Closing the recycling circle
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news feature Credit: Getty Images / eleonora galli Closing the recycling circle Capturing the value back from plastic waste has been the holy grail of recyclers. Biotechnology is taking us closer to a solution. Laura DeFrancesco I n April, the French industrial biotech glycol and terephthalic acid (benzene- accumulating in landfills and oceans as company Carbios published work that 1,4-dicarboxylic acid) — and reform them plastic waste, remains to be seen (Fig. 1). signals a new era in the world of plastic into a bottle that matched bottles made from In the meantime, the hunt is on to find and recycling. In a public–private partnership virgin PET, completing the cycle. hone enzymes, organisms or even consortia with the Toulouse Biotechnology Institute, What preceded this accomplishment was of organisms that can take a bite out of the company researchers reported in Nature decades of work by the research community plastic waste problem. the recycling of plastic waste into fully scouring landfills for microbes that could functional plastic bottles. Most recycled live on plastic, likely because they have Getting after PET plastic bottle waste — roughly 35 million enzymatic pathways capable of metabolizing PET, a semicrystalline polyester made up tons per year — is turned into pellets or these xenobiotics. Reports of such bacteria of repeating units of ethylene glycol and material of lesser value, or “downcycled” date at least as far back as the 1990s. dimethyl terephthalate is an important in industry parlance. Applying some clever This is the easy part. Going from there industrial polymer, used in packaging genetic engineering tricks to leaf compost to an activity that works efficiently and and textiles. Although it currently is the cutinase (LCC), the research team was able on a large scale is not. most recycled of the big five polymers to enzymatically reduce plastic waste made Whether Carbios’s approach for (polyethylene, polypropylene, PET, polyvinyl from poly(ethylene terephthalate) (PET) into PET will work for the tons of other, chloride and polystyrene), industrial its two component molecules — ethylene harder-to-degrade olefins and polyesters, processes for recycling PET degrade the Nature Biotechnology | VOL 38 | June 2020 | 665–668 | www.nature.com/naturebiotechnology 665
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Poly(ethylene terephthalate) According to John McGeehan, director of
Polyurethane the Centre for Enzyme Innovation at the
University of Portsmouth, it was in part
Polystyrene
due to the timing of their publication,
Polyvinyl chloride
which coincided with the airing of the BBC
Polyethylene, high density series Blue Planet. By several accounts,
Polyethylene, low density this program was a turning point, at least
Polypropylene in Europe, capturing the attention of the
Others
public to the problem of plastic waste. It
had immediate benefits to McGeehan, as
0 5 10 15 20 25 GlaxoSmithKline, located a few miles up the
European plastic demand (%)
road from his institute, reached out to offer
their fermenters for producing the enzyme.
Fig. 1 | The demand for plastic resins in Europe during 2017 as a percentage of total plastic demand. This connection in turn led to an important
Source: PlasticsEurope. industrial insight that the researchers might
have missed. “The first thing they said
was ‘What is the product of this reaction,
ethylene glycol and terephthalic acid?
monomers, leading to the loss of potential other known PET-degrading hydrolases Do you know the market price of this?’”
value of the byproducts. So researchers and the MHET hydrolase being perhaps he recalls. He was told this would “markedly
have been looking at enzymes as a less totally unique. Also remarkable is that impact the viability of this system.”
destructive, more ecofriendly way to recycle the bacteria appear to have evolved to use Reports of other promising enzymes
PET, to both spare the environment and PET as an energy source, even though were to follow. Wolfgang Zimmermann
recapture the plastic’s value. Whereas the material entered into wide use as and coworkers at Leipzig University
activities against ester bonds exist in nature, recently as the 1970s. described naturally occurring hydrolases
in order to work on something like a bottle, The Japanese group and others have from a thermophilic actinomycete,
enzymes would have to thermostable, as drilled down to learn more about the Thermobifida fusca KW3 (TfCut), and an
they must function at temperatures at or I. sakaiensis enzymes. Several groups have LCC isolated from a compost metagenome.
above the glass transition of the polymer. solved the crystal structures of the two They improved on the activity at high
This is the point at which semi-crystalline, enzymes. A group of collaborators from temperatures of TfCut by replacing several
largely impenetrable structures become National Renewables Energy Laboratory amino acids in the substrate binding
partially amorphous, allowing enzymes in Golden, Colorado (NREL) and the pocket to make it more closely resemble
access to the polymers. University of Portsmouth in the United a cutinase. Also notable is the 2018
The first real breakthrough in the Kingdom have improved upon the activity publication by Richard Gross at Rensselaer
modern era from enzymatic approaches to of the PETase by engineering the catalytic Polytechnic Institute describing a LCC that
recycling PET was reported in 2016, when site to resemble that of previously studied, outperforms the I. sakaiensis enzyme at
Japanese scientists discovered a bacterium more active cutinases. By modifying just two high temperature, although its activity was
that could both degrade and assimilate PET. amino acids, they improved the enzyme’s still too low. However, they discovered that
Through analyzing hundreds of samples ability to degrade crystalline PET, suggesting low activity at high temperature was due
containing consortia of microorganisms a path to creating industrially useful tools. enzyme aggregation, a problem they solved
living on plastic bottles in a recycling bin, This was a heady time for the team, used by expressing the protein in Pichia pastoris,
they isolated a novel bacterial species they to working in relative obscurity, garnering which glycosylated the enzyme at its natural
named Ideonella sakaiensis 201-F6 that them a spotlight in the popular press. glycosylation sites.
can degrade plastic film made of PET.
After attaching itself to the plastic, this bug
secretes a PET-degrading enzyme called
PETase; takes up the main byproduct,
mono-2-hydroxyethyl terephthalate
(MHET); and degrades it further into usable
metabolites by means of a second enzyme,
MHET hydrolase (Fig. 2).
Although this report garnered a lot of
attention from the press, the dirty little
secret was that the enzymes performed
poorly, in comparison to previously
described enzymes from bacteria and fungi
capable of degrading the ester bonds holding
the PET polymer together. It took six weeks
for these bacteria to fully degrade a PET
film, which itself is not the major form of
PET found in landfills. Nonetheless, the
finding was noteworthy for its novelty: the Fig. 2 | Structure of PETase from I. sakaiensis. The three-amino-acid catalytic site is in cyan and the
bacteria and both activities were unique, the substrate 2-H(MHET)4 molecule in orange at the active site. Reprinted with permission from S. Joo et al.
PETase showing only modest homology to Nat. Commun. 9, 382 (2018).
666 Nature Biotechnology | VOL 38 | June 2020 | 665–668 | www.nature.com/naturebiotechnologynews feature
Box 1 | Funding ramps up
Whether it was the airing of the series in that all the program release dates are national labs, Bio-Optimized Technologies
Blue Planet or the horrifying images from 2019 or later. to keep Thermoplastics out of Landfills
of sea life engorged with plastic, In the United States, for example, the and the Environment (https://www.bottle.
funding agencies around the globe final act of former Secretary of Energy in org), whose mission includes “efficient,
are finally putting money where their the Trump administration Rick Perry as scalable technologies to deconstruct and up
collective mouths have been. Numerous he departed in late 2019 was establishing cycle plastic.” The group brought together
pronouncements about the plastic the Plastics Innovative Initiative, the stakeholders from all segments in the
problem have been made, especially first major overarching initiative at DOE supply chain to brainstorm the problem of
since the Chinese stopped accepting it addressing the problem of plastic waste. plastic waste. Companies “have heartburn
(as if that were a solution), but it appears Under this rubric, Nichole Fitzgerald, a over the issue of packaging something as
that, until recently, not much was behind technology manager at DOE’s Bioenergy transient as food with something that
the pronouncements. The funding Technologies Office, has helped establish a will be in a landfill for hundreds of years,”
opportunities (see table) are notable consortium of three universities and three says Fitzgerald.
Funding initiatives for recycling plastic waste
Name of initiative Funding or sponsoring Amount of funding Date announced Goals
agency available
Plastics Innovation Challenge DOE $50 million November 2019 Umbrella agency
Bottle.org DOE $2 million December 2019 Keep plastics out of landfills
BOTTLE Funding Opportunity DOE $25 million March 2020 Create new recyclable plastics and a circular
economy for recycling plastic waste
REUSE ARPA-E Not specified August 2019 Create feedstock from plastic waste
REMADE DOE $12 million (not all November 2019 Reduce embodied energy and decrease
for plastic waste) emissions
SBIR DOE Office of $1 million for December 2019 Develop novel utilization strategies for ocean
Energy Efficiency and phase 1 plastic waste
Renewable Energy
Horizon 2020 European Union €100 million 2020 Improve recycling possibilities
($109 million)
Enabling Research in Smart UKRI, UK National £8 million February 2020 Research recycling technologies and other
Sustainable Plastic Packaging Environmental ($10 million) topics relating to plastic packaging
Research Council
Emerging Frontiers in Research NSF $30 million November 2019 Engineer the elimination of end-of-life plastics
and Innovation
ARPA-E, Advanced Research Projects Agency–Energy; NSF, US National Science Foundation; REUSE, Recycle Underutilized Solids to Energy; REMADE, Reducing Embodied Energy and Decreasing
Emissions; SBIR, Small Business Innovation Research; UKRI, UK Research & Innovation; DOE, US Department of Energy.
Getting cute with PET and Sophie Duquesne, researchers at the disulfide bridge. With these modifications,
Carbios and its collaborators had a different Toulouse Biotechnology Institute, they they could achieve, in only ten hours, a
goal in mind. According to the company’s evolved a cutinase (chosen as a good starting 90% depolymerization of amorphous PET,
CSO, Alain Marty, they wanted not only point as its native activity bested several something others in the field consider
to improve the ability of an enzyme to other hydrolases that they considered) remarkable. “Kudos to them. It’s really
depolymerize PET, but also to recover that eventually led to a modified LCC that quite impressive,” says Jennifer Le Roy,
the two monomers (ethylene glycol and was thermostable at PET’s glass-transition chief technical officer of a California-based
terephthalic acid) and use them to recreate temperature (75–78˚C). They did this by startup, BioCellection, a recycling and green
plastic bottles made from PET. Marty says using computer-aided targeted mutagenesis materials company.
when Carbios CEO Jean-Claude Lumaret to design mutants with changes at 11 sites Although pretreatment was still
tasked him with recycling PET, his initial in the substrate binding groove, did required to make the polymers accessible
reaction was that it would be impossible to site-specific saturation mutagenesis, — the researchers used a combination of
do economically. And his results for the first tested 209 thus-generated variants on extrusion and micronization, which Marty
few years seemed to prove him right, as they commercially available amorphous PET, says is “industrially relevant” — the usual
were getting but a few percent conversion and isolated several with activity that was mechano-thermal methods employed for
after weeks of degradation. But the discovery 75% higher. They further enhanced the recycling PET leave the monomers degraded
of the enzyme from I. sakaiensis gave them stability at high temperature of one such to the point that they can only be used to
some hope. With collaborators Isabel André variant by introducing one stabilizing create lower value products, like carpet
Nature Biotechnology | VOL 38 | June 2020 | 665–668 | www.nature.com/naturebiotechnology 667news feature
liners. But using recovered and purified ecofriendly chemical method when they hopefully of value. I think that’s where
terephthalate monomers, the Carbios team realized their process would never be biotech has a really cool role to play,”
was able to reconstitute a bottle with the price competitive. says Beckham.
mechanical properties of virgin PET, closing Furthermore, the Carbios method still It may be premature to rule chemistry
the proverbial loop. requires an energy-intensive pretreatment out of the picture, as it might take a
Several partnerships with large step and, at least as described in their paper, combination of chemistry and biology to
commercial players will both enable uses petrochemical-based ethylene glycol solve the mixed waste issue. At NREL, for
Carbios’s team to scale up enzyme monomers in the rebuilding phase. “When example, they are using a combination of
production (Novozymes) and assist with companies claim that they can fully degrade biological funneling with chemical catalysis
the creation of an industrial-scale platform biodegrade PET bottle, it’s not completely to convert the waste from lignin (another
(Pepsico, L’Oréal and Suntory, among true. They have to do some chemical or aromatic polymer) produced when the
others). Carbios sees their process becoming mechanical pretreatment. It’s not fully polymer is used to make biofuels into
industrialized by 2024, with actual recycled biological,” says Zimmerman. He calls such a several intermediates, including adipic acid,
bottles hitting the market in 2025. process “semi-green.” a precursor to nylon.
Carbios has another iron in the fire: And that’s just PET; there are the other Another interesting possibility is creating
a self-degrading plastic. In collaboration big four plastics. “It would be really exciting consortia of microbes, rather than focusing
with the group at Toulouse, they created to see the same enthusiasm to enzymatically on individual enzymes. Zimmerman’s
an extremely thermostable enzyme, recycling PET to more challenging plastics,” group at Leipzig had focused on enzymes,
another feat that Marty originally thought says Le Roy. but recently started taking a closer look at
impossible. This enables the enzyme to Gregg Beckham, who is leader of the organisms and the possibility of expanding
withstand the extrusion temperature of group at NREL and is also heading up a new them into consortia. “That’s what happens in
polylactic acid (170 degrees Celsius), so they US Department of Energy (DOE) program, nature. It’s probably not just one bug doing
can embed the enzyme during manufacture. the Bottle.org consortium (Box 1), that but it may have synergistic activities,” he
A joint venture with Limagrain and is working to fill that gap. He and his says. A new European Union project under
BpiFrance called Carbiolice is taking this collaborators are on the hunt for dozens the auspices of European Research Area’s
project forward. to hundreds more enzymes, to “offer a CoBioTech program called MIPLACE will
bigger buffet,” he says. They are using a be working on this possibility.
Not the end of the story combination of standard bioinformatics Ultimately, industrializing the process
Although Carbios received accolades for screening techniques to fish out of the of enzyme-based recycling of plastic waste
their paper in Nature, which was featured billions of extant microbial sequences those means making it price competitive with
on the cover of the journal, a daunting road that resemble the 25 known activities, thermomechanical processes. At least for
lies ahead. For one thing, PET is one of the followed by machine-learning hidden now, mechanical size reduction as well as
best recycled of the major sources of plastic Markov models to determine those most decrystallization may still be required to
waste, with 50% of it recycled presently. And likely to be thermophilic. “We only fish out create industrially relevant technologies.
technology exists to convert PET into its two the thermophilic ones,” he says. Carbios calculated that the price of the
monomers and create food-grade plastic Another challenge is mixed plastic, enzyme will represent 4% of the price of
containers. However, the challenge is to do which includes materials that blend plastics the virgin PET, but that’s only one part of
it at a price that can compete with virgin as well as streams of mixed plastic consumer the picture. Beckman sums it up: “Whether
PET, according to Le Roy. That’s why so few materials that all get tossed together. To get all that is scalable and economical is the
of these technologies have been adopted, at this problem, researchers are developing $64,000 question.” ❐
she says: “It’s not because the technology methods for biological funneling to separate
doesn’t exist; it’s because they can’t compete out usable monomers from a mixture. Laura DeFrancesco
on price.” Le Roy would know. Her company “You can take mixed plastics and bust them Senior Editor, Nature Biotechnology.
started out working on enzymatic platforms apart into little [bits] and use the selectivity
to depolymerize polyethylene film — a more of biology to convert the mixture of stuff Published online: 21 May 2020
recalcitrant polluter — but pivoted to an to a single molecule that is interesting and https://doi.org/10.1038/s41587-020-0541-0
668 Nature Biotechnology | VOL 38 | June 2020 | 665–668 | www.nature.com/naturebiotechnologyYou can also read
