The unique salt bridge network in GlacPETase: a key to its stability.

The extensive accumulation of polyethylene terephthalate (PET) has become a critical environmental issue. PET hydrolases can break down PET into its building blocks. Recently, we identified a glacial PET hydrolase GlacPETase sharing less than 31% amino acid identity with any known PET hydrolases. In this study, the crystal structure of GlacPETase was determined at 1.8 Å resolution, revealing unique structural features including a distinctive N-terminal disulfide bond and a specific salt bridge network. Site-directed mutagenesis demonstrated that the disruption of the N-terminal disulfide bond did not reduce GlacPETase's thermostability or its catalytic activity on PET. However, mutations in the salt bridges resulted in changes in melting temperature ranging from -8°C to +2°C and the activity on PET ranging from 17.5% to 145.5% compared to the wild type. Molecular dynamics simulations revealed that these salt bridges stabilized the GlacPETase's structure by maintaining their surrounding structure. Phylogenetic analysis indicated that GlacPETase represented a distinct branch within PET hydrolases-like proteins, with the salt bridges and disulfide bonds in this branch being relatively conserved. This research contributed to the improvement of our comprehension of the structural mechanisms that dictate the thermostability of PET hydrolases, highlighting the diverse characteristics and adaptability observed within PET hydrolases.IMPORTANCEThe pervasive problem of polyethylene terephthalate (PET) pollution in various terrestrial and marine environments is widely acknowledged and continues to escalate. PET hydrolases, such as GlacPETase in this study, offered a solution for breaking down PET. Its unique origin and less than 31% identity with any known PET hydrolases have driven us to resolve its structure. Here, we report the correlation between its unique structure and biochemical properties, focusing on an N-terminal disulfide bond and specific salt bridges. Through site-directed mutagenesis experiments and molecular dynamics simulations, the roles of the N-terminal disulfide bond and salt bridges were elucidated in GlacPETase. This research enhanced our understanding of the role of salt bridges in the thermostability of PET hydrolases, providing a valuable reference for the future engineering of PET hydrolases.

Glacier as a source of novel polyethylene terephthalate hydrolases.

Polyethylene terephthalate (PET) is a major component of microplastic contamination globally, which is now detected in pristine environments including Polar and mountain glaciers. As a carbon-rich molecule, PET could be a carbon source for microorganisms dwelling in glacier habitats. Thus, glacial microorganisms may be potential PET degraders with novel PET hydrolases. Here, we obtained 414 putative PET hydrolase sequences by searching a global glacier metagenome dataset. Metagenomes from the Alps and Tibetan glaciers exhibited a higher relative abundance of putative PET hydrolases than those from the Arctic and Antarctic. Twelve putative PET hydrolase sequences were cloned and expressed, with one sequence (designated as GlacPETase) proven to degrade amorphous PET film with a similar performance as IsPETase, but with a higher thermostability. GlacPETase exhibited only 30% sequence identity to known active PET hydrolases with a novel disulphide bridge location and, therefore may represent a novel PET hydrolases class. The present work suggests that extreme carbon-poor environments may harbour a diverse range of known and novel PET hydrolases for carbon acquisition as an environmental adaptation mechanism.

Degradation of polyvinyl chloride by a bacterial consortium enriched from the gut of Tenebrio molitor larvae.

Polyvinyl chloride (PVC), a carbon backbone synthetic plastic containing chlorine element, is one of six widely used plastics accounting for 10% global plastics production. PVC wastes are recalcitrant to be broken down in the environment but release harmful chlorinated compounds, causing damage to the ecosystem. Although biodegradation represents a sustainable approach for PVC reduction, virtually no efficient bacterial degraders for additive-free PVC have been reported. In addition, PVC depolymerization by Tenebrio molitor larvae was suggested to be gut microbe-dependent, but to date no additive-free PVC degraders have been isolated from insect guts. In this study, a bacterial consortium designated EF1 was newly enriched from the gut of Tenebrio molitor larvae, which was capable of utilizing additive-free PVC for its growth with the PVC-mass reduction and dechlorination of PVC. PVC films inoculated with consortium EF1 for 30 d were analyzed by diverse polymer characterization methods including atomic force microscopy, scanning electron microscope, water contact angle, time-of-flight secondary ion mass spectrometry, Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis technique, and ion chromatography. It was found that bio-treated PVC films were covered with tight biofilms with increased -OH and -CC- groups and decreased chlorine contents, and erosions and cracks were present on their surfaces. Meanwhile, the hydrophilicity of bio-treated films increased, but their thermal stability declined. Furthermore, Mw, Mn and Mz values were reduced by 17.0%, 28.5% and 16.1% using gel permeation chromatography, respectively. In addition, three medium-chain aliphatic primary alcohols and their corresponding fatty acids were identified as PVC degradation intermediates by gas chromatography-mass spectrometry. Combing all above results, it is clear that consortium EF1 is capable of efficiently degrading PVC polymer, providing a unique example for PVC degradation by gut microbiota of insects and a feasibility for the removal of PVC wastes.

Biodegradation of additive-free polypropylene by bacterial consortia enriched from the ocean and from the gut of Tenebrio molitor larvae.

Accumulation of highly recalcitrant PP wastes has caused a serious environmental pollution. We evaluated the biodegradation of two types of additive-free PP polymers by microbial degraders from different environments. Two bacterial consortia, designated as PP1M and PP2G, were enriched from the ocean and from the guts of Tenebrio molitor larvae. Both consortia were able to utilize each of two different additive-free PP plastics with relatively low molecular weights (low molecular weight PP powder and amorphous PP pellets) as the sole carbon source for growth. After a 30-day incubation, several plastic characterization methods, including high-temperature gel permeation chromatography, scanning electron microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry, were used to characterize the PP samples. The bio-treated PP powder was covered with tight biofilms and extracellular secretions with significantly increased hydroxyl and carbonyl groups and slightly decreased methyl groups. This suggested that degradation and oxidation had occurred. The altered molecular weights and the increased melting enthalpy and average crystallinity of the bio-treated PP samples all suggested that both consortia preferred to depolymerize and degrade the fractions with molecular weights of ≤34 kDa and the amorphous phase fractions of the two types of PP. Furthermore, low molecular weight PP powder was more susceptible to bacterial degradation compared to amorphous PP pellets. This study provides a unique example of different types of additive-free PP degradation by different culturable bacteria from the ocean and insect guts as well as a feasibility of PP waste removal in different environments.

Characterization of Two Marine Lignin-Degrading Consortia and the Potential Microbial Lignin Degradation Network in Nearshore Regions.

Terrestrial organic carbon such as lignin is an important component of the global marine carbon. However, the structural complexity and recalcitrant nature of lignin are deemed challenging for biodegradation. It has been speculated that bacteria play important roles in lignin degradation in the marine system. However, the extent of the involvement of marine microorganisms in lignin degradation and their contribution to the oceanic carbon cycle remains elusive. In this study, two bacterial consortia capable of degrading alkali lignin (a model compound of lignin), designated LIG-B and LIG-S, were enriched from the nearshore sediments of the East and South China Seas. Consortia LIG-B and LIG-S mainly comprised of the Proteobacteria phylum with Nitratireductor sp. (71.6%) and Halomonas sp. (91.6%), respectively. Lignin degradation was found more favorable in consortium LIG-B (max 57%) than in LIG-S (max 18%). Ligninolytic enzymes laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) capable of decomposing lignin into smaller fragments were all active in both consortia. The newly emerged low-molecular-weight aromatics, organic acids, and other lignin-derived compounds in biotreated alkali lignin also evidently showed the depolymerization of lignin by both consortia. The lignin degradation pathways reconstructed from consortium LIG-S were found to be more comprehensive compared to consortium LIG-B. It was further revealed that catabolic genes, involved in the degradation of lignin and its derivatives through multiple pathways via protocatechuate and catechol, are present not only in lignin-degrading consortia LIG-B and LIG-S but also in 783 publicly available metagenomic-assembled genomes from nine nearshore regions. IMPORTANCE Numerous terrigenous lignin-containing plant materials are constantly discharged from rivers and estuaries into the marine system. However, only low levels of terrigenous organic carbon, especially lignin, are detected in the global marine system due to the abundance of active heterotrophic microorganisms driving the carbon cycle. Simultaneously, the lack of knowledge on lignin biodegradation has hindered our understanding of the oceanic carbon cycle. Moreover, bacteria have been speculated to play important roles in the marine lignin biodegradation. Here, we enriched two bacterial consortia from nearshore sediments capable of utilizing alkali lignin for cell growth while degrading it into smaller molecules and reconstructed the lignin degradation network. In particular, this study highlights that marine microorganisms in nearshore regions mostly undergo similar pathways using protocatechuate and catechol as ring-cleavage substrates to drive lignin degradation as part of the oceanic carbon cycle, regardless of whether they are in sediments or water column.

Biodegradation of bisphenol-A polycarbonate plastic by Pseudoxanthomonas sp. strain NyZ600.

Bisphenol-A polycarbonate (PC) is a widely used engineering thermoplastic and its release has caused damage to the ecosystem. Microbial degradation of plastic represents a sustainable approach for PC reduction. In this study, a bacterial strain designated Pseudoxanthomonas sp. strain NyZ600 capable of degrading PC was isolated from activated sludge by using diphenyl carbonate as a surrogate substrate. Within a 30-day period of incubating with strain NyZ600, PC films were analyzed with atomic force microscopy, scanning electron microscope, water contact angle, X-ray photoelectron spectroscopy, fourier transform infrared spectroscopy, differential scan calorimeter and thermogravimetric analysis technique. The analyses results indicated that the treated PC films were bio-deteriorated and formed some "corrosion pits" on the PC film surface. In addition, strain NyZ600 performed broad depolymerization of PC indicated by the reduction of Mn from 23.55 to 16.75 kDa and Mw from 45.67 to 31.97 kDa and two degradation products bisphenol A and 4-cumylphenol (the two monomers of PC) were also found, which established that PC were biodegraded by strain NyZ600. Combing all above results, it is clear that the strain NyZ600 can degrade PC which provides a unique example for bacterial degradation of PC and a feasibility for the removal of PC waste.

[Microbial degradation of petroleum-based plastics].

Synthetic plastics include polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyurethane (PUR), etc. Because of their large molecular weights, high hydrophobic characters and high chemical bond energies, they are difficult to be degraded by microbes. More and more plastic products are widespread consumed and gradually accumulated in the environment, so that the "white pollution" has become a global concern. Therefore, safe and economic microbial degradation of synthetic plastics is an option. This article reviews microbial degradation of six petroleum-based plastics, including polyethylene, polystyrene, polypropylene, polyurethane, polyethylene terephthalate and polyvinyl chloride, from the aspects of microbial strains involved and the related enzymatic studies. This paper provides clues for the further study of the microbial degradation of synthetic plastics, including the screening of a variety of plastic degrading bacteria and microflora, and the functional identification of their degradation mechanism at the genetic, molecular and biochemical levels. Hopefully, efficient resolutions for complete biodegradation of plastics together with production of high added-value products can then be materialized in the future.

Biodegradation of mixture of plastic films by tailored marine consortia.

This work sheds light on the physicochemical changes of naturally weathered polymer surfaces along with changes of polymer buoyancy due to biofilm formation and degradation processes. To support the degradation hypothesis, a microcosm experiment was conducted where a mixture of naturally weathered plastic pieces was incubated with an indigenous pelagic community. A series of analyses were employed in order to describe the alteration of the physicochemical characteristics of the polymer (FTIR, SEC and GPC, sinking velocity) as well as the biofilm community (NGS). At the end of phase II, the fraction of double bonds in the surface of microbially treated PE films increased while changes were also observed in the profile of the PS films. The molecular weight of PE pieces increased with incubation time reaching the molecular weight of the virgin pieces (230,000 g mol-1) at month 5 but the buoyancy displayed no difference throughout the experimental period. The number-average molecular weight of PS pieces decreased (33% and 27% in INDG and BIOG treatment respectively), implying chain scission; accelerated (by more than 30%) sinking velocities compared to the initial weathered pieces were also measured for PS films with biofilm on their surface. The orders Rhodobacterales, Oceanospirillales and Burkholderiales dominated the distinct platisphere communities and the genera Bacillus and Pseudonocardia discriminate these assemblages from the planktonic counterpart. The functional analysis predicts overrepresentation of adhesive cells carrying xenobiotic and hydrocarbon degradation genes. Taking these into account, we can suggest that tailored marine consortia have the ability to thrive in the presence of mixtures of plastics and participate in their degradation.