A Practical Fluorescence-Based Screening Protocol for Polyethylene Terephthalate Degrading Microorganisms

Abstract

We propose a practical, low-cost and selective fluorescence-based protocol adapted to identify polyethylene terephthalate (PET) degrading microorganisms. The microbial hydrolysis of PET nanoparticles was monitored by 2-hydroxyterephthalate, a fluorophore produced in situ after radical hydroxylation of terephthalic acid (TPA), the final hydrolysis product, by the Fenton reaction. Seven fungi presenting promising PET hydrolytic potential using the proposed microscale screening assay were identified. The strains evaluated presented a substantial increase of up to 18-fold in PET nanoparticles conversion, such as obtained by the fungus Trichoderma sp. C70, after their cultivation in a PET-enriched medium. The formation of other hydrolysis products, along with TPA, was observed using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).

Hydrocarbon-associated substrates reveal promising fungi for poly (ethylene terephthalate) (PET) depolymerization

“…Other investigations also observed high hydrolytic activities (lipase/esterase) in strains isolated from hydrocarbon-associated environments [57]. This hypothesis is based on the theory of directed evolution when the substrate of contact to which a strain or a wild enzyme is exposed, leading to genotypic alterations that allow the assimilation of previously unassimilated molecules, due to the increase in the enzymatic activities carried out by modifications in certain proteins [47]. Thereby, Crecy et al [58] obtained strains of Metarhizium anisopliae with a recognized thermotolerance increment, desired for the application of the strain as a biocontrol agent.…”

“…Fungi with promising conversion results obtained from HTS assays were evaluated regarding the potential of PET nanoparticles biodegradation based on a methodology developed by Chaves et al [47]. Briefly, fungal cell suspensions (100 μL, 1.0 mg.mL −1 in borate buffer (pH 7.8), resulted from colonies with 7 day-growth were incubated with a solution of PET nanoparticles (30 μL, 0.11 mg.mL The study of each isolate was delimited per line in the 96-well plate.…”

“…This is the first report of application of M. arundinis strains (CBMAI 2109 and CBMAI 2110) in studies of PET biodegradation/depolymerization from commercial bottles (real condition). Chaves et al [47] obtained promising results with strains of these fungi on PET nanoparticles and reported the importance of subsequent studies with these strains. The analyses showed that the isolates can be considered sources of enzymes with potential application in PET degradation.…”

Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate

“…This assay protocol can be performed both in cuvettes (3 mL assay final volume) or in microplate wells (150 μL assay final volume); its fluorescence signal is linear from 0.01 to 0.075 m m TPA (in 0.1 m phosphate buffer, pH 8.5) [ 58 ]. Neither proteins nor microorganisms interfere in fluorescence emission in the sample assays; thus, this method can be used to assay PET hydrolytic reactions without requiring any preliminary treatment of the sample [ 60 ]. A further major advantage of this method, in comparison with the spectrophotometric detection of the PET hydrolysis products at ˜ 240 nm, is its specificity for detecting TPA as both MHET and BHET do not react with the hydroxyl radical [ 58 ].…”

“…This approach was recently exploited for screening a microbial library for enzymes active on PET on microplates (final volume of 200 μL). By adding exogenous H 2 O 2 to the assay mixture (130 m m ), the limiting starting O 2 concentration (0.24 m m ) could be overcome: Owing to the increase in H 2 O 2 concentration, the linear detection range of TPA could be extended up to 0.5 m m [ 60 ].…”