This report details the work carried out for the development of a biotechnological process for the conversion of polyethylene terephthalate (PET) plastic bottles into biodegradable plastic project (2005-ET-LS-9-M3). Plastic waste is and will continue to be a major problem for Irish society as a whole. Our need to reduce the amounts of waste produced are of utmost importance, however due to the quick pace of modern society plastic waste will be a burning issue for the foreseeable future. In tackling the problem of waste plastic there is an opportunity to develop new and innovative ways to deal with excess waste that is not currently reused or recycled but incinerated or dumped. This goal of this project was to try to develop a technology that could take plastic waste namely PET (which is most commonly associated with plastic drinks bottles) and convert this waste to a new biodegradable polymer.
Success in this project represents an important step towards developing a technology with a broad significance to the global community. In our ever changing world which is under server threat from climate change the value of this indigenous novel technology which can help deal with one of the world?s waste problems will be of great advantage to Ireland both environmentally and economically. A large amount of development is still required to make this goal a reality but this research has shown that not only is this process possible for PET but other plastic wastes. This expands the horizons of opportunity to for a green environmentally friendly technology designed, researched and firmly rooted in Ireland
The first task of this project was to develop a biological process that could convert terephthalic acid into the biodegradable polymer polyhydroxyalkanoate (PHA). The thermal treatment of PET in the absence of air (pyrolysis) generates terephthalic acid (TA) as the major product. The TA generated is potentially a feedstock for the microbial synthesis of the value added biodegradable polymer PHA. Despite the knowledge that bacteria have been reported to degrade TA and other bacterial strains can accumulate PHA the production of PHA from TA has never been reported and thus the conversion of PET to a biodegradable plastic has not been achieved. Known TA degraders were tested for PHA accumulation but all strains failed to accumulate PHA. Consequently new strains were isolated from soil exposed to PET granules and screened for PHA accumulation. 32 strains were isolated; three of these strains were capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 also accumulates 3-hydroxydecanoic acid as the predominant monomer but with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium.
Having investigated the conversion of PET to PHA using soil isolates the next part of the project aimed to use molecular techniques in order to convert TA to polyhydroxybutyrate (PHB) and to enhance TA conversion to PHA. The first strategy targeted PHB accumulation and involved the cloning of an operon containing the genes necessary for the production of PHB into a selection of organisms known to utilize TA as the sole source of carbon and energy along with a range of bacteria that were isolated during this project from PET contaminated soil for their ability to grow on TA but failed to accumulate PHB or mclPHA. The genes responsible for PHB accumulation in the gram-negative soil bacterium Ralstonia eutropha are organised on a gene cluster phaCAB. Here we have successfully created a broad-host range plasmid containing these genes and expressed them in a target bacterium producing 4% of cell dry weight CDW as PHB. The second strategy was targeted at mcl-PHA and involved two different approaches, one aimed at cloning the genes responsible for TA degradation from Comamonas testosteroni and expressing these genes in two organisms that have been extensively studied and shown to produce mcl-PHA Pseudomonas putida CA-3 and Pseudomonas putida KT2440, but that do not possess the ability to utilize TA as the sole source of carbon and energy. Unfortunately no TA degradation was achieved with these recombinant organisms during this sturdy. The other approach involved cloning the phaG gene from Pseudomonas putida CA-3 which encodes a (R)-3-hydroxyacyl-ACP-CoA transferase into Pseudomonas putida GO10 a gram negative soil organism isolated on TA as a sole source of carbon and energy during the course of this study. The wildtype strain GO10 was able to accumulate PHA from alkanoic acid (e.g octanoic acid) but could not accumulate PHA from TA. The recombinant GO10_ pJB861-phaG was recorded to produce up to 12% PHA of CDW from TA when the phaG gene was expressed in this strain.
Since the wild type strains isolated in this study were better than the recombinant strains we decided to proceed with the wild type strains for further development of mclPHA production from PET. The improvement of this process by enhancing the fermentation production was then undertaken. Bioprocess manipulation has been successfully employed in the production of PHA from a variety of both PHA related and unrelated substrates previously. However, this is the first study to employ TA as the source of carbon and energy in a fermentor to produce mcl-PHA. Bioprocess manipulation allows for increased control over growth as well as PHA production, due to the enhanced ability to closely monitor and control some of the various physical parameters involved in microbial growth. Specific feeding strategies were employed in order to increase the biomass and PHA volumetric productivity (g/l∙h) over a 48 h period, and increase the amount of TA that is utilized during this period. In addition to manipulating the feeding of TA, a co-substrate; glycerol (a waste from the biodiesel industry) was used to further enhance the process. The co-feeding of TA and waste glycerol in our recycling process enabled us to provide higher biomass and PHA volumetric productivity (g/l?h), a route for another waste material and allows us to produce and study mcl-PHAs with varying monomer compositions. The ability to alter the monomer composition of PHA allows us to alter the material properties, thus offering greater diversity and flexibility for PHA production
In the final portion of the project, we applied the technology developed for PET conversion to PHA for a major portion of the products from the pyrolysis of mixed plastic waste, which are benzene, toluene, ethylbenzene, p-xylene and styrene. We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX), and styrene as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% of CDW for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA accumulated to 24% and to 36% of the CDW of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350C and a glass transition temperature of −48.5°C. This project has been successful in its goal to convert waste PET to PHA, In addition to this another form of plastic waste reuse has been identified and investigated in the conversion of mixed plastic waste to PHA. During this research two patents have been secured protecting this technology and while both of these processes require further investigation to achieve commercial viability the results of this project have laid the foundations for a new and innovative green technology that may prove valuable to an Ireland striving towards a greener future.

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Kenny, S. O'Connor, K.D. Nikodinovic-Runic, J. Kaminksy, W.   "An investigation of the conversion of waste polyethylene terephthalate to the biodegradable polymer polyhydroxyalkanoate". Associated datasets and digitial information objects connected to this resource are available at: Secure Archive For Environmental Research Data (SAFER) managed by Environmental Protection Agency Ireland https://eparesearch.epa.ie/safer/resource?id=02dd49da-b6f7-102e-a0a4-f81fb11d7d1c (Last Accessed: 2024-10-11)

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SAFER-Data Display URL	https://eparesearch.epa.ie/safer/iso19115/display?isoID=204
Resource Keywords	Recycling, Polyhydroxalkanoate, Polyethylene terephthalate, Pseudomonas putida, Green technology, Pyrolysis, Glycerol
EPA/ERTDI/STRIVE Project Code	2005-ET-LS-9-M3
EPA/ERTDI/STRIVE Project Theme	Environmental Technologies
Resource Availability:	Public-Open
Limitations on the use of this Resource	Any 3rd party usage must properly cite the original authors of this project. A citation is provided below. The original authors can be contacted in relation to other data and outputs associated with this project which are mentioned in the report
Number of Attached Files (Publicly and Openly Available for Download):	2
Project Start Date	Friday 1st September 2006 (01-09-2006)
Earliest Recorded Date within any attached datasets or digital objects	Friday 1st September 2006 (01-09-2006)
Most Recent Recorded Date within any attached datasets or digital objects	Thursday 1st April 2010 (01-04-2010)
Published on SAFER	Wednesday 13th April 2011 (13-04-2011)
Date of Last Edit	Thursday 6th October 2011 at 14:37:09 (06-10-2011)
Datasets or Files Updated On	Thursday 6th October 2011 at 14:37:09 (06-10-2011)

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