Exploring PhaZ depolymerase sequence space for the bio-cyclable loop for biopolymers

Polyhydroxyalkanoates (PHA) are a green substitute for conventional plastics, owing to their biological origin, biodegradability, biocompatibility and structural diversity. However, environmental biodegradation of PHA is achieved in a time frame of several months to several years, depending on environmental conditions, and properties of both PHA and PHA degrading enzymes (PhaZ) [1]. Taking into account the high production cost of PHA, landfilling at the end of life is not likely to be cost-effective, so enzymatic biodegradation as an alternative offers an ecofriendly bio-cyclable route to cost-effective PHA. Our study aims to tailor PhaZ properties to create suitable biocatalysts for the industrially relevant time frame and operating conditions. In order to do so, we decided to randomize PhaZ sequences and functionally screen enzyme variants for accelerated PHA degradation and improved biocatalyst stability. Up to this day, various phaZ genes have been mutated solely for mechanistic purposes eg. Catalytic residue identification, and elucidation of the substrate recognition process [2,3,4,5,6,7].KNJIGA IZVODA: 9. simpozijum Hemija i zaštita životne sredine Kladovo, 4-7. jun 2023. BOOK OF ABSTRACTS : 9th Symposium Chemistry and Environmental Protection Kladovo, 4-7th June 202

Groundwater and soil as a reservoir for polyurethane-degrading bacteria

Plastic waste is a global environmental burden. Polyurethanes (PU), toxic and ubiquitous synthetic polymers, do not biodegrade quickly, leading to their rapid accumulation in the soil and water environments. Highly efficient PU-degrading microorganisms are rare in nature and are of fundamental importance for achieving circular plastic economy. Bacterial isolates from groundwater, originating from magmatogenic massif and Tertiary basin within metamorphic area, as well as soil isolates collected from various pristine (PS) and contaminated sites (CS), were screened using PU model compound Impranil® DLN-SD (IMP) as sole C source to identify PU-degrading isolates. Phylogenetic analysis of 16S rRNA gene sequences from IMP-degrading isolates was performed using the neighbor-joining method to observe their clustering. Thirty one of 96 isolates (32.3 %) from groundwater and 18 of 220 isolates (8.2%) from soil produced prominent IMP-clearing zones. Thirteen IMPdegrading isolates from each type of environment, belonging to 8 genera (Pseudomonas, Proteus, Enterobacter, Flavobacterium, Serratia, Pantoea, Acinetobacter and Stenotrophomonas) for groundwater and to 6 genera (Streptomyces, Pseudomonas, Rhodococcus, Achromobacter, Bacillus and Paenibacillus) for soil environment, were included in phylogenetic analysis. No clear grouping of groundwater and soil isolates was observed, indicating that isolates are too distinct. Stronger clustering was observed for groundwater compared to soil isolates. For groundwater, strongest clustering was observed for 2 isolates belonging to Proteus genus, 2 belonging to Flavobacterium and 2 to Pseudomonas. For soil samples, strongest clustering was observed for 3 isolates belonging to genus Streptomyces. There was no clear grouping within isolates from CS and PS. In the future, wider range of environmental niches should be included in screening efforts for development of biocatalytic processes for management of plastic waste. Subterranean ecosystems, which are not readily accessible for sampling and represent largely unexplored reservoir of biotechnologically relevant enzymatic activities, should also be more represented in such screenings.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Progressing Plastics Circularity: A Review of Mechano-Biocatalytic Approaches for Waste Plastic (Re)valorization

Inspirational concepts, and the transfer of analogs from natural biology to science and engineering, has produced many excellent technologies to date, spanning vaccines to modern architectural feats. This review highlights that answers to the pressing global petroleum-based plastic waste challenges, can be found within the mechanics and mechanisms natural ecosystems. Here, a suite of technological and engineering approaches, which can be implemented to operate in tandem with nature's prescription for regenerative material circularity, is presented as a route to plastics sustainability. A number of mechanical/green chemical (pre)treatment methodologies, which simulate natural weathering and arthropodal dismantling activities are reviewed, including: mechanical milling, reactive extrusion, ultrasonic-, UV- and degradation using supercritical CO2. Akin to natural mechanical degradation, the purpose of the pretreatments is to render the plastic materials more amenable to microbial and biocatalytic activities, to yield effective depolymerization and (re)valorization. While biotechnological based degradation and depolymerization of both recalcitrant and bioplastics are at a relatively early stage of development, the potential for acceleration and expedition of valuable output monomers and oligomers yields is considerable. To date a limited number of independent mechano-green chemical approaches and a considerable and growing number of standalone enzymatic and microbial degradation studies have been reported. A convergent strategy, one which forges mechano-green chemical treatments together with the enzymatic and microbial actions, is largely lacking at this time. An overview of the reported microbial and enzymatic degradations of petroleum-based synthetic polymer plastics, specifically: low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), polyethylene terephthalate (PET), polyurethanes (PU) and polycaprolactone (PCL) and selected prevalent bio-based or bio-polymers [polylactic acid (PLA), polyhydroxyalkanoates (PHAs) and polybutylene succinate (PBS)], is detailed. The harvesting of depolymerization products to produce new materials and higher-value products is also a key endeavor in effectively completing the circle for plastics. Our challenge is now to effectively combine and conjugate the requisite cross disciplinary approaches and progress the essential science and engineering technologies to categorically complete the life-cycle for plastics

Revalorization of biodegradable polymers to valuable bacterial nanocellulose

Large amounts of polymers are discarded worldwide each year, leading to a significant polymer waste in natural environment. The upcycling has been found as an efficient way to transform polymer waste into high-value biomaterials meeting the conditions required for circularity by being indefinitely recyclable, without reduction in value or usability. The presented study refers to the upcycling of commercial biopolymers into bacterial nanocellulose. Polymer blends, consisted of biodegradable polymers, such as poly(lactic acid), PLA, poly(butylene succinate), PBS, and poly(ε-caprolactone), PCL. Polymers were hydrolyzed and the obtained hydrolysates were investigated as potential carbon source for K. medellinensis ID13488 growth and nanocellulose production. Degradation products were analyzed using HPLC analysis. Different growth media, including tap water, HS medium, absence / presence of glucose, were tested and bacterial nanocellulose growth was confirmed under the most of the tested conditions. Once the BNC growth was set up, the BNC production was scaled up and the obtained material was investigated in terms of structure confirmation (FTIR analysis), thermal properties (DSC/TG analysis), morphology (optical microscopy, AFM analysis) and crystallinity (XRD analysis). Finally, the full life cycle of mixed biopolymers: from biodegradation to revalorization of end products into bacterial nanocellulose appeared as perfect model approach to plastic circularity.Biotechnology for a circular bioeconomy: 28 -29 march 2023. AFOB-EFB Virtual conferenc

Medium chain length polyhyoxyalkanoates (mcl-PHA) model compounds for the discovery of novel PHA depolymerases

PHAs are naturally made microbial polyesters that have been commercialized as biodegradable plastics. However, it has been shown that these materials are not so easily biodegraded in natural environments [1]. PHA depolymerases are key PHA degrading enzymes and their identification and characterization is of great interest and importance. Currently, screening is done on polymeric substrates using techniques such as clear zone assays on agar or weight loss measurements. Results obtained using these different methods cannot be directly compared, since they depend highly on the polymer used, PHA granules preparation and assay conditions [2]. In order to design a more specific test for the determination of PHA depolymerase activity, we synthesized 3-hyoxyalkanoate monomers (3-HA monomer) and 3-hyoxyalkanoic acid dimers (3-HA dimer) and their respective p-nitrophenyl esters, allowing for spectrophotometric determination of their activity [3]. Compounds were characterized using N and FTIR. Para-nitrophenyl labeled substrates were then used in the enzymatic activity assay with the benchmark polyhyoxyoctanoate (PHO) depolymerase from Pseudomonas fluorescens GK13 expressed in Escherichia coli CodonPlus-RIPL hosts. This activity was compared to recombinantly expressed leaf-branch compost cutinase (LCC cutinase) and polyethyleneterephtalate (PET) hyolyzing esterase from Ideonella sakaiensis (IsPETase). Our initial results revealed increased specificity of PHO depolymerase towards newly synthetized substrates, suggesting their suitability for specific screens and isolation of new mcl-PHA depolymerases, as well as in high throughput screening assays designed for guiding their directed evolution.10th International Conference of MIKROBIOKOSMOS, Larissa from 30 Novewmber to 2 December 2023

Proteomic examination of polyester-polyurethane degradation by Streptomyces sp. PU10: Diverting polyurethane intermediates to secondary metabolite production

Global plastic waste accumulation has become omnipresent in public discourse and the focus of scientific research. Ranking as the sixth most produced polymer globally, polyurethanes (PU) significantly contribute to plastic waste and environmental pollution due to the toxicity of their building blocks, such as diisocyanates. In this study, the effects of PU on soil microbial communities over 18 months were monitored revealing that it had marginal effects on microbial diversity. However, Streptomyces sp. PU10, isolated from this PU-contaminated soil, proved exceptional in the degradation of a soluble polyester-PU (Impranil) across a range of temperatures with over 96% degradation of 10 g/L in 48 h. Proteins involved in PU degradation and metabolic changes occurring in this strain with Impranil as the sole carbon source were further investigated employing quantitative proteomics. The proposed degradation mechanism implicated the action of three enzymes: a polyester-degrading esterase, a urethane bond-degrading amidase and an oxidoreductase. Furthermore, proteome data revealed that PU degradation intermediates were incorporated into Streptomyces sp. PU10 metabolism via the fatty acid degradation pathway and subsequently channelled to polyketide biosynthesis. Most notably, the production of the tri-pyrrole undecylprodigiosin was confirmed paving the way for establishing PU upcycling strategies to bioactive metabolites using Streptomyces strains

DEGRADATION OF POLYAMIDE/POLYURETHANE TEXTILE BLEND BY STREPTOMYCES SP. R1

The increasing production and utilization of synthetic polymers in the textile industry over the past five decades has raised concerns about the environmental impact of the industry. The recalcitrant nature of synthetic fibers hampers the biodegradation of these textiles in the environment and leads to the accumulation of textile waste. Effective solutions for recycling and proper disposal of textile waste are lacking, however, the use of microorganisms and enzymes has emerged as a promising approach. The genus Streptomyces has been well studied as a producer of different hydrolytic enzymes, several of which have found use in industrial settings as well. As an integral part of the soil microbiome, Streptomyces species have been shown to interact with different textile materials in soil and may play a role in the degradation of these materials. This study aimed to examine the interaction of Streptomyces sp. R1, isolated from the rhizosphere of Cotinus coggygria, with polyamide/polyurethane textile, and identify potential enzymes involved in the biodegradation of synthetic textiles. The degradation of the textile was tested in liquid cultures (minimal salt medium) and model compost, bio-augmented with Streptomyces sp. R1 for 4 months. After the incubation, morphological, and changes in the functional groups of the textiles were analysed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The surface of the textile showed noticeable cracks and fissures after 4 months of burial in the bioaugmented model compost, alongside changes in the functional groups of the polyamide/polyurethane textile, which indicates biodegradation of the synthetic fibers. Searching the genome of Streptomyces sp. R1, several enzymes involved in the degradation of synthetic polymers were identified, including an esterase homologous to highly efficient plastic degrading depolymerases. Overall, the results presented here indicate Streptomyces sp. R1 has the potential for synthetic textile degradation and bioremediation.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

Microbial degradation of bis (2-hydroxyethyl) terephthalate

U poslednje vreme, bis-(2-hidroksietil)-tereftalat (BHET) se često koristi kao model jedinjenje za identifikovanje novih biokatalizatora za degradaciju polietilen-tereftalat (PET). Stoga, u radu je predstavljeno ispitivanje mehanizma degradacije BHET-a pomoću mikroorganizama

Microbial degradation of bis (2-hydroxyethyl) terephthalate

Proizvodnja plastike i zamena staklene i keramičke ambalaže plastičnim materijalima doveli su do nagomilavanja plastičnog otpada. Neophodno je naći povoljan sistem za degradaciju plastičnog otpada, bez nastanka toksičnih produkata ili dodatnog zagađenja životne sredine. Polietilen-tereftalat (PET) je jedan od najčešće proizvedenih plastičnih polimera. Proizvodnja PET-a započinje esterifikacijom tereftalne kiseline i etilen glikola, pri čemu nastaje bis-(2-hidroksietil)-tereftalat (BHET), koji se dalje polikondenzuje do polimera. U poslednje vreme, BHET se često koristi kao model jedinjenje za identifikovanje novih biokatalizatora za degradaciju PET-a [1,2]. Cilj ovog rada bio je ispitivanje mehanizma degradacije BHET-a pomoću mikroorganizama. U preliminarnom testu na čvrstim podlogama, kapacitet za degradaciju BHET-a je testiran kod stotinak mikroorganizama, nakon čega su odabrani najefikasniji sojevi, koji su identifikovani sekvenciranjem gena za 16s rRNK. Dalje, ispitivana je degradacija u tečnoj podlozi gde je BHET bio glavni izvor ugljenika. Eksperiment je trajao 7 dana, a degradacija je praćena nakon drugog, petog i sedmog dana upotrebom tečne hromatografije (HPLC). Kao najefikasniji sojevi pokazali su se pripadnici roda Pseudomonas. Oni su u potpunosti transformisali BHET do različitih intermedijera. Rezultati su pokazali da ispitivani sojevi mogu da transformišu BHET, korišćenjem najmanje dva različita puta, pa će se naredni eksperimenti usmeriti na identifikaciju intermedijera degradacije. Takođe, radi optimizacije degradacije, ispitivaće se simbiotsko i sinergističko dejstvo različitih konzorcijuma, kako bi se obezbedila potpuna degradacija ovog model jedinjenja.KNJIGA IZVODA: 9. simpozijum Hemija i zaštita životne sredine Kladovo, 4-7. jun 2023. BOOK OF ABSTRACTS : 9th Symposium Chemistry and Environmental Protection Kladovo, 4-7th June 202

Upcycling biodegradable pva/starch film to a bacterial biopigment and biopolymer

Meeting the challenge of circularity for plastics requires amenability to repurposing post-use, as equivalent or upcycled products. In a compelling advancement, complete circularity for a biodegradable polyvinyl alcohol/thermoplastic starch (PVA/TPS) food packaging film was demonstrated by bioconversion to high-market-value biopigments and polyhydroxybutyrate (PHB) polyesters. The PVA/TPS film mechanical properties (tensile strength (σu), 22.2 ± 4.3 MPa; strain at break (εu), 325 ± 73%; and Young’s modulus (E), 53–250 MPa) compared closely with low-density polyethylene (LDPE) grades used for food packaging. Strong solubility of the PVA/TPS film in water was a pertinent feature, facilitating suitability as a carbon source for bioprocessing and microbial degradation. Biodegradability of the film with greater than 50% weight loss occurred within 30 days of incubation at 37◦C in a model compost. Up to 22% of the PVA/TPS film substrate conversion to biomass was achieved using three bacterial strains, Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699), Streptomyces sp. JS520, and Bacillus subtilis ATCC6633. For the first time, production of the valuable biopigment (undecylprodigiosin) by Streptomyces sp. JS520 of 5.3 mg/mL and the production of PHB biopolymer at 7.8% of cell dry weight by Ralstonia eutropha H16 from this substrate were reported. This low-energy, low-carbon post-use PVA/TPS film upcycling model approach to plastic circularity demonstrates marked progress in the quest for sustainable and circular plastic solutions