Towards bio-upcycling of polyethylene terephthalate.

Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, the sequential conversion of post-consumer polyethylene terephthalate (PET) into two types of bioplastics is presented: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films are hydrolyzed by a thermostable polyester hydrolase yielding highly pure terephthalate and ethylene glycol. The obtained hydrolysate is used directly as a feedstock for a terephthalate-degrading Pseudomonas umsongensis GO16, also evolved to efficiently metabolize ethylene glycol, to produce PHA. The strain is further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which are used as monomers for the chemo-catalytic synthesis of bio-PU. In short, a novel value-chain for PET upcycling is shown that circumvents the costly purification of PET monomers, adding technological flexibility to the global challenge of end-of-life management of plastics.

Biodegradable Plastic Blends Create New Possibilities for End-of-Life Management of Plastics but They Are Not a Panacea for Plastic Pollution.

Plastic waste pollution is a global environmental problem which could be addressed by biodegradable plastics. The latter are blended together to achieve commercially functional properties, but the environmental fate of these blends is unknown. We have tested neat polymers, polylactic acid (PLA), polyhydroxybutyrate, polyhydroxyoctanoate, poly(butylene succinate), thermoplastic starch, polycaprolactone (PCL), and blends thereof for biodegradation across seven managed and unmanaged environments. PLA is one of the world's best-selling biodegradable plastics, but it is not home compostable. We show here that PLA when blended with PCL becomes home compostable. We also demonstrate that the majority of the tested bioplastics and their blends degrade by thermophilic anaerobic digestion with high biogas output, but degradation times are 3-6 times longer than the retention times in commercial plants. While some polymers and their blends showed good biodegradation in soil and water, the majority of polymers and their blends tested in this study failed to achieve ISO and ASTM biodegradation standards, and some failed to show any biodegradation. Thus, biodegradable plastic blends need careful postconsumer management, and further design to allow more rapid biodegradation in multiple environments is needed as their release into the environment can cause plastic pollution.

Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity.

Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess > 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 µg/ml for Escherichia coli, 50 µg/ml for Bacillus subtilis, 100 µg/ml for Salmonella typhimurium, 200 µg/ml for Pseudomonas aeruginosa and 400 µg/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus.

Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum.

The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO2-containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter PcooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHAMCL), enhanced gene expression through the PcooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Plac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHAMCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition. IMPORTANCE: Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.

Polyhydroxyalkanoate-based 3-hydroxyoctanoic acid and its derivatives as a platform of bioactive compounds.

A library of 18 different compounds was synthesized starting from (R)-3-hydroxyoctanoic acid which is derived from the bacterial polymer polyhydroxyalkanoate (PHA). Ten derivatives, including halo and unsaturated methyl and benzyl esters, were synthesized and characterized for the first time. Given that (R)-3-hydroxyalkanoic acids are known to have biological activity, the new compounds were evaluated for antimicrobial activity and in vitro antiproliferative effect with mammalian cell lines. The presence of the carboxylic group was essential for the antimicrobial activity, with minimal inhibitory concentrations against a panel of bacteria (Gram-positive and Gram-negative) and fungi (Candida albicans and Microsporum gypseum) in the range 2.8-7.0 mM and 0.1-6.3 mM, respectively. 3-Halogenated octanoic acids exhibited the ability to inhibit C. albicans hyphae formation. In addition, (R)-3-hydroxyoctanoic and (E)-oct-2-enoic acids inhibited quorum sensing-regulated pyocyanin production in the opportunistic pathogen Pseudomonas aeruginosa PAO1. Generally, derivatives did not inhibit mammalian cell proliferation even at 3-mM concentrations, while only (E)-oct-2-enoic and 3-oxooctanoic acid had IC50 values of 1.7 and 1.6 mM with the human lung fibroblast cell line.

Understanding the physiological roles of polyhydroxybutyrate (PHB) in Rhodospirillum rubrum S1 under aerobic chemoheterotrophic conditions.

Polyhydroxybutyrate (PHB) is an important biopolymer accumulated by bacteria and associated with cell survival and stress response. Here, we make two surprising findings in the PHB-accumulating species Rhodospirillum rubrum S1. We first show that the presence of PHB promotes the increased assimilation of acetate preferentially into biomass rather than PHB. When R. rubrum is supplied with (13)C-acetate as a PHB precursor, 83.5 % of the carbon in PHB comes from acetate. However, only 15 % of the acetate ends up in PHB with the remainder assimilated as bacterial biomass. The PHB-negative mutant of R. rubrum assimilates 2-fold less acetate into biomass compared to the wild-type strain. Acetate assimilation proceeds via the ethylmalonyl-CoA pathway with (R)-3-hydroxybutyrate as a common intermediate with the PHB pathway. Secondly, we show that R. rubrum cells accumulating PHB have reduced ribulose 1,5-bisphosphate carboxylase (RuBisCO) activity. RuBisCO activity reduces 5-fold over a 36-h period after the onset of PHB. In contrast, a PHB-negative mutant maintains the same level of RuBisCO activity over the growth period. Since RuBisCO controls the redox potential in R. rubrum, PHB likely replaces RuBisCO in this role. R. rubrum is the first bacterium found to express RuBisCO under aerobic chemoheterotrophic conditions.

High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply.

High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L(-1) h(-1) ) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L(-1) was achieved in 33 h with a biomass productivity of 3.1 g L(-1) h(-1) which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L(-1) h(-1) ) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA(-1) .

Identification and characterization of an acyl-CoA dehydrogenase from Pseudomonas putida KT2440 that shows preference towards medium to long chain length fatty acids.

Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make Pseudomonas putida KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of P. putida is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (ß-oxidation) in this strain. In silico analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in P. putida KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5-7.

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate.

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.

Fed-batch strategies using butyrate for high cell density cultivation of Pseudomonas putida and its use as a biocatalyst.

A mathematically based fed-batch bioprocess demonstrated the suitability of using a relatively cheap and renewable substrate (butyric acid) for Pseudomonas putida CA-3 high cell density cultivation. Butyric acid fine-tuned addition is critical to extend the fermentation run and avoid oxygen consumption while maximising the biomass volumetric productivity. A conservative submaximal growth rate (µ of 0.25 h(-1)) achieved 71.3 g L(-1) of biomass after 42 h of fed-batch growth. When a more ambitious feed rate was supplied in order to match a µ of 0.35 h(-1), the volumetric productivity was increased to 2.0 g L(-1) h(-1), corresponding to a run of 25 h and 50 g L(-1) of biomass. Both results represent the highest biomass and the best biomass volumetric productivity with butyrate as a sole carbon source. However, medium chain length polyhydroxyalkanoate (mcl-PHA) accumulation with butyrate grown cells is low (4 %). To achieve a higher mcl-PHA volumetric productivity, decanoate was supplied to butyrate grown cells. This strategy resulted in a PHA volumetric productivity of 4.57 g L(-1) h(-1) in the PHA production phase and 1.63 g L(-1) h(-1)over the lifetime of the fermentation, with a maximum mcl-PHA accumulation of 65 % of the cell dry weight.

Medium chain length polyhydroxyalkanoate (mcl-PHA) production from volatile fatty acids derived from the anaerobic digestion of grass.

A two step biological process for the conversion of grass biomass to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) was achieved through the use of anaerobic and aerobic microbial processes. Anaerobic digestion (mixed culture) of ensiled grass was achieved with a recirculated leach bed bioreactor resulting in the production of a leachate, containing 15.3 g/l of volatile fatty acids (VFAs) ranging from acetic to valeric acid with butyric acid predominating (12.8 g/l). The VFA mixture was concentrated to 732.5 g/l with a 93.3 % yield of butyric acid (643.9 g/l). Three individual Pseudomonas putida strains, KT2440, CA-3 and GO16 (single pure cultures), differed in their ability to grow and accumulate PHA from VFAs. P. putida CA-3 achieved the highest biomass and PHA on average with individual fatty acids, exhibited the greatest tolerance to higher concentrations of butyric acid (up to 40 mM) compared to the other strains and exhibited a maximum growth rate (µMAX = 0.45 h⁻¹). Based on these observations P. putida CA-3 was chosen as the test strain with the concentrated VFA mixture derived from the AD leachate. P. putida CA-3 achieved 1.56 g of biomass/l and accumulated 39 % of the cell dry weight as PHA (nitrogen limitation) in shake flasks. The PHA was composed predominantly of 3-hydroxydecanoic acid (>65 mol%).

Carbon-rich wastes as feedstocks for biodegradable polymer (polyhydroxyalkanoate) production using bacteria.

Research into the production of biodegradable polymers has been driven by vision for the most part from changes in policy, in Europe and America. These policies have their origins in the Brundtland Report of 1987, which provides a platform for a more sustainable society. Biodegradable polymers are part of the emerging portfolio of renewable raw materials seeking to deliver environmental, social, and economic benefits. Polyhydroxyalkanoates (PHAs) are naturally-occurring biodegradable-polyesters accumulated by bacteria usually in response to inorganic nutrient limitation in the presence of excess carbon. Most of the early research into PHA accumulation and technology development for industrial-scale production was undertaken using virgin starting materials. For example, polyhydroxybutyrate and copolymers such as polyhydroxybutyrate-co-valerate are produced today at industrial scale from corn-derived glucose. However, in recent years, research has been undertaken to convert domestic and industrial wastes to PHA. These wastes in today's context are residuals seen by a growing body of stakeholders as platform resources for a biobased society. In the present review, we consider residuals from food, plastic, forest and lignocellulosic, and biodiesel manufacturing (glycerol). Thus, this review seeks to gain perspective of opportunities from literature reporting the production of PHA from carbon-rich residuals as feedstocks. A discussion on approaches and context for PHA production with reference to pure- and mixed-culture technologies is provided. Literature reports advocate results of the promise of waste conversion to PHA. However, the vast majority of studies on waste to PHA is at laboratory scale. The questions of surmounting the technical and political hurdles to industrialization are generally left unanswered. There are a limited number of studies that have progressed into fermentors and a dearth of pilot-scale demonstration. A number of fermentation studies show that biomass and PHA productivity can be increased, and sometimes dramatically, in a fermentor. The relevant application-specific properties of the polymers from the wastes studied and the effect of altered-waste composition on polymer properties are generally not well reported and would greatly benefit the progress of the research as high productivity is of limited value without the context of requisite case-specific polymer properties. The proposed use of a waste residual is advantageous from a life cycle viewpoint as it removes the direct or indirect effect of PHA production on land usage and food production. However, the question, of how economic drivers will promote or hinder advancements to demonstration scale, when wastes generally become understood as resources for a biobased society, hangs today in the balance due to a lack of shared vision and the legacy of mistakes made with first generation bioproducts.

Pilot-scale production of mcl-PHA by Pseudomonas citronellolis using acetic acid as the sole carbon source.

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biobased materials with promising properties for environmentally friendly applications. Due to high production costs, which are related to the cost of the carbon sources combined with conversion insufficiencies, currently only small quantities are produced. This results in a lack of reliable data on properties and application potential for the variety of polymers from different types of production strains. This study investigated the potential for the production of mcl-PHA from volatile fatty acids (VFA) at a larger scale, given their potential as low-cost and sustainable raw material within a carboxylate-platform based biorefinery. Pseudomonas citronellolis (DSMZ 50332) was chosen as the production strain, and acetic acid was selected as the main carbon and energy source. Nitrogen was limited to trigger polymer production, and a fed-batch process using a pH-stat feeding regime with concentrated acid was established. We report successful production, extraction, and characterization of mcl PHA, obtaining a total of 1.76 kg from two 500-litre scale fermentations. The produced polymer was identified as a copolymer of 3-hydroxydecanoate (60.7%), 3-hydroxyoctanoate (37.3%), and 3-hydroxyhexanoate (2.0%) with a weight average molecular weight (Mw) of 536 kDa. NMR analysis indicates the presence of unsaturated side chains, which may offer additional possibilities for modification. The results confirm that there is a potential to produce significant amounts of mcl-PHA with interesting rubber-like properties from waste-derived VFA.

Development of a bioprocess to convert PET derived terephthalic acid and biodiesel derived glycerol to medium chain length polyhydroxyalkanoate.

Sodium terephthalate (TA) produced from a PET pyrolysis product and waste glycerol (WG) from biodiesel manufacture were supplied to Pseudomonas putida GO16 in a fed-batch bioreactor. Six feeding strategies were employed by altering the sequence of TA and WG feeding. P. putida GO16 reached 8.70 g/l cell dry weight (CDW) and 2.61 g/l PHA in 48 h when grown on TA alone. When TA and WG were supplied in combination, biomass productivity (g/l/h) was increased between 1.3- and 1.7-fold and PHA productivity (g/l/h) was increased 1.8- to 2.2-fold compared to TA supplied alone. The monomer composition of the PHA accumulated from TA or WG was predominantly composed of 3-hydroxydecanoic acid. PHA monomers 3-hydroxytetradeeanoic acid and 3-hydroxytetradecenoic acid were not present in PHA accumulated from TA alone but were present when WG was supplied to the fermentation. When WG was either the sole carbon source or the predominant carbon source supplied to the fermentation the molecular weight of PHA accumulated was lower compared to PHA accumulated when TA was supplied as the sole substrate. Despite similarities in data for the properties of the polymers, PHAs produced with WG present in the PHA accumulation phase were tacky while PHA produced where TA was the sole carbon substrate in the polymer accumulation phase exhibited little or no tackiness at room temperature. The co-feeding of WG to fermentations allows for increased utilisation of TA. The order of feeding of WG and TA has an effect on TA utilisation and polymer properties.

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies.

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l-1 in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l-1 in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l-1 containing 65% PHA at the end of a 25-h incubation.

Process analysis of the conversion of styrene to biomass and medium chain length polyhydroxyalkanoate in a two-phase bioreactor.

The improvement and modeling of a process for the supply of the volatile aromatic hydrocarbon, styrene, to a fermentor for increased biomass production of the medium chain length polyhydroxyalkanoate (mcl-PHA) accumulating bacterium Pseudomonas putida CA-3 was investigated. Fed-batch experiments were undertaken using different methods to provide the styrene. Initial experiments where styrene was supplied as a liquid to the bioreactor had detrimental effects on cell growth and inhibited PHA polymer accumulation. By changing the feed of gaseous styrene to liquid styrene through the air sparger a 5.4-fold increase in cell dry-weight was achieved (total of 10.56 g L(-1)) which corresponds to a fourfold improvement in PHA production (3.36 g L(-1)) compared to previous studies performed in our laboratory (0.82 g L(-1)). In addition this final improved feeding strategy reduced the release of styrene from the fermentor 50-fold compared to initial experiments (0.12 mL total styrene released per 48 h run). An unstructured kinetic model was developed to describe cell growth along with substrate and oxygen utilization. The formation of dispersed gas (air) and liquid (styrene) phases in the medium and the transfer of styrene between the aqueous and dispersed liquid droplet phases was also modeled. The model provided a detailed description of these phase transitions and helped explain how the feeding strategy led to improved process performance in terms of final biomass levels. It also highlighted the key factors to be considered during further process improvement.

Robust process for high yield conversion of non-degradable polyethylene to a biodegradable plastic using a chemo-biotechnological approach.

In this study, the optimisation of a process for producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) by Pseudomonas putida KT2440 when fed with a polyethene (PE)-derived fatty acid mixture was investigated. The PE was pyrolysed to produce a hydrocarbon wax that was subsequently oxidised to produce a mixture of fatty acids, purified, and used as a PHA substrate for the growth and selection of microorganisms. Based on the shaken flask screening, a production strain, i.e., Pseudomonas putida KT2440, was selected for conducting bioreactor studies. Feeding PE-derived fatty acids in a 20-L setup resulted in high mcl-PHA yields (83.0 g L-1 CDW with 65% PHA in 25 h). Furthermore, life-cycle assessment (LCA) was conducted to determine the environmental advantages of the proposed process and its impacts compared to those of other technologies for treating PE-derived waste streams. We conclude that processing waste PE into PHA, rather than incineration, produces biodegradable material while also reducing the additional emissions that arise from traditional PE waste treatment processes, such as incineration to gain energy.

Genome analysis of the metabolically versatile Pseudomonas umsongensis GO16: the genetic basis for PET monomer upcycling into polyhydroxyalkanoates.

The throwaway culture related to the single-use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET-derived terephthalic acid (TA) into PHA and performed an in-depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.

Unraveling 1,4-Butanediol Metabolism in Pseudomonas putida KT2440.

Plastics, in all forms, are a ubiquitous cornerstone of modern civilization. Although humanity undoubtedly benefits from the versatility and durability of plastics, they also cause a tremendous burden for the environment. Bio-upcycling is a promising approach to reduce this burden, especially for polymers that are currently not amenable to mechanical recycling. Wildtype P. putida KT2440 is able to grow on 1,4-butanediol as sole carbon source, but only very slowly. Adaptive laboratory evolution (ALE) led to the isolation of several strains with significantly enhanced growth rate and yield. Genome re-sequencing and proteomic analysis were applied to characterize the genomic and metabolic basis of efficient 1,4-butanediol metabolism. Initially, 1,4-butanediol is oxidized to 4-hydroxybutyrate, in which the highly expressed dehydrogenase enzymes encoded within the PP_2674-2680 ped gene cluster play an essential role. The resulting 4-hydroxybutyrate can be metabolized through three possible pathways: (i) oxidation to succinate, (ii) CoA activation and subsequent oxidation to succinyl-CoA, and (iii) beta oxidation to glycolyl-CoA and acetyl-CoA. The evolved strains were both mutated in a transcriptional regulator (PP_2046) of an operon encoding both beta-oxidation related genes and an alcohol dehydrogenase. When either the regulator or the alcohol dehydrogenase is deleted, no 1,4-butanediol uptake or growth could be detected. Using a reverse engineering approach, PP_2046 was replaced by a synthetic promotor (14g) to overexpress the downstream operon (PP_2047-2051), thereby enhancing growth on 1,4-butanediol. This work provides a deeper understanding of microbial 1,4-butanediol metabolism in P. putida, which is also expandable to other aliphatic alpha-omega diols. It enables the more efficient metabolism of these diols, thereby enabling biotechnological valorization of plastic monomers in a bio-upcycling approach.

Conversion of waste cooking oil into medium chain polyhydroxyalkanoates in a high cell density fermentation.

Biodegradable and biocompatible polymers polyhydroxyalkanoates (PHAs) have a wide range of applications from packaging to medical. For the production of PHA at scale it is necessary to develop a high productivity bioprocess based on the use of a cheap substrate. The objective of the current study was to develop a high cell density bioreactor-based process for the production of medium chain length polyhydroxyalkanoate (mclPHA) with waste cooking oil as the sole carbon and energy source. A number of substrate feeding strategies for bacterial growth and polymer production were investigated. Pseudomonas chlororaphis 555 achieved high biomass of 73 g/l medium and a good biomass yield (including PHA in the cell) of 0.52 g/g substrate. P. chlororaphis 555 accumulated 13.9 g mclPHA/L and achieved polymer productivity of 0.29 g mclPHA/(L h). The mclPHA contained predominantly (R)-3-hydroxyoctanoic acid and (R)-3-hydroxydecanoic acid monomers, with a high fraction of (R)-3-hydroxydodecanoic acid monomers. This polymer is of low molecular weight (18 324 kDa), low polydispersity, it is amorphous, and has a glass transition temperature of -64 °C.