Screening of polyurethane-degrading microbes using a quenching fluorescence probe by microfluidic droplet sorting.

Excessive use of polyurethane (PU) polymers has led contributed to serious environmental pollution. The plastic recycling technology using microorganisms and enzymes as catalysts offers a promising green and low-carbon approach for managing plastic waste. However, current methods for screening PU-degrading strains suffer from drawbacks such as being time-consuming and inefficient. Herein, we present a novel approach for screening PU-degrading microorganisms using a quenching fluorescent probe along with the fluorescence-activated droplet sorting (FADS). The FPAP could specifically recognize the 4,4'-methylenedianiline (MDA) derivates released from PU degradation, with fluorescence quenching as a response. Based on the approach, we successfully screen two PU-degrading strains (Burkholderia sp. W38 and Bacillus sp. C1). After 20 d of cultivation, strain W38 and C1 could degrade 41.58% and 31.45% of polyester-PU film, respectively. Additionally, three metabolites were identified during the degradation of PU monomer (2,4-toluene diamine, 2,4-TDA) and a proposed degradation pathway was established. Consequently, the fluorescence probe integrated with microfluidic droplet systems, demonstrates potential for the development of innovative PU-biocatalysts. Furthermore, the identification of the 2,4-TDA degradation pathway provides valuable insights that can propel advancements in the field of PU biodegradation.

Microbial biofilms as a platform for diverse biocatalytic applications.

Microbial biofilms have gained significant traction in commercial wastewater treatment due to their inherent resilience, well-organized structure, and potential for collaborative metabolic processes. As our understanding of their physiology deepens, these living catalysts are finding exciting applications beyond wastewater treatment, including the production of bulk and fine chemicals, bioelectricity generation, and enzyme immobilization. While the biological applications of biofilms in different biocatalytic systems have been extensively summarized, the applications of artificially engineered biofilms were rarely discussed. This review aims to bridge this gap by highlighting the untapped potential of engineered microbial biofilms in diverse biocatalytic applications, with a focus on strategies for biofilms engineering. Strategies for engineering biofilm-based systems will be explored, including genetic modification, synthetic biology approaches, and targeted manipulation of biofilm formation processes. Finally, the review will address key challenges and future directions in developing robust biofilm-based biocatalytic platforms for large-scale production of chemicals, pharmaceuticals, and biofuels.

Maximizing the benefits of biofilms in fermentation processes.

Biofilm-based fermentation has great potential, as it possesses inherent characteristics such as self-immobilization, high resistance to reactants, and long-term activity. This forum focuses on research targets for promoting biofilm engineering to maximize the beneficial features of biofilms and to effectively utilize them in biofilm-mediated fermentation.

Attractant and repellent induce opposing changes in the four-helix bundle ligand-binding domain of a bacterial chemoreceptor.

Motile bacteria navigate toward favorable conditions and away from unfavorable environments using chemotaxis. Mechanisms of sensing attractants are well understood; however, molecular aspects of how bacteria sense repellents have not been established. Here, we identified malate as a repellent recognized by the MCP2201 chemoreceptor in a bacterium Comamonas testosteroni and showed that it binds to the same site as an attractant citrate. Binding determinants for a repellent and an attractant had only minor differences, and a single amino acid substitution in the binding site inverted the response to malate from a repellent to an attractant. We found that malate and citrate affect the oligomerization state of the ligand-binding domain in opposing way. We also observed opposing effects of repellent and attractant binding on the orientation of an alpha helix connecting the sensory domain to the transmembrane helix. We propose a model to illustrate how positive and negative signals might be generated.

Enhancing rhamnolipid production through a two-stage fermentation control strategy based on metabolic engineering and nitrate feeding.

Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.

Integration of ARTP Mutation and Adaptive Laboratory Evolution to Reveal 1,4-Butanediol Degradation in Pseudomonas putida KT2440.

Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol (BDO) is one of the major depolymerization monomers of many plastics polymers. BDO valorization presents great significance for waste plastic up-recycling and fermenting feedstock exploitation. In the present study, atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the BDO utilization capability of Pseudomonas putida KT2440. The excellent mutant P. putida NB10 was isolated and stored in the China Typical Culture Preservation Center (CCTCC) with the deposit number M 2021482. Whole-genome resequencing and transcriptome analysis revealed that the BDO degradation process consists of ß-oxidation, glyoxylate carboligase (GCL) pathway, glyoxylate cycle and gluconeogenesis pathway. The imbalance between the two key intermediates (acetyl-CoA and glycolyl-CoA) and the accumulation of cytotoxic aldehydes resulted in the weak metabolism performance of KT2440 in the utilization of BDO. The balance of the carbon flux and enhanced tolerance to cytotoxic intermediates endow NB10 with great BDO degradation capability. This study deeply revealed the metabolic mechanism behind BDO degradation and provided an excellent chassis cell for BDO further up-cycling to high-value chemicals. IMPORTANCE Plastic waste represents not only a global pollution problem but also a carbon-rich, low-cost, globally renewable feedstock for industrial biotechnology. BDO is the basic material for polybutylene terephthalate (PBT), poly butylene adipate-co-terephthalate (PBAT), poly (butylene succinate) (PBS), etc. Herein, the construction of BDO valorization cell factory presents great significance for waste plastic up-recycling and novel fermentation feedstock exploitation. However, BDO is hard to be metabolized and its metabolic pathway is unclear. This study presents a P. putida mutant NB10, obtained through the integration of ARTP and ALE, displaying significant growth improvement with BDO as the sole carbon source. Further genome resequencing, transcriptome analysis and genetic engineering deeply revealed the metabolic mechanism behind BDO degradation in P. putida, this study offers an excellent microbial chassis and modification strategy for plastic waste up-cycling.

Future focuses of enzymatic plastic degradation.

Enzyme-based plastic degradation and valorization of the plastic-derived monomers has emerged as a potent option to address the plastic waste dilemma. Obstacles in implementing the enzymatic degradation of plastics in industry are here summarized, and strategies to overcome these obstacles are discussed to exploit the full potential of enzymatic plastic degradation toward a sustainable plastic economy.

Biodegradation of polyester polyurethane by Cladosporium sp. P7: Evaluating its degradation capacity and metabolic pathways.

Microorganisms capable of decomposing polyurethane (PU) and other plastics have the potential to be used in bio-recycling processes. In this study, 20 PU-degrading strains were isolated, including 11 bacteria and 9 fungi, using a synthesized poly(1,4-butylene adipate)-based PU (PBA-PU) as the screening substrate. Three PU substrates with increasing structure complexities were used for a thorough evaluation of microbial degradation capacity: Impranil® DLN-SD, PBA-PU film and PU foam waste. After 4 days, the best fungal PBA-PU degrader, Cladosporium sp. P7, could degrade 94.5% of Impranil® DLN-SD. After 28 days of cultivation, 32.42% and 43.91% of solid PBA-PU film was converted into soluble small molecules when used as the sole carbon source or in a medium with other co-carbon sources, respectively. Accordingly, the weight loss of PU foam waste after 15 days was 15.3% for the sole carbon condition and 83.83% for the co-carbon conditions. Furthermore, PBA-PU was used for metabolic pathway analysis because of its known composition and chemical structure. Six metabolites were identified during the degradation process of PBA-PU, including adipic acid (AA), 1,4-butanediol (BDO), and 4,4'-methylenedianiline (MDA), which can also be used as the sole carbon source to grow the fungal strain P7, resulting in the discovery of two MDA metabolites during the cultivation processes. Based on the presence of these eight metabolites, we hypothesized that PBA-PU is first depolymerized by the fungal strain P7 via ester and urethane bond hydrolysis, followed by intracellular metabolism and mineralization of the three monomers to CO2 and H2O.

Application of a novel fluorogenic polyurethane analogue probe in polyester-degrading microorganisms screening by microfluidic droplet.

Application of polyester-degrading microorganisms or enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. However, current impranil DLN-based screening of polyester-degrading microorganisms is time-consuming, labour-intensive and unable to distinguish polyesterases from other protease- or amidase-like enzymes. Herein, we present an approach that combined a novel synthetic fluorescent polyurethane analogue probe (FPAP), along with the droplet-based microfluidics to screen polyurethane-degrading microorganisms through fluorescence-activated droplet sorting (FADS) pipeline. The fluorescent probe FPAP exhibited a fluorescence enhancement effect once hydrolysed by polyesterases, along with a strong specificity in discriminating polyesterases from other non-active enzymes. Application of FPAP in a microfluidic droplet system demonstrated that this probe exhibited high sensitivity and efficiency in selecting positive droplets containing leaf-branch compost cutinase (LCC) enzymes. This novel fluorogenic probe, FPAP, combined with the droplet microfluidic system has the potential to be used in the exploitation of novel PUR-biocatalysts for biotechnological and environmental applications.

Bioengineering Comamonas testosteroni CNB-1: a robust whole-cell biocatalyst for efficient PET microplastic degradation.

The escalating crisis of polyethylene terephthalate (PET) microplastic contamination in biological wastewater treatment systems is a pressing environmental concern. These microplastics inevitably accumulate in sewage sludge due to the absence of effective removal technologies. Addressing this urgent issue, this study introduces a novel approach using DuraPETase, a potent enzyme with enhanced PET hydrolytic activity at ambient temperatures. Remarkably, this enzyme was successfully secreted from Comamonas testosteroni CNB-1, a dominant species in the active sludge. The secreted DuraPETase showed significant hydrolytic activity toward p-NPB and PET nanoplastics. Furthermore, the CNB-1 derived whole-cell biocatalyst was able to depolymerize PET microplastics under ambient temperature, achieving a degradation efficiency of 9% within 7 days. The CNB-1-based whole biocatalysts were also capable of utilizing PET degradation intermediates, such as terephthalic acid (TPA) and ethylene glycol (EG), and bis(2-hydroxyethyl)-TPA (BHET), for growth. This indicates that it can completely mineralize PET, as opposed to merely breaking it down into smaller molecules. This research highlights the potential of activated sludge as a potent source for insitu microplastic removal.

Priority changes between biofilm exopolysaccharides synthesis and rhamnolipids production are mediated by a c-di-GMP-specific phosphodiesterase NbdA in Pseudomonas aeruginosa.

The synthesis of biofilm exopolysaccharides and rhamnolipids (RLs) are two interrelated processes in Pseudomonas aeruginosa, but how bacteria coordinate these two processes remains unclear. We collected a P. aeruginosa KT1115 with rugose small colony variant (RSCV) phenotype from soil, and used it to study the dynamic regulation mechanism of biofilm polysaccharide and RLs synthesis. The results showed that the overproduction of biofilm exopolysaccharides at biofilm stage ultimately contributed the surge of RLs production at RLs stage. This phenomenon was further verified by comparing PAO1 with its engineered RSCV mutant, PAO1ΔwspF. Further genomic, transcriptomic analyses and gene deletion revealed that downregulation of c-di-GMP level was the key to switch biofilm exopolysaccharides accumulation to RLs surge, by transcriptionally upregulating a c-di-GMP phosphodiesterase NbdA. Overall, this study demonstrates the importance of c-di-GMP in coordinating biofilm exopolysaccharides and RLs synthesis, and provides an inspiration for enhancing RLs production through regulating c-di-GMP level.

Toward a unified nomenclature for strains with hyper-biofilm phenotypes.

Hyper-biofilm strains form robust biofilms, are highly adaptable, and form highly tolerant subpopulations in biofilms grown in vivo and in vitro. Such subpopulations are formed by a wide range of bacteria and thus have been given different names in different species. This situation calls for the establishment of a unified nomenclature for strains with hyper-biofilm phenotypes.

Construction and comparison of synthetic microbial consortium system (SMCs) by non-living or living materials immobilization and application in acetochlor degradation.

The microbial degradation of pesticides by pure or mixed microbial cultures has been thoroughly explored, however, they are still difficult to apply in real environmental remediation. Here, we constructed a synthetic microbial consortium system (SMCs) through the immobilization technology by non-living or living materials to improve the acetochlor degradation efficiency. Rhodococcus sp. T3-1, Delftia sp. T3-6 and Sphingobium sp. MEA3-1 were isolated for the SMCs construction. The free-floating consortium with the composition ratio of 1:2:2 (Rhodococcus sp. T3-1, Delftia sp. T3-6 and Sphingobium sp. MEA3-1) demonstrated 94.8% degradation of acetochlor, and the accumulation of intermediate metabolite 2-methyl-6-ethylaniline was decreased by 3 times. The immobilized consortium using composite materials showed synergistic effects on the acetochlor degradation with maximum degradation efficiency of 97.81%. In addition, a novel immobilization method with the biofilm of Myxococcus xanthus DK1622 as living materials was proposed. The maximum 96.62% degradation was obtained in non-trophic media. Furthermore, the immobilized SMCs showed significantly enhanced environmental robustness, reusability and stability. The results indicate the promising application of the immobilization methods using composite and living materials in pollutant-contaminated environments.

[Microplastics in wastewater treatment: current status and future trends].

Microplastics (MPs) have been detected in many ecosystems, such as the ocean, land and the atmosphere. A large number of MPs in urban sewage are trapped in the activated sludge by sewage treatment plants, but tens of thousands of MPs 'escape' the treatment and are discharged into the nature. Meanwhile, most of the MPs are transferred into the activated sludge during sewage treatment, and the sludge will be further used in agriculture, leading to secondary pollution of the MPs. Through literature research, we summarized the sources, distribution and hazards of MPs in the environment, the treatment of MPs with activated sludge, and the treatment methods of residual MPs in activated sludge, and summed up the potentials of biotechnology and synthetic biology in the genetic modification of key bacteria in activated sludge to endow them with MPs-degrading ability. The conclusion is expected to serve as a reference for optimizing the biodegradation of MPs in wastewater treatment plants.

Biodegradation of polyether-polyurethane foam in yellow mealworms (Tenebrio molitor) and effects on the gut microbiome.

Polyurethane (PU) is one of the mass-produced recalcitrant plastics with a high environmental resistance but extremely low biodegradability. Therefore, improperly disposed PU waste adds significantly to plastic pollution, which must be addressed immediately. In recent years, there has been an increasing number of reports on plastic biodegradation in insect larvae, especially those that can feed on polyethylene and polystyrene. This study revealed that yellow mealworm (Tenebrio molitor) larvae can chew and ingest polyether-PU foams efficiently, resulting in a significant mass loss of nearly 67% after 35 days at a similar survival rate compared to when fed on bran. However, polyether-PU fragments were found in the frass of T. molitor, indicating that polyether-PU biodegradation and bioconversion in intestinal tracts were not complete. The scission of ether and urethane bonds in the polyether-PU can be evidenced by comparing polymer fragments recovered from frass with the pristine ones using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Gel permeation chromatography suggested the release of low-molecular-weight oligomers as a result of the biodegradation, which also resulted in poor thermal stability of the polyether-PU foam as determined by thermogravimetric analysis. High-throughput sequencing of the gut microbiome revealed significant changes in the microbial community populations due to the polyether-PU diet, for example, an increase in the families Enterobacteriaceae and Streptococcaceae, suggesting that these microorganisms may contribute to the polyether-PU biodegradation.

Transcription-Associated Fluorescence-Activated Droplet Sorting for Di-rhamnolipid Hyperproducers.

Rhamnolipids (RLs) are biosurfactants with great economic significance that have been used extensively in multiple industries. Pseudomonas aeruginosa is a promising microorganism for sustainable RL production. However, current CTAB-MB based screening of RL-producing strains is time-consuming, labor-intensive, and unable to distinguish mono- and di-RL. In this study, we developed a novel transcription-associated fluorescence-activated droplet sorting (FADS) method to specifically target the di-RL hyperproducers. We first investigated critical factors associated with this method, including the specificity and sensitivity for discriminating di-RL overproducers from other communities. Validation of genotype-phenotype linkage between the GFP intensity, rhlC transcription, and di-RL production showed that rhlC transcription is closely correlated with di-RL production, and the GFP intensity is responsive to rhlC transcription, respectively. Using this platform, we screened out ten higher di-RL producing microorganisms, which produced 54-208% more di-RL than the model P. aeruginosa PAO1. In summary, the droplet-based microfluidic platform not only facilitates a more specific, reliable, and rapid screening of P. aeruginosa colonies with desired phenotypes, but also shows that intracellular transcription-associated GFP intensity can be used to measure the yield of di-RL between populations of droplets containing different environmental colonies. This method also can be integrated with transposon mutation libraries to target P. aeruginosa mutants.

Mutations in surface-sensing receptor WspA lock the Wsp signal transduction system into a constitutively active state.

Pseudomonas aeruginosa rugose small-colony variants (RSCVs) are frequently isolated from chronic infections, yet, they are rarely reported in environmental isolates. Here, during the comparative genomic analysis of two P. aeruginosa strains isolated from crude oil, we discovered a spontaneous in-frame deletion, wspAΔ280-307 , which led to hyper-biofilm and RSCV phenotypes. WspA is a homologue of methyl-accepting chemotaxis proteins (MCPs) that senses surfaces to regulate biofilm formation by stimulating cyclic-di-guanosine monophosphate (c-di-GMP) synthesis through the Wsp system. However, the methylation sites of WspA have never been identified. In this study, we identified E280 and E294 of WspA as methylation sites. The wspAΔ280-307 mutation enabled the Wsp system to lock into a constitutively active state that is independent of regulation by methylation. The result is an enhanced production of c-di-GMP. Sequence alignment revealed three conserved repeat sequences within the amino acid residues 280-313 (aa280-313) region of WspA homologues, suggesting that a spontaneous deletion within this DNA encoding region was likely a result of intragenic recombination and that similar mutations might occur in several related bacterial genera. Our results provide a plausible explanation for the selection of RSCVs and a mechanism to confer a competitive advantage for P. aeruginosa in a crude-oil environment.

[Isolation and characterization of a polyurethane-degrading bacterium].

Biodegradation of polyurethane (PUR) pollutants by microorganisms has received widespread attention currently. Identification of microorganisms capable of efficiently degrading PUR plastics is a key point. In this study, a strain P10 capable of degrading PUR was isolated from the plastic wastes, and identified as a bacterium belonging to the genus of Brevibacillus based on colony morphology and 16S rDNA phylogenetic analysis. Brevibacillus sp. P10 was capable of degrading 71.4% of waterborne polyurethane (Impranil DLN) after 6 days growth in MSM medium with DLN as a sole carbon source. In addition, strain P10 can use commercial PUR foam as the sole carbon source for growth. Brevibacillus sp. P10 can degrade 50 mg PUR foam after 6 days growth in MSM medium supplemented with 5% (V/V) LB after optimization of degradation conditions. This indicates that Brevibacillus sp. P10 has potential to be used in biodegradation of PUR waste.

Rugose small colony variant and its hyper-biofilm in Pseudomonas aeruginosa: Adaption, evolution, and biotechnological potential.

One of the hallmarks of the environmental bacterium Pseudomonas aeruginosa is its excellent ecological flexibility, which can thrive in diverse ecological niches. In different ecosystems, P. aeruginosa may use different strategies to survive, such as forming biofilms in crude oil environment, converting to mucoid phenotype in the cystic fibrosis (CF) lung, or becoming persisters when treated with antibiotics. Rugose small colony variants (RSCVs) are the adaptive mutants of P. aeruginosa, which can be frequently isolated from chronic infections. During the past years, there has been a renewed interest in using P. aeruginosa as a model organism to investigate the RSCVs formation, persistence and pathogenesis, as RSCVs represent a hyper-biofilm formation, high adaptability, high-tolerance sub-population in biofilms. This review will briefly summarize recent advances regarding the phenotypic, genetic and host interaction associated with RSCVs, with an emphasis on P. aeruginosa. Meanwhile, some non-pathogenic bacteria such as Pseudomonas fluorescence, Pseudomonas putida and Bacillus subtilis will be also included. Remarkable emphasis is given on intrinsic functions of such hyper-biofilm formation characteristic as well as its potential applications in several biocatalytic transformations including wastewater treatment, microbial fermentation, and plastic degradation. Hopefully, this review will attract the interest of researchers in various fields and shape future research focused not only on evolutionary biology but also on biotechnological applications related to RSCVs.