Bio-Recycling Plastic and New Bio-Packaging—Recent Advances Using Nucleic Acid-Based Tools

Posted in: Nucleic Acids

  • By 2050, There Will Be More Plastic in the Ocean Than Fish 
  • Bio-Recycling Uses Newly Discovered Microorganisms to Reduce Plastics Pollution
  • Nucleic Acid-Based Tools Enable Progress in Reducing Pollution

Introduction

Recently, the media headline “German Scientists Identify New Strain of Plastic-Eating Bacteria” piqued my interest. To learn more, I researched the literature on the utilization of nucleic acid-based tools for bio-recycling. This blog is a synopsis of the current bio-recycling approaches that are being used to target the global plastics problem, including existing types of petroleum-derived (“chemical”) plastics largely used for packaging. We will also discuss bio-packaging, which seeks safe, ecofriendly replacements for problematic chemical plastics. Achieving sustainable plastics for cleaner air, “greener” land, and purer water are all key drivers of these initiatives. 

Before diving into these topics, it’s worth mentioning that an estimated ~900 billion pounds [~400 million metric tons (~400 Mt)>

of plastic will be produced in 2020, and much of it is destined for burial or trash in waterways and oceans. This a huge problem—if not a crisis—that needs to be addressed immediately. According to another estimate, by 2050, there will be more plastic in the ocean than fish by weight. How did we get to this point? A short but informative article titled Plastic Packaging History: Innovations Through the Decades spans from 1860 to the present, and states that the first synthetic plastic, introduced in 1862, was derived from cellulose. Ironically, this ecofriendly material was superseded by chemical plastics until 2000, when polylactic acid (PLA) packaging brought back bio-plastics.

How Slowly Do Plastics Degrade in the Environment?

To put the problem of this massive accumulation of chemical plastics into a sobering context, it is estimated that ~80% of the ~400 Mt is discarded and degrades very slowly. The recalcitrance and impermeability of these plastics, which make them ideal for applications such as food packaging, sterile medical uses, construction, etc., also make them long-lived after being discarded. The exact definition of “long-lived” has been studied by Chamas et al., who used a metric called the specific surface degradation rate (SSDR) to extrapolate half-lives, as summarized in the chart shown here.

Taken from Chamas et al. ACS Sustainable Chem. Eng. 2020, 8, 3494−3511, an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

SSDRs were measured for the 7 standard types of plastic under accelerated and nonaccelerated conditions in land, marine, biological, and sun environments. Results for all plastics varied widely due to differences in chemical composition, shape, and condition. Each SSDR value was then converted into an estimated half-life for the rate of decomposition (depicted below). The resultant half-lives, summarized in Table 1 in Chamas et al., show a correspondingly wide range of values. For example, polyethylene terephthalate (PET) (e.g. single-use water bottle) has an amazingly long half-life of >2,500 years buried in land, but “only” 2.3 years in accelerated sunny conditions, while high-density polyethylene (HDPE) (e.g. pipes) has a half-life of 5,000 years or 530 years under these respective conditions. These millennia half-lives are remarkable, if not shocking. 

Bio-Recycling of Polyesters

Granules of colored polyethylene terephthalate (PET) used as feedstock for manufacturing a wide variety of consumer products.

Broadly speaking, bio-recycling refers to processes in which biological organisms convert old or waste materials into new and useful ones. For example, human waste from community wastewater treatment plants is now commonly converted into plant nutrients and organic soil matter, which serves as the base of the food chain for all life. Bio-recycling in plastics is a relatively new concept, and it is much more challenging given the refractory nature of plastics. Nonetheless, a case involving the polyester PET, pictured here, serves a promising example of what can be achieved.

Carbios, headquartered in France, has developed a scalable enzymatic process in which pulverized PET plastic waste—the kind of plastic found in drink bottles and polyester clothing—is mixed with water and enzymes, heated, and stirred in a bioreactor similar to the one pictured here. In a matter of hours, the enzymes decompose the plastic into terephthalic acid, the material’s basic building block. This can then be separated, purified, and used to make new plastic that is identical to the virgin material. 

Actinomycetes, gram-positive anaerobic bacteria that resemble fungi, 3D illustration.

Interested readers can search the term Carbios in Google Scholar and obtain access to numerous patents. A sub-search of these for PCR or sequencing led to an informative example in a pending patent. Briefly, Thermobifida cellulosilytica, an actinomyces bacterial strain discovered in 2002 in the overheated region of manure compost, was used for expression of cutinase (Thc_Cut1), a hydrolytic enzyme known to degrade aromatic and aliphatic polyesters. PCR-based amplification allowed for isolation of the cutinase gene, with introduction of a 6xHis-Tag at the C-terminus for cloning into a plasmid. 

The resultant plasmid vector was used to transform a commercially available strain of Escherichia coli (E. coli), and the sequence of the Thc_Cut1 gene was confirmed by primer-based DNA sequencing. The transformed E. coli were then grown in culture in order to obtain cell lysate, from which the cutinase enzyme was immobilized using the His-Tag. A 1-g sample of PET plastic was incubated with 5 uM cutinase in 1 L of Tris/HCl buffer, pH 7.0 for 7 days at 60° C with 300 rpm shaking. The amount of terephthalic acid produced was quantified by reversed-phase C18 HPLC, as was the amount of ethylene glycol, using a standard assay. It was found that, under these conditions, 10% hydrolysis of PET occurred (see Addendum). 

The above-mentioned patent applies to other types of polyesters, meaning that it has broader applicability for bio-recycling. This and other advances made by Carbios have led to significant partnerships. Among these, Carbios and L’Oréal founded a Consortium in 2017 to bring enhanced recycling technology to market on an industrial scale. Committed to supporting sustainable development with innovative solutions, Nestlé Waters, PepsiCo, and Suntory Beverage & Food Europe joined the Consortium in April 2019 to help support the “circular plastics economy” recycling technology. 

Bio-Recycling of Polyurethanes 

Reaction of toluene-2,4-diisocyanate (TDI) with ethylene glycol (EG) to form a polyurethane (TDI-EG-Pu). PubChem Sketcher drawing by Jerry Zon.

Background: Readers familiar with organic chemistry will recall that ester linkages, such as those in PET and other polyesters, hydrolyze more readily than amide linkages, such as those in polyurethanes (PUs). PUs are commonly synthesized from diisocyanates and diols, as exemplified with toluene-2,4-diisocyanate (TDI) and ethylene glycol (EG). The hydrolytic stability of amide vs. ester bonds is consistent with degradation of polyurethanes by water being slow compared to polyesters (see type 7 vs. type 1 in Chamas et al. above). A widely used application of PUs is spray insulation, as pictured here.

2,4-Diaminotoluene (DAT). PubChem Sketcher drawing by Jerry Zon.

Previous studies of the hydrolysis of TDI-containing PUs analogous to TDI-EG-PU have confirmed the expected formation of 2,4-diaminotolune (DAT), the structure of which is shown below. The U.S. National Toxicology Program (NTP) Division of the National Institute of Environmental Health Sciences (NIEHS) has issued a report concluding that, based on animal experiments, DAT is “reasonably anticipated to be a human carcinogen” and is therefore subject to strict Environmental Protection Agency (EPA) guidelines. 

Bio-Recycling Discovery: To investigate the fate of DAT released from a TDI-containing PU by degradation, a team of collaborators (Cárdenas-Espinosa et al.) in Germany screened for bacteria capable of degrading both DAT and a representative TDI-containing PU (“Polyurethane diol solution”). As briefly summarized below, a Pseudomonas bacterial species isolated from soil samples taken from “a site rich in brittle plastic waste” was found to grow on this PU oligomer as the sole source of carbon and energy, and consume the DAT released during degradation of this PU oligomer. 

Electron micrograph of Pseudomonas putida (P. putida) taken from wikimedia.org and free to use.

For the isolation of novel bacteria from soil rich in brittle plastic waste (Paunsdorf, Leipzig, Germany), 1-g of each of three samples was dissolved in 9 mL of NaCl 0.9% m/V and diluted in a series of 10–1, 10–2, and 10–3. Aliquots (150 μL) of the diluted soil solutions were added to agar plates containing different concentrations of DAT (2, 5, and 10 mM) as the sole carbon and energy source. After 5 days of incubation at 30°C, bacteria were transferred to fresh plates, and the complete 16S rRNA gene sequence was obtained from the TDA1 genome. The sequence was then aligned with other known Pseudomonas species using the Ribosomal Database Project (RDP) Classifier database. According to Wang et al., the strain was identified as a Pseudomonas sp. strain that showed high similarity to P. oryzihabitans and various P. putida strains. The strain TDA1 was referred to as Pseudomonas sp. TDA1. 

Proposed Degradation Pathway: According to Cárdenas Espinosa et al., this is the first report on the isolation of a pure bacterial culture for growth on DAT, which can also degrade a PU building block. With supporting information from the literature, the researchers then go into a detailed discussion about a series of nine possible metabolic processes (1-9) that could be involved, as shown here. 

Proposed degradation pathway for DAT including extradiol cleavage (5) of 4-aminocatechol in the putative Pseudomonas sp. TDA1. Cárdenas Espinosa et al. Front. Microbiol., 27 March 2020. Copyright © 2020 Authors. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

Conclusions: Cárdenas Espinosa et al. conclude that “[t>

he majority of the enzymes involved in the proposed pathway must be promiscuous regarding their substrate specificity, i.e., they need to accept especially amino substituted analogs. Due to the low steric hindrance of an additional amino group, substrate promiscuity might be favored”. According to the researchers, enzymes involved in aromatics degradation that exhibit significant activity with substituted substrate analogs have been previously reported. Identifying the key enzymes for the degradation of both DAT as the putative degradation product, as well as for DAT as a precursor of PUs (via DAT generation of TDI, see above) could lead to “future development of a novel two-step bio-recycling processes.

Plasticizer Bio-Degradation by Marine Bacterial Isolates

Background: Oceans, lakes, and rivers cover over 70% of the earth’s surface, and they house a massive reservoir of aquatic microbes that have long been screened for uniquely structured compounds as possible new drugs. Portions of these novel chemical structures oftentimes have no known counterparts in terrestrially produced natural products, the enzymes and metabolic pathways are unique to marine microorganisms. Whether these pathways might also play a serendipitous role in the bio-degradation of plastics is an important question.

Many plastics contain plasticizers as additives. These alter the physical and chemical properties of the material, and can represent up to 10-70% of the material’s weight. Plasticizers are not chemically bound to the plastic and are therefore able to leach out, either during consumer use (a safety issue) or after disposal (an environmental issue). Bis(2-ethyl hexyl) phthalate (DEHP) and dibutyl phthalate (DBP) are considered toxic, and they are the two most widely used plasticizers. Newer acetyl tributyl citrate (ATBC) is thought to be a safer alternative.

Bis(2-ethyl hexyl) phthalate (DEHP), dibutyl phthalate (DBP) and acetyl tributyl citrate (ATBC). PubChem Sketcher drawing by Jerry Zon.

Case Study: In a 2020 report in the journal of Environmental Science & Technology, Wright et al. investigated marine bacterial isolates with regard to the degradation of six plasticizers, including DEHP, DBP, and ATBC. Briefly, a natural microbial community obtained from marine plastic debris (shown here) was enriched with each of the six different plasticizers. Forty-two strains were isolated and cultured several times under different conditions. The ten isolates that showed the highest potential for growth with several of the plasticizers were identified by primer-mediated PCR amplification and sequencing of the 16S rRNA gene.

Picture of plastics that the inoculum used for this study came from showing a mixture of plastics. Wright et al. Environ. Sci. Technol. 2020, 54, 2244−2256. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

BLASTN searches of the NCBI database were used to identify the resulting 16S rRNA gene sequences. These ten isolates were then narrowed down to two: Mycobacterium sp. DBP42 and Halomonas sp. ATBC28, based on their robust growth on a variety of plasticizers and diverse phylogenetic origin. The complete genome sequences of the two selected strains were then obtained, followed by extensive proteomic (LC-MS/MS) and metabolic (LC-MS) analyses, as detailed in the full paper.

According to Wright et al., “[t>

his study presents, for the first time, a comprehensive characterization of the plasticizer biodegrading potential extant in biofilms on marine plastic debris, from the enrichment and isolation of microbes to the proteogenomic and metabolomic analysis of degradation.” Molecular analyses of biodegradation by Mycobacterium sp. DBP42 and Halomonas sp. ATBC28) have revealed:

  • an array of esterases involved in the first steps of DBP and ATBC degradation;
  • the use of different mechanisms for removal of the ester side chains from DBP and DEHP;
  • the complexity of induction of the catabolic pathways involved in the degradation of such compounds and;
  • a number of strategies used by the microbes to deal with these toxic compounds. 

Wright et al. concluded that noncovalently bound plasticizers in plastics are likely to be leached into the surrounding environment. Microbial biofilms that grow on these materials are the first to encounter such chemicals and, as shown here, they are likely able to catabolize them, reducing their release into their aquatic surroundings.

Synthetic Biology for Biodegradable Plastics

In synthetic biology, the original genetic engineering approaches using enzymatic “cut-and-paste” or oligo-mediated “mutation” manipulations of existing genes have been superseded by “synthesize from scratch.” Complete de novo synthesis of any desired gene construct or sets of multiple genes has been enabled by low-cost high-throughput synthesis and sequencing. Applications of synthetic biology are limited only by imagination. Next, we will discuss an example of how synthetic biology has been applied to improved production of biodegradable polymers as a replacement for conventional chemical manufacturing.

Polyhydroxyalkanoates (PHA) are biodegradable polyesters produced by microorganisms. They can also be manufactured by chemical synthesis, as reviewed elsewhere. Compared to chemical processing, complications with microbial or enzymatic processes make industrial biotech products less competitive with chemical sources. However, Chen et al. have developed synthetic biology approaches that have allowed for certain improvements, making a bioprocess competitive with chemistry. 

Bacterial production of PHA is challenged by complex biochemical pathways that make it difficult to control its structures, which include homopolymers, random copolymers, block copolymers, and ratios of monomers in the copolymers. Chen et al. therefore designed an approach for highly efficient PHB pathway optimization in E. coli, based on PHB synthesis of the phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha), a gram-negative bacterium that has two flagella, rather than a single one as shown here.

Workflow for PHB Pathway Optimization. Drawing by Jerry Zon.

The optimization approach summarized in the workflow diagram below starts with rationally designed Ribosomal Binding Sites (RBS). This method is predictive for synthetic RBS, enabling rational control of the production rates of multiple proteins in a genetic circuit. The resultant libraries for three genes of interest were constructed based on high or low copy number plasmids in a one-pot reaction, using the Oligo-Linker Mediated Assembly (OLMA) method. The number of possible variations in a library can be reduced from random combinations to a smaller number of combinations (103). On-plate visual selection to identify pathways accumulating PHB reduced the library complexity to 102. High-throughput screening via fluorescence activated cell sorting (FAC) to screen PHB producers further reduced the pathways of interest to about 101. Further detailed analysis involved gas chromatography (GC), gel permeation chromatography (GPC), and more. With this approach, strains accumulating up to 92% PHB content in cell dry-weight (CDW) were obtained. 

Waste materials or waste water as input can then be used to produce PHA, reducing costs. For example, Pittmann & Steinmetz reported the production of PHAs as a side-stream process on a municipal waste water treatment plant in different operation conditions. Tests were conducted on high PHA production and stable PHA composition as functions of input concentration, temperature, pH, and cycle time of an installed “feast/famine-regime.” It was found that lower input concentration, 20 °C, neutral pH, and a 24-h cycle time are preferable for a high PHA production of up to 28 % of CDW. PHA composition was only influenced by cycle time, and a stable PHA composition was reached.

Biodegradable Natural Polymers for Packaging

In the introduction of this blog, I noted that in 1862, the first-ever plastic packaging was made from cellulose, a natural product, while superseding plastics were made from synthetic, nonrenewable chemicals, up until the introduction of polylactic acid (PLA) in 2000. Fortunately, there has been an increasing interest in natural, renewable polymers as safer and readily biodegradable options for packaging.  

As pointed out in a review by Ashok et al., biodegradable plastics can be developed from the synergic combinations of agricultural biology and microbiology. Starch and cellulose-based bio degradable zero-waste plastics can replace nonrenewable plastics with comparable packaging properties. Packaging industries have wide applications, and the requirement for each area is unique. Food packaging industries requires shelf-life improving characters, while industrial packaging requires high mechanical properties that can resist mechanical damaging. Ashok et al. review suitability factors and emerging techniques for improving the packaging properties of bioplastics. These points are echoed in a more recent review by Ivonkovic et al., who also discuss societal factors, including consumer behavior.

As conscientious consumers, it is a important to be mindful of the types of plastic we purchase. Favoring eco-friendly bio-plastics and biodegradable plastics allows us to collectively help with sustainability and reduce pollution.

 Your comments are welcomed.

Addendum

After this blog was written, a breakthrough report was published by Carbios and collaborators in Nature on April 8, describing details for the production of a much more effective enzyme to degrade PET. These researchers mass produced mutant enzymes in bacteria and then screened these enzymes for PET cleavage efficiency. A mutant enzyme was found to be 10,000 times more efficient at PET bond-breaking than the native enzyme. This mutant also works without breaking down at 72°C, which is close to the temperature at which PET becomes molten. 

In a small reactor designed to test the enzyme, the team found that the mutant enzyme could break down 90% of 200 grams of PET in 10 hours. Moreover, the researchers then used the terephthalic acid and ethylene glycol building blocks generated by the enzyme to make new PET and produce plastic bottles that were just as strong as those made from conventional starting materials.

Another breakthrough involves MarinaTex, a bioplastic material designed to serve as an alternative to single-use plastic in a variety of applications. It is translucent and stronger than low-density polyethylene. This biodegradable bioplastic is made from red algae and organic waste from the fishing industry, decomposing within only 4-6 weeks in a home compostable environment. MarinaTex was created as a final year project by Lucy Hughes, a product designer graduate from the University of Sussex who was also recently granted a prestigious award.

2 years ago
44 view(s)
Did you like this post?
0
0
""