Plastic substrate and residual time of microplastics in the urban river shape the composition and structure of bacterial communities in plastisphere.

The widespread secondary microplastics (MPs) in urban freshwater, originating from plastic wastes, have created a new habitat called plastisphere for microorganisms. The factors influencing the structure and ecological risks of the microbial community within the plastisphere are not yet fully understood. We conducted an in-site incubation experiment in an urban river, using MPs from garbage bags (GB), shopping bags (SB), and plastic bottles (PB). Bacterial communities in water and plastisphere incubated for 2 and 4 weeks were analyzed by 16S high-throughput sequencing. The results showed the bacterial composition of the plastisphere, especially the PB, exhibited enrichment of plastic-degrading and photoautotrophic taxa. Diversity declined in GB and PB but increased in SB plastisphere. Abundance analysis revealed distinct bacterial species that were enriched or depleted in each type of plastisphere. As the succession progressed, the differences in community structure was more pronounced, and the decline in the complexity of bacterial community within each plastisphere suggested increasing specialization. All the plastisphere exhibited elevated pathogenicity at the second or forth week, compared to bacterial communities related to natural particles. These findings highlighted the continually evolving plastisphere in urban rivers was influenced by the plastic substrates, and attention should be paid to fragile plastic wastes due to the rapidly increasing pathogenicity of the bacterial community attached to them.

The plastisphere of biodegradable and conventional microplastics from residues exhibit distinct microbial structure, network and function in plastic-mulching farmland.

The inhomogeneity of plastisphere and soil may result in different microbial communities, thus potentially affecting soil functions. Biodegradable plastics offer an alternative to conventional plastics, nevertheless, the inadequate end-of-life treatment of biodegradable plastics may release more microplastics. Herein, we collected PE and PBAT/PLA microplastics in plastic-mulching farmland in Hebei, China. The bacterial communities of soil, PE and PBAT/PLA plastisphere were investigated using 16 S high-throughput sequencing. We found that the structure of bacterial communities in PBAT/PLA plastisphere were significantly distinct from PE plastisphere and soil. The alpha diversities in PBAT/PLA plastisphere were significantly lower than PE plastisphere and soil. Statistical analysis of differentially ASVs suggested that PBAT/PLA microplastics act as a filter, enriching taxa with the capability to degrade plastic polymers such as Proteobacteria and Actinobacteria. Compared to PE plastisphere, PBAT/PLA plastisphere has networks of less complexity, lower modularity, and more competitive interactions. Predicted metabolic pathways involved in human diseases, carbohydrate metabolism, amino acid metabolism, and xenobiotic biodegradation and metabolism were promoted in PBAT/PLA plastisphere, along with the facilitation in abundance of genes associated with carbon and nitrogen cycling. Our results highlighted the uniqueness of plastisphere of biodegradable microplastics from conventional microplastics and their potential impact on soil functions.

Co-occurrence of light microplastics and phthalate esters in soils of China.

The terrestrial environment is both a critical source and sink for microplastics (MPs). However, further efforts into the risk assessment, management, and mitigation activities of MPs in the terrestrial environment were limited by the scant data on their occurrence. In this study, we investigated the co-occurrence and correlations of light MPs and phthalate esters (PAEs) in the soils of China's hotspots and non-hotspot regions. Light MPs and PAEs were detected in all agricultural and urban soils (n = 125). In soils from hotspots (Shihezi, Xinjiang) where intense plastic mulching was used, the concentrations of MPs and phthalate diesters (di-PAEs) were 650-36,450 pcs kg-1 and 55.60-1236.64 µg kg-1, respectively. In hotspots but not in non-hotspot regions of China, a positive correlation between MPs and PAEs was established, suggesting PAEs may serve as an indicator of MP contamination in hotspots. High quantities of MPs (1143-5911 pcs kg-1) and PAEs (67.3-1236.64 µg kg-1) were also detected in urban park soils, demonstrating a need for future research on MP in urban soils. In addition, the ubiquitous co-occurrence of MPs and PAEs in all 125 investigated soils revealed that potential joint toxicity, co-transformation, and co-transportation of MPs and PAEs should not be disregarded.

Automated identification and quantification of invisible microplastics in agricultural soils.

Microplastics in agricultural soils have become the research hotspot in recent years, however, the quantitative methods based on the traditional visual inspection may have a high false detection rate. Here we combined the laser direct infrared (LDIR) and Fourier-transform infrared (FTIR) methods to investigate the microplastics in farmland with long-term agricultural activities. The results showed that the total abundance of microplastics reached 1.98 ± 0.41 × 105, 1.57 ± 0.28 × 105, 1.78 ± 0.27 × 105, and 3.20 ± 0.41 × 105 particles/kg soil in cotton fields with film mulching of 5, 10, 20, and >30 years, respectively. LDIR results indicated that microplastics ranging from 10 to 500 µm accounted for 96.5-99.9 % of the total microplastic amounts in the soils. Additionally, a total of 26 polymer types of microplastics were detected, among which polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyamide (PA), and polytetrafluoroethylene (PTFE) were dominantly observed. For the microplastics detected by FTIR (500 µm-5 mm), PE polymer was majorly observed (88.0-98.9 %). Most microplastics were films (88.2 %), while fibers and pellets were also found. The reclaimed water from sewage treatment plants, the drip irrigation utilities, and the residual plastic film are the potential sources of microplastics in the farmland soils. By using the automated quantitative and identifiable approaches, this study suggested that the commonly used visual counting method may underestimate the microplastic contamination in agricultural soils.

Contribution of stochastic processes to the microbial community assembly on field-collected microplastics.

A growing body of evidence suggests that microplastics may be colonized with a unique microbiome, termed 'plastisphere', in aquatic environments. However, the deep mechanisms (deterministic and/or stochastic processes) underlying the community assembly on microplastics are still poorly understood. Here, we took the estuary of Hangzhou Bay (Zhejiang, China) as an example and examined the assembly mechanisms of bacterial communities in water and microplastic samples. Results from high-throughput sequencing showed that Proteobacteria, Firmicutes, and Actinobacteria were the dominant phyla across all samples. Additionally, microorganisms from plastisphere and planktonic communities exhibited contrasting taxonomic compositions, with greater within-group variation for microplastic samples. The null model analysis indicated the plastisphere bacterial communities were dominantly driven by the stochastic process of drift (58.34%) and dispersal limitation (23.41%). The normalized stochasticity ratio (NST) also showed that the community assembly on microplastics was more stochastic (NST > 50%). Based on the Sloan neutral community model, the migration rate for plastisphere communities (0.015) was significantly lower than that for planktonic communities (0.936), potentially suggesting that it is the stochastic balance between loss and gain of bacteria (e.g., stochastic births and deaths) critically shaping the community assembly on microplastics and generating the specific niches. This study greatly enhanced our understanding of the ecological patterns of microplastic-associated microbial communities in aquatic environments.

Dissolved organic matter (DOM) quality drives biogeographic patterns of soil bacterial communities and their association networks in semi-arid regions.

It is of great interest to elucidate the biogeographic patterns of soil microorganisms and their driving forces, which is fundamental to predicting alterations in microbial-mediated functions arising from environment changes. Although dissolved organic matter (DOM) represents an important resource for soil microorganisms, knowledge of how its quality affects microbial biogeography is limited. Here, we characterized soil bacterial communities and DOM quality in 45 soil samples collected from a 1500-km sampling transect through semi-arid regions in northern China which are currently suffering great pressure from climate change, using Illumina Miseq sequencing and fluorescence spectroscopy, respectively. We found that DOM quality (i.e. the source of DOM and the humification degree of DOM) had profound shaping influence on the biogeographic patterns exhibited by bacterial diversity, community composition and association networks. Specifically, the composition of bacteria community closely associated with DOM quality. Plant-derived DOM sustained higher bacterial diversity relative to microbial-derived DOM. Meanwhile, bacterial diversity linearly increased with increasing humification degree of DOM. Additionally, plant-derived DOM was observed to foster more complex bacterial association networks with less competition. Together, our work contributes to the factors underlying biogeographic patterns not only of bacterial diversity, community composition but also of their association networks and reports previously undocumented important role of DOM quality in shaping these patterns.

Spatial Patterns of Soil Fungal Communities Are Driven by Dissolved Organic Matter (DOM) Quality in Semi-Arid Regions.

Soil fungi are ecologically important as decomposers, pathogens, and symbionts in nature. Understanding their biogeographic patterns and driving forces is pivotal to predict alterations arising from environmental changes in ecosystem. Dissolved organic matter (DOM) is an essential resource for soil fungi; however, the role of its quality in structuring fungal community patterns remains elusive. Here using Illumina MiSeq sequencing, we characterized total fungi and their functional groups in 45 soil samples collected from a 1500-km sampling transect through semi-arid regions in northern China, which are currently suffering great pressure from climate change. Total fungi and their functional groups were all observed to exhibit significant biogeographic patterns which were primarily driven by environmental variables. DOM quality was the best and consistent predictor of diversity of both total fungi and functional groups. Specifically, plant-derived DOM was associated with greater diversity relative to microbe-dominated origins. In addition, fungal diversity linearly increased with increases in degree of humification in DOM. Similarly, among all measured environmental variables, DOM quality had the strongest effects on the community composition of total fungi and functional groups. Together, our work contributes to the factors underlying fungal biogeographic patterns and adds detail to the importance of DOM quality in structuring fungal communities.

Effects of Rose Bengal- and Methylene Blue-Mediated Potassium Iodide-Potentiated Photodynamic Therapy on Enterococcus faecalis: A Comparative Study.

BACKGROUND AND OBJECTIVES: This study was performed to compare the use of methylene blue (MB) and rose bengal (RB) in antimicrobial photodynamic therapy (PDT) targeting Enterococcus faecalis (E. faecalis) bacteria in planktonic and biofilm forms with potassium iodide (KI) potentiation. STUDY DESIGN/MATERIALS AND METHODS: E. faecalis bacteria in planktonic form were exposed to antimicrobial PDT protocols activating MB and RB, with or without KI potentiation, following laser irradiation with different exposure times, 60 mW/cm2 laser power, and different photosensitizer agent (PS)/potentiator concentrations to observe relationships among the variables. Two continuous-wave diode lasers were used for irradiation (red light: λ = 660 nm and green light: λ = 565 nm). The pre-irradiation time was 10 minutes. The vitality of E. faecalis biofilm was assessed by confocal laser scanning microscopy, and the morphology was determined by scanning electron microscopy. The effects on the proliferation of stem cells from the apical papilla (SCAPs) were analyzed by cell counting kit-8 assay. The staining effect of antimicrobial PDT on dentin slices was investigated. Statistical analysis using a one-way analysis of variance was done. RESULTS: KI-potentiated RB and MB antimicrobial PDT both effectively eradicated E. faecalis bacteria in planktonic and biofilm forms. The minimum bactericidal concentrations of PSs (±100 mM KI) were obtained through PDT on planktonic E. faecalis, and the optimal light parameters were 60 mW/cm2 , 6 J/cm2 for 100 seconds. KI-potentiated PDT effectively strengthened the ability to inhibit E. faecalis biofilm with 86.50 ± 5.78% for MB (P = 0.0015 < 0.01) and 91.50 ± 1.75% for RB (P = 0.0418 < 0.05) of bactericidal rate, with less toxicity for SCAPs (P < 0.001) and less staining. KI could reduce the staining induced by antimicrobial PDT on dentin slices. CONCLUSION: A combination of KI and antimicrobial PDT may be a useful alternative to conventional disinfection methods in endodontic treatment. MB and RB antimicrobial PDT at much lower concentrations with KI could hopefully achieve disinfection effects comparable with those of 1.5% NaClO while causing few adverse effects on SCAPs. KI helps to avoid staining problems associated with high concentrations of photosensitizer agents. Lasers Surg. Med. © 2020 Wiley Periodicals, LLC.

Deciphering the archaeal communities in tree rhizosphere of the Qinghai-Tibetan plateau.

BACKGROUND: The Qinghai-Tibetan Plateau represents one of the most important component of the terrestrial ecosystem and a particularly vulnerable region, which harbouring complex and diverse microbiota. The knowledge about their underground microorganisms have largely been studied, but the characteristics of rhizosphere microbiota, particularly archaeal communities remains unclear. RESULTS: High-throughput Illumina sequencing was used to investigate the rhizosphere archaeal communities of two native alpine trees (Picea crassifolia and Populus szechuanica) living on the Qinghai-Tibetan Plateau. The archaeal community structure in rhizospheres significantly differed from that in bulk soil. Thaumarchaeota was the dominant archaeal phylum in all soils tested (92.46-98.01%), while its relative abundance in rhizospheres were significantly higher than that in bulk soil. Ammonium nitrogen, soil organic matter, available phosphorus and pH were significantly correlated with the archaeal community structure, and the deterministic processes dominated the assembly of archaeal communities across all soils. In addition, the network structures of the archaeal community in the rhizosphere were less complex than they were in the bulk soil, and an unclassified archaeal group (Unclassified_k_norank) was identified as the keystone species in all archaeal networks. CONCLUSIONS: Overall, the structure, assembly and co-occurrence patterns of archaeal communities are significantly affected by the presence of roots of alpine trees living on the Qinghai-Tibetan Plateau. This study provides new insights into our understanding of archaeal communities in vulnerable ecosystems.

Evidence of selective enrichment of bacterial assemblages and antibiotic resistant genes by microplastics in urban rivers.

The ubiquitous presence of microplastics in aquatic environments has recently drawn considerable attention due to their potential threat to the entire ecosystem. The colonization of bacterial communities on microplastics is an important ecological linkage for microplastics in aquatic ecosystems, which is yet poorly understood. In this study, microplastic particles were sampled in two urbanized rivers in Jiaxing, Zhejiang, China, and the differences between bacterial assemblages colonizing microplastics and planktonic bacteria were estimated. Results from high-throughput sequencing showed that the bacterial communities on microplastics were less rich and diverse compared to those from the freshwater samples, with a significantly distinct taxonomic composition. The predicted functional profiles also indicated significant differences between microplastic and water samples. The functions related to biofilm formation and human diseases were relatively higher for the bacterial communities on the microplastics. Network analyses suggested that microplastic bacterial communities possessed higher average path length, clustering coefficient, and modularity compared to those in water samples. Additionally, quantitative PCR results showed microplastics selectively enriched antibiotic resistant genes (ARGs), and a good-fit correlation between ARG profiles and bacterial community composition was observed. The relative abundances of integron-integrase gene classes 1 and 2 were greater on microplastics, potentially suggesting a higher level of horizontal gene transfer. Findings of this study suggested microplastics are a novel microbial niche and may serve as hotspots for microbial interaction, potentially increasing risks to freshwater ecosystems and human health.

Agricultural plastic mulching as a source of microplastics in the terrestrial environment.

Plastic mulching is suspected to be a significant source of microplastics in terrestrial environments owing to its intensive application and improper disposal. However, there has been a comparative lack of studies examining this hypothesis. In this study, the occurrence of macroplastics in agricultural soils was investigated by analysing 384 soil samples collected from 19 provinces across China. Additionally, the abundance of microplastics was investigated in potential hotspots that have carried out plastic mulching for over 30 years. Macroplastic concentrations in the soil samples ranged from 0.1 to 324.5 kg/ha, with an average of 83.6 kg/ha; the concentrations were higher in western China than in eastern China. A highly significant linear correlation (R2 = 0.61) was found between the consumption of mulching film and the plastic residue in soils, indicating plastic film mulching may be a major source of macroplastics. The abundances of microplastic particles increased over time in the locations where plastic mulching was continuously employed, with concentrations of 80.3 ± 49.3, 308 ± 138.1, and 1075.6 ± 346.8 pieces/kg soil in fields with 5, 15, and 24 y of continuous mulching, respectively. Fourier transform infrared analyses revealed that the composition of the microplastics matched that of the mulching films, suggesting the microplastic particles originated from the mulching films. These findings confirm that plastic mulching is an important source of macroplastic and microplastic contamination in terrestrial environments. Further studies to investigate the microplastic hazards in soils are thus necessary.

LDPE microplastic films alter microbial community composition and enzymatic activities in soil.

Concerns regarding microplastic contamination have spread from aquatic environments to terrestrial systems with a growing number of studies have been reported. Notwithstanding, the potential effects on soil ecosystems remain largely unexplored. In this study, the effects of polyethylene microplastics on soil enzymatic activities and the bacterial community were evaluated, and the microbiota colonizing on microplastics were also investigated. Microplastic amendment (2000 fragments per kg soil) significantly increased the urease and catalase activities in soil after 15 days, and no discernible alteration of invertase activities was detected. Results from high-throughput sequencing of 16S rRNA revealed that the alpha diversities (richness, evenness, and diversity) of the microbiota in soil were not obviously changed by the PE amendment, whereas the diversity indexes of microbiota on plastic fragments were significantly lower than those in the control and amended soils. Different taxonomic composition was observed in between the control and amended soils after 90 days of incubation. Bacterial assemblages with distinct community structure colonized the PE microplastics. Additionally, several taxa including plastic-degrading bacteria and pathogens were more abundant on microplastics. Simultaneously, the predicted functional profiles showed that the pathways of amino acid metabolism and xenobiotics biodegradation and metabolism were higher on the microplastics. These results indicated that microplastics in soil, compared with those in aquatic environments, can also act as a distinct microbial habitat, potentially altering the ecological functions of soil ecosystems.

Microplastics from mulching film is a distinct habitat for bacteria in farmland soil.

Microplastics, as an emerging pollutant of global importance, have been well documented in aquatic ecosystems. However, little is known about the effects of microplastics on agroecosystems, particularly for soil microbial communities. Herein, microplastics collected from cotton fields in Xinjiang, China, were analysed with a scanning electron microscope (SEM) and high-throughput sequencing to investigate the attached bacterial communities. Microplastic surfaces, especially pits and flakes, were colonized by various microorganisms, suggesting active hydrolysis of plastic debris. The bacterial communities colonizing microplastics were significantly different in structure from those in the surrounding soil, plant litter and macroplastics. In addition, statistical analysis of differentially abundant OTUs showed that microplastics serve as a "special microbial accumulator" in farmland soil, enriching some taxa that degrade polyethylene, such as Actinobacteria, Bacteroidetes and Proteobacteria. Co-occurrence network analysis revealed that the biotic interactions between microorganisms on microplastics are as complex as those in soil, and Acidobacteria, Chloroflexi, Gemmatimonadetes, and Bacteroidetes are considered keystone species in bacterial communities. Collectively, the findings imply that microplastics acted as a distinct habitat for bacteria in farmland soil, which increases our understanding of microplastic pollution.

Simvastatin Promotes Dental Pulp Stem Cell-induced Coronal Pulp Regeneration in Pulpotomized Teeth.

INTRODUCTION: Pulpotomy is a specific treatment used to save root pulp, in which only the inflamed coronal pulp is removed and capping materials are placed. Our study aims to study the effect of simvastatin (1) on the proliferation and differentiation of dental pulp stem cells (DPSCs) and (2) on DPSC-induced pulp regeneration after pulpotomy. METHODS: DPSCs were treated with different concentrations of simvastatin. Cell counting kit-8 activity was examined to test cell proliferation, and alkaline phosphatase assays and alizarin red S staining were conducted to examine differentiation. In addition, DPSCs pretreated with simvastatin were transplanted into the dorsum of CB-17 severe combined immunodeficiency mice. Areas of mineralized tissue were compared. Eighteen immature premolars from 2 beagle dogs were divided into 4 groups and treated by pulpotomy: the mineral trioxide aggregate, absorbable gelatin sponge, canine DPSCs (cDPSCs), and simvastatin groups. The teeth were extracted after 10 weeks, and the areas of regenerated pulp and dentin were calculated and compared. RESULTS: Simvastatin at 1 µmol/L suppressed cDPSCs proliferation but significantly increased alkaline phosphatase activity and mineral nodule formation. In addition, cDPSCs pretreated with 1 µmol/L simvastatin formed significantly more mineralized tissue in CB-17 severe combined immunodeficiency mice. In the in vivo study, the ratios of the areas of pulp and dentin regeneration were 47.3% ± 2.5%, 76.8% ± 4.3%, and 85.8% ± 0.9%, respectively, in the absorbable gelatin sponge, cDPSCs, and simvastatin groups. CONCLUSIONS: Simvastatin stimulates cDPSCs mineralization both in vivo and in vitro. It also promotes DPSC-induced pulp and dentin regeneration after pulpotomy.

Preliminary study on dental pulp stem cell-mediated pulp regeneration in canine immature permanent teeth.

INTRODUCTION: The health of human teeth depends on the integrity of the hard tissue and the activity of the pulp and periodontal tissues, which are responsible for nutritional supply. Without the nourishing of the pulp tissue, the possibility of tooth fracture can increase. In immature permanent teeth, root development may be influenced as well. This study explored the potential of using autologous dental pulp stem cells (DPSCs) to achieve pulp regeneration in a canine pulpless model. METHODS: The establishment of the pulpless animal model involved pulp extirpation and root canal preparation of young permanent incisor teeth in beagles. Autologous DPSCs were obtained from extracted first molars and expanded ex vivo to obtain a larger number of cells. The biological characteristics of canine DPSCs (cDPSCs) were analyzed both in vitro and in vivo by using the same method as used in human DPSCs. cDPSCs were transplanted into the pulpless root canal with Gelfoam as the scaffold, and root development was evaluated by radiographic and histologic analyses. RESULTS: cDPSCs with rapid proliferation, multiple differentiation capacity, and development potential were successfully isolated and identified both in vitro and in vivo. After they were transplanted into the pulpless root canal with Gelfoam as the scaffold, DPSCs were capable of generating pulp-like tissues containing blood vessels and dentin-like tissue. Thickening of the root canal wall was also observed. CONCLUSIONS: This study demonstrates the feasibility of using stem cell-mediated tissue engineering to realize pulp regeneration in immature teeth.

Effect of skimmed pasteurized milk and Hank's balanced salt solution on viability and osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND/AIM: The purpose of this study was to compare the effect of skimmed pasteurized milk and Hank's balanced salt solution on the viability and osteogenic differentiation potential of the human periodontal ligament stem cells at room temperature in vitro. MATERIAL AND METHODS: Human periodontal ligament stem cells were obtained from extracted healthy third molars and conserved in skimmed pasteurized milk and Hank's balanced salt solution for 1, 2, and 4 h at room temperature to detect the viability of the cells and their osteogenic differentiation potential. RESULTS: The efficacy of skimmed pasteurized milk on cell viability at 4 h was significantly higher than that of HBSS (P < 0.05), and cells stored in skimmed pasteurized milk showed significantly higher levels of mineralization than those in HBSS at 2 and 4 h (P < 0.05). CONCLUSIONS: Skimmed pasteurized milk was more effective than Hank's balanced salt solution in maintaining the viability and osteogenic differentiation potential of PDLSCs at room temperature in vitro.

[Isolation, culture and biological characterization of Beagle stem cells from apical papilla].

OBJECTIVE: To isolate and characterize the Beagle stem cells from apical papilla. METHODS: Apical papilla was severed from the end of freshly extracted Beagle's young permanent upper anterior teeth, and digested by collagenase type I and dispase for cell culture. The isolated cells were investigated for stem cell properties by analyzing their colony-forming efficiency, growth characteristics and the expression of mesenchymal stem cell markers; and evaluating their multidifferentiation potentials including osteogenic, adipogenic, and chondrogenic potentials. Additionally, the cells were transplanted subcutaneously into immunocompromised mice to observe the mineral tissue formation. RESULTS: Our study showed that a clonogenic, rapidly proliferative population of cells existed in Beagle's apical papilla, and these cells had a significantly higher colony-forming rate than the stem cells from apical papilla derived from humans (P<0.001). These cells had multilineage differentiation ability including osteogenic, adipogenic and chondrogenic potentials. Mineralized nodules were formed after osteogenic induction, lipid droplets were found after adipogenic induction, and the pellets showed positive immunohistochemical staining for collagen II after chondrogenic induction. These cells also expressed the mesenchymal stem cell markers including STRO-1 and CD146, while negative for CK. Moreover, these cells transplanted with hydroxyapatite in immunocompromised mice could form mineral tissue and pulp-dentin complex-like tissue. CONCLUSION: There are stem cells from the apical papilla which have high proliferation ability and multilineage differentiation potential existing in the Beagle's apical papilla.