochman et al., 2013).
However, research into the biodegradation of PET and other plastics should continue and be stimulated. By funding biodegradation research, genetic libraries can be expanded to an extent where enzymatic behaviour can be predicted according to genetic code or amino acid sequence. This will speed up research to find more and new enzymes to biorecycle PET but, perhaps more importantly, other plastics as well. PET is the most widely recycled plastic in the world but there are several other plastics that are used to a larger extent than PET, but which are hardly recycled.
There might be a use for complete biodegradation of PET in landfills, which is to speed up the natural selection of bacteria to degrade PET faster as well as to speed up the natural selection of bacteria that degrade other types of plastics. A such, on a small scale, the landfills could be used incidentally to cultivate new microbial species to degrade PET and other plastics which would aid bioengineering research.
Discussion on the Biorecycling of PET
Driving research into the biorecycling of PET seems more promising than biodegradation as multiple constraints can be omitted and CO2
emissions can be reduced. With more research, PHE’s can provide the industry with simpler, safer and more environmentally friendly recycling processes. In addition, biorecycling can facilitate the recycling of colored PET waste for which no recycling strategy was established yet to produce high quality PET products.
Plastic recycling is the most sustainable way of disposing of plastics only if it replaces the
use of virgin plastics. If we continue to use plastics where alternatives should be found, a successful plastic recycling system might even stimulate the use of plastics instead, which would lead to a continued stream of unavoidable unintended losses of plastic to the environment.
An example of an industry that conventionally uses enzymes is the paper and pulp industry. The paper and pulp industry started using enzymes that were figuratively ‘low-hanging fruit’, but bioengineering allows us to try and use ‘high-hanging fruit’ as well (Goldsmith & Tawfik, 2012). To find enzymes with desirable characteristics through bioengineering, two methods may be considered: selection or screening. Screening will allow for the parallel testing of 10^6 to 10^12 different variant, where selection will only allow to test 10^2 to 10^4 variants individually. Screening might seem quick, but it uses random mutations which might be handy if one is lacking a large library of enzymes with the desired activity. Though, if such a library of enzymes is already available, then site directed mutagenesis would be wiser to apply as it uses mutations directed to alter specific areas of the wild type enzyme that are expected to improve its performance. Research using site directed mutagenesis has so far shown great potential for optimizing the efficiency of PET biodegradation on a lab scale (Austin et al., 2018).
The French company CARBIOS has committed itself to researching and scaling up the biorecycling of PET and other polymers, and plans to start the biorecycling of PET with a pilot plant in 2021. It has recently patented a novel biorecycling process using a bioengineered group of esterases. Press releases inform that the company has reached a staggering 97% conversion of PET in 16hrs. Recently, a paper from this same research group was published in Nature, informing that 90% could be reached in 10 hours. CARBIOS has planned to build a full scale PET biorecycling facility in 2023 (Maddox, 2019).
Although this sounds promising, our estimates from Table X show that there is still
16 some uncertainty to the economic viability of this
process.
These landfills could be a large resource for biorecycling plants as enzymes are highly selective towards these substrates. This means that a waste stream could be treated with an enzyme that hydrolyzes only one type of plastic, removing it from the waste mixture. If another type of plastic hydrolyzing enzyme is available, that same waste stream can be treated with the second enzyme to remove another type of plastic and a third, fourth, fifth enzyme etc. until all plastics are removed from the waste stream. In this way, plastics can be hydrolyzed as well as purified.
Earlier market studies already predicted that the global market for enzymes would grow to be worth over US$ 10 billion by the year 2024, but more recent market reports sound even more optimistic (Chapman, Ismail, & Dinu, 2018b). All segments of the enzyme market are expected to grow to a similar extent including the polymerases sector in which we could categorize PETase. Though, with the outbreak of COVID-19, the demand for enzymes is predicted to increase as a result of a growing food and beverage market as well as a growing demand for enzymes typically used for the production of drugs in the pharmaceutical industry (Grand View Research, 2020).
Although the demand for industrial enzymes will increase as a result of food and beverage consumption, this does not guarantee that more funds will become available for research in plastic biodegradation. On the contrary, a threat is posed if sustainability goals are considered to be a threat to the recovery of our economy.
The political decisions made to tackle short term threats to our existence will have to consider the damage they do or prevent for serious long term threats to our global society. Oil has now been proven to be an unreliable investment, whereas plastics are used intensively and daily by the vast majority of our society.
Conclusions
PET Biodegradation should be researched for the final purpose of removing PET microparticles from waste water in WWTPs. In addition, PET biodegradation research will expand the genetic libraries available to researches that wish to bioengineer existing microbes or enzymes for the purpose of biodegradation and/or biorecycling. Biodegrading plastics in organic compost is time-consuming, expensive and does not significantly improve the quality of the final fertilizer. As such, the latter would not result profitable investments.
Furthermore, biorecycling of PET seems quite far advanced in a select group of researchers and should be developed further. If the recycled TA can be sold for 1500 euros per tonne and if operational costs can be lowered to 1300 euros per tonne, PET biorecycling could be profitable. However, operating times need to be communicated to determine economic viability. Geographical factors and market prices for reactants seem to greatly affect the final production costs of recycled TA. If biorecycling of other plastics could be developed as well, cheaper waste plastics have a chance to be recycled into high quality plastics also instead of being burned, landfilled, or downcycled. Perhaps, biorecycling could be researched for the purpose of treating mixed plastic waste, allowing for separation and depolymerization into highly pure recycled input streams for plastic production. Acknowledgements
I would like to thank Plastic Recycling Amsterdam for sharing their knowledge on the plastic recycling industry in the Netherlands. Furthermore, I would like to thank Jan van Maarseveen for reading and grading this thesis and for attending the presentation of this thesis. Also, I would like to thank Gert-Jan Gruter for guiding me through the writing of this thesis as my supervisor, his knowledge about the plastic industry helped a lot throughout the process of writing this thesis.
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