Epiphytic Biofilms Research Articles

Interactions between SDBS and Hydrilla verticillata − epiphytic biofilm in wetland receiving STPs effluents: Nutrients removal and epiphytic microbial assembly

The fate and effects of sodium dodecyl benzene sulfonate (SDBS) in sewage treatment plants (STPs) effluents on nutrients and submerged macrophytes are far from clear in wetlands. This study conducted a 24-day experiment to investigate changes in nutrients and epiphytic biofilm of Hydrilla verticillata in wetlands receiving effluents with 0.5, 2 and 5 mg L−1 SDBS. The decrease of SDBS in overlying water followed pseudo-first-order kinetic equation, with over 80 % of SDBS removal achieved. 2 and 5 mg L−1 SDBS decreased nutrient removal efficiency, induced oxidative stress response and damaged cells of H. verticillata. SDBS altered bacterial and eukaryotic community diversity. 0.5 mg L−1 SDBS can promote carbon fixation and methane oxidation of microorganisms. Network analysis revealed that 0.5 mg L−1 SDBS decreased the stability of epiphytic ecosystems. Mantel tests indicated significant influences of SDBS, temperature, and total nitrogen on epiphytic microbial communities.

Response mechanisms of submerged macrophytes and epiphytic biofilms to single and coexisting per-and poly-fluoroalkyl substances and antibiotics stress

Combined pollutants of per- and poly-fluoroalkyl substances (PFAS) and antibiotics are commonly found in aquatic environments. This study aimed to investigate the effects of PFAS (perfluorobutyric acid (PFBA) and perfluorooctanoic acid (PFOA)) and antibiotics (tetracycline (TC) and chloramphenicol (CAP)) on Vallisneria natans (V. natans) and their epiphytic biofilms. The changes in photosynthetic pigments and oxidative damage in V. natans were altered by these pollutants. For example, the total chlorophyll was increased from 4.64 to 8.10 mg/L due to the combined pollutant of PFOA and TC. When exposed to the combined pollutant of PFBA and TC, a concentration of malondialdehyde of 28.4 nmol/g was measured, which was significantly higher than that in the control group. For combined toxicity, the PFAS and antibiotics primarily exhibited antagonistic effects within V. natans. The glycine, serine, and threonine metabolism pathways were mainly affected by the combined stress of PFBA and TC. Single pollutants of TC and CAP were found to inhibit the uptake of nutrients by V. natans, while the coexisting antibiotics enhanced the inhibitory effects of PFBA and PFOA in combined pollutant treatments. The growth of microorganisms in the epiphytic biofilms was significantly promoted by PFBA and PFOA, whereas their growth was notably inhibited by CAP. The composition of biofilms could be altered by single and combined pollutants, with antibiotics being more likely than PFAS to change the microbial communities within the biofilms. When exposed to TC and CAP, the ACE indices decreased from 1171.5 to 466.4 and 703.8, respectively.

Chlormequat inhibits Vallisneria natans growth and shapes the epiphytic biofilm microbial community.

Submerged macrophytes can overgrow and negatively affect freshwater ecosystems. This study aimed to investigate the use of chlormequat (CQ) to regulate submerged Vallisneria natans growth as well as its impact on the microbial community of epiphytic biofilms. V.natans height under CQ dosages of 20, 100, and 200 mg/L decreased within 21 days by 12.57%, 30.07%, and 44.62%, respectively, while chlorophyll content increased by 1.94%, 20.39%, and 38.83%. At 100 mg/L, CQ reduced the diversity of bacteria in the biofilm attached to V.natans leaves but increased the diversity of the eukaryotic microbial community. CQ strongly inhibited Cyanobacteria; compared with the control group, the treatment group experienced a significant reduction from 36.54% to 2.61%. Treatment significantly inhibited Gastrotricha and Rotifera, two dominant phyla of eukaryotes in the leaf biofilm, reducing their relative abundances by 17.41% and 6.48%, respectively. CQ significantly changed the leaf biofilm microbial community correlation network. The treatment group exhibited lower modularity (2.012) compared with the control group (2.249); however, the central network of the treated group contained a higher number of microbial genera (13) than the control group (4), highlighting the significance of eukaryotic genera in the network. The results obtained from this study provide invaluable scientific context and technical understanding pertinent to the restoration of submerged macrophytes within aquatic ecosystems. PRACTITIONER POINTS: Chlormequat reduced the plant height but increased leaf chlorophyll content. Chlormequat reduced biofilm bacterial diversity but increased eukaryotic diversity. Chlormequat affected the bacterial-fungal association networks in biofilms.

Tetracycline and quinolone contamination mediate microbial and antibiotic resistant gene composition in epiphytic biofilms of mesocosmic wetlands

The fate and ecological impact of antibiotics on aquatic ecosystems have not been properly elucidated in mesocosm wetlands scale. This study explored how tetracyclines (TCs, including tetracycline TC and oxytetracycline) and fluoroquinolones (QNs, including ciprofloxacin CIP and levofloxacin) affect mesocosm wetlands vegetated by V. spiralis, focusing on their impact on epiphytic biofilm microbial communities and antibiotic resistance genes (ARGs). Results showed that submerged plants absorbed more antibiotics than sediment. Both TCs and QNs disrupted microbial communities in different ways and increased eukaryotic community diversity in a concentration-dependent manner (2-4 mg/L for CIP, 4-8 mg/L for TC). TCs mainly inhibited epiphytic bacteria, while CIP increased bacterial phyla abundance. TC reduced Cyanobacteriota, Acidobacteriota, and Patescibacteria but increased Bacillota, Bacteroidota, and Armatimonadota. In contrast, CIP reduced Bacteroidota, Cyanobacteriota, and Gemmatimonadota but increased Bacillota, Planctomycetota, and Acidobacteriota. Significant differences in ARG profiles were observed between QNs and TCs, with TCs having a more substantial effect on ARGs due to their stronger impact on bacterial communities. Both antibiotics raised ARG levels with higher concentrations, particularly for multidrug resistance, tetracyclines, trimethoprim, sulfonamides, aminoglycosides, and fosfomycin, emphasizing their role in antimicrobial resistance. The study suggests that antibiotics can either stimulate or inhibit ARGs depending on their effects on bacterial communities. This study provides key evidence on the ecological mechanisms underlying the impact of TCs and QNs on epiphytic microbes of mesocosm wetlands.

Glufosinate-ammonium increased nitrogen and phosphorus content in water and shaped microbial community in epiphytic biofilm of Hydrilla verticillata

Glufosinate-ammonium (GLAM) can be released into adjacent water bodies with rainfall runoff and return water from farmland irrigation. However, impacts of GLAM on aquatic organisms remain unclear. In this study, changes in water quality, plant physiological parameters and epiphytic microbial community were investigated in wetlands with Hydrilla verticillata exposed to GLAM for 24 days. We found GLAM addition damaged cell and reduced chlorophyll a content in Hydrilla verticillata leaves, and increased ammonium and phosphorus in water (p < 0.001). The α-diversity increased in bacterial community but decreased in eukaryotic community with GLAM exposure. Neutral community models explained 62.3 % and 55.0 % of the variance in bacterial and eukaryotic communities, respectively. Many GLAM micro-biomarkers were obtained, including some clades from Proteobacteria, Bacteroidete, Actinobacteriota, Phragmoplastophyta, Annelida and Arthropoda. Redundancy analysis revealed that GLAM concentration was positively correlated to Flavobacterium, Gomphonema and Closterium but negatively to Methyloglobulus and Methylocystis. Network analysis revealed that 15 mg/L GLAM disturbed the interactions among phytoplankton, protozoa, metazoan and bacteria and reduced the stability of the microbial communities compared to 8 mg/L GLAM. GLAM shaped the nitrogen and phosphorus cycle related bacterial genes. This study highlights that herbicides are non-neglectable factors affecting the efficiency of aquatic ecological restoration in agricultural areas to control agricultural non-point source pollution.

Unravelling the mechanisms of PFAS toxicity to submerged macrophytes and epiphytic biofilms at metabolic and molecular levels

Per- and poly-fluoroalkyl substances (PFAS) are an emerging class of persistent organic pollutants that are widespread in aquatic ecosystems and pose a serious threat to aquatic organisms. It is thus crucial to explore the toxicity mechanisms of PFAS to submerged macrophytes and biofilms. In this study, Vallisneria natans (V. natans) was exposed to environmentally relevant concentrations of perfluorooctanoic acid (PFOA) and perfluorooctane sulphonate (PFOS). Results showed that PFAS induced the excessive production of reactive oxygen species, triggering antioxidant responses. V. natans exhibited an improved stress tolerance by altering the biosynthesis of several plant secondary metabolites and the histidine, arginine, proline pathways in response to PFAS exposure. Moreover, PIP1–1, PIP2–2, SLAH1 and SLAH2 genes were upregulated, indicating the activation of aquaporins and slow-type anion channels. The uptake of PFOA and PFOS by V. natans was 41.74 % and 52.31 %, respectively. Notably, PFAS bound to functional proteins (GSTF10), promoting the detoxification of plants. Exposure to PFAS also altered the structure of biofilms by inducing the synthesis of large amounts of polysaccharides and proteins. The diversity and richness of the microbial community within periphytic biofilms changed significantly. These results provide a comprehensive description of the responses of aquatic plants and periphytic biofilms to PFAS and the removal mechanism of PFAS, contributing to the environmental risk assessments and removal of PFAS in aquatic ecosystems.

Ecological succession of eukaryotic community in epiphytic biofilms on Hydrilla verticillata in wetlands under norfloxacin stress

Eukaryotic communities play critical roles in the diversity and functions of aquatic ecosystems. However, the fate of norfloxacin (NOR) and its impact on eukaryotic community succession in epiphytic biofilm remain unclear. This study investigated the effects of NOR on the aquatic environment, eukaryotic diversity, community assembly and microbial interactions in microcosmic wetlands dominated by Hydrilla verticillata. About 95 % of NOR was removed but exerted serious negative impacts on ecological environment. High concentrations of antibiotics decreased eukaryotic community richness but increased diversity. The assembly process of the eukaryotic community was shaped by stochastic processes, primarily involving dispersal limitation, whereas more positive co-occurrence correlations were triggered in microbial networks (from 57.1 % to 64.6 %), reducing community stability. In addition, the shift in keystones from Bacillariophyta to Metazoa with increased antibiotic concentrations, accompanied by a functional transformation. The partial least squares path modeling showed antibiotics positively affected nutrients (0.693) and network (0.5), but negatively affected β-diversity (-0.288). These data highlight that the ecological impacts of NOR on phylogenetic turnover in eukaryotes and offer references for promoting phytoremediation.

Unraveling the ecological mechanisms of Aluminum on microbial community succession in epiphytic biofilms on Vallisneria natans leaves: Novel insights from microbial interactions

The extensive use of aluminum (Al) poses an escalating ecological risk to aquatic ecosystems. The epiphytic biofilm on submerged plant leaves plays a crucial role in the regulation nutrient cycling and energy flow within aquatic environments. Here, we conducted a mesocosm experiment aimed at elucidating the impact of different Al concentrations (0, 0.6, 1.2, 2.0 mg/L) on microbial communities in epiphytic biofilms on Vallisneria natans. At 1.2 mg/L, the highest biofilms thickness (101.94 µm) was observed. Al treatment at 2.0 mg/L significantly reduced bacterial diversity, while micro-eukaryotic diversity increased. Pseudomonadota and Bacteroidota decreased, whereas Cyanobacteriota increased at 1.2 mg/L and 2.0 mg/L. At 1.2 and 2.0 mg/L. Furthermore, Al at concentrations of 1.2 and 2.0 mg/L enhanced the bacterial network complexity, while micro-eukaryotic networks showed reduced complexity. An increase in positive correlations among microbial co-occurrence patterns from 49.51% (CK) to 57.05% (2.0 mg/L) was indicative of augmented microbial cooperation under Al stress. The shift in keystone taxa with increasing Al concentration pointed to alterations in the functional dynamics of microbial communities. Additionally, Al treatments induced antioxidant responses in V. natans, elevating leaf reactive oxygen species (ROS) content. This study highlights the critical need to control appropriate concentration Al concentrations to preserve microbial diversity, sustain ecological functions, and enhance lake remediation in aquatic ecosystems.

Interactions between water quality and microbes in epiphytic biofilm and superficial sediment of lake in trophic agriculture area

Epiphytic and superficial sediment biofilm-dwelling microbial communities play a pivotal role in water quality regulation and biogeochemical cycling in shallow lakes. However, the interactions are far from clear between water physicochemical parameters and microbial community on aquatic plants and in surface sediments of lake in trophic agriculture area. This study employed Illumina sequencing, Partial Least Squares Path Modeling (PLS-PM), and physico-chemical analytical methods to explore the interactions between water quality and microbes (bacteria and eukaryotes) in three substrates of trophic shallow Lake Cyohoha North, Rwanda. The Lake Cyohoha was significantly polluted with total phosphorus (TP), total nitrogen (TN), nitrate nitrogen (NO3-N), and ammonia nitrogen (NH3−N) in the wet season compared to the dry season. PLS-PM revealed a strong positive correlation (+0.9301) between land use types and physico-chemical variables in the rainy season. In three substrates of the trophic lake, Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were dominant phyla in the bacterial communities, and Rotifers, Platyhelminthes, Gastrotricha, and Ascomycota dominated in microeukaryotic communities. As revealed by null and neutral models, stochastic processes predominantly governed the assembly of bacterial and microeukaryotic communities in biofilms and surface sediments. Network analysis revealed that the microbial interconnections in Ceratophyllum demersum were more stable and complex compared to those in Eichhornia crassipes and sediments. Co-occurrence network analysis (|r| > 0.7, p < 0.05) revealed that there were complex interactions among physicochemical parameters and microbes in epiphytic and sediment biofilms, and many keystone microbes on three substrates played important role in nutrients removal, food web and microbial community stable. These findings emphasize that eutrophic water influence the structure, composition, and interactions of microbes in epiphytic and surface sediment biofilms, and provided new insights into the interconnections between water quality and microbial community in presentative substrates in tropical lacustrine ecosystems in agriculturally polluted areas. The study provides useful information for water quality protection and aquatic plants restoration for policy making and catchment management.

Microbial associates of an endemic Mediterranean seagrass enhance the access of the host and the surrounding seawater to inorganic nitrogen under ocean acidification

Seagrasses are important primary producers in oceans worldwide. They live in shallow coastal waters that are experiencing carbon dioxide enrichment and ocean acidification. Posidonia oceanica, an endemic seagrass species that dominates the Mediterranean Sea, achieves high abundances in seawater with relatively low concentrations of dissolved inorganic nitrogen. Here we tested whether microbial metabolisms associated with P. oceanica and surrounding seawater enhance seagrass access to nitrogen. Using stable isotope enrichments of intact seagrass with amino acids, we showed that ammonification by free-living and seagrass-associated microbes produce ammonium that is likely used by seagrass and surrounding particulate organic matter. Metagenomic analysis of the epiphytic biofilm on the blades and rhizomes support the ubiquity of microbial ammonification genes in this system. Further, we leveraged the presence of natural carbon dioxide vents and show that the presence of P. oceanica enhanced the uptake of nitrogen by water column particulate organic matter, increasing carbon fixation by a factor of 8.6–17.4 with the greatest effect at CO2 vent sites. However, microbial ammonification was reduced at lower pH, suggesting that future ocean climate change will compromise this microbial process. Thus, the seagrass holobiont enhances water column productivity, even in the context of ocean acidification.

Quorum sensing bacteria in microplastics epiphytic biofilms and their biological characteristics which potentially impact marine ecosystem

Microplastics (MPs) have been shown to be a new type of pollutant in the oceans, with complex biofilms attached to their surfaces. Bacteria with quorum sensing (QS) systems are important participants in biofilms. Such bacteria can secrete and detect signal molecules. When a signal molecule reaches its threshold level, bacteria with QS systems can perform several biological functions, such as biofilm formation and antibiotic metabolite production. However, the ecological effects of QS bacteria in biofilm as MPs distribute globally with ocean currents are not to be elucidate yet. In this study, polypropylene and polyvinyl chloride were selected for on-site enrichment to acquire microplastics with biofilms. Eight culturable QS bacteria in the resulting biofilm were isolated by using biosensor assays, and their biodiversity was analyzed. The profiles of the N-acyl-homoserine lactones (AHLs) produced by these bacteria were analyzed by using thin-layer chromatography (TLC)–bioautography and gas chromatography and mass spectrometry (GC–MS). Biofilm-forming properties and several biological characteristics, such as bacteriostasis, algal inhibition, and dimethylsulfoniopropionate (DMSP) degradation, were explored along with QS quenching. Results showed that QS bacteria were mainly affiliated with class Alphaproteobacteria, particularly Rhodobacteraceae, followed by class Gammaproteobacteria. TLC–bioautography and GC–MS analyses revealed that seven AHLs, namely, C6-HSL, C8-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, and two unidentified AHLs were produced. The QS system equipped bacteria with strong biofilm-forming capacity and may contribute to the keystone roles of Rhodobacteraceae. In addition, QS bacteria may exacerbate the adverse environmental effects of MPs, such as inducing the misfeeding of planktons on MPs. This study elucidated the diversity of QS bacteria in MP-associated biofilms and provided a new perspective of the effect of key membrane-forming bacteria on the marine ecological environment.

Coupling of submerged macrophytes and epiphytic biofilms reduced methane emissions from wetlands: Evidenced by an antibiotic inhibition experiment

Wetlands are the largest natural methane source, but how submerged macrophytes affect methane emission remains controversial. In this study, the impacts of submerged macrophytes on methane fluxes, water purification, and epiphytic microbial community dynamics were investigated in simulated wetlands (with and without Hydrilla verticillata) treated with norfloxacin (NOR) for 24 days. Mean methane fluxes were significantly lower in treatments with Hydrilla verticillata (56.84–90.94 mg/m2/h) than bulks (65.96–113.21 mg/m2/h) (p < 0.05) during the experiment regardless of NOR. The relative conductivity (REC) values, H2O2, and malondialdehyde (MDA) contents increased in plant leaves, while water nutrients removal rates decreased with increasing NOR concentration at the same sampling time. The partial least squares path model analysis revealed that plant physiological indices and water nutrients positively affected methane fluxes (0.72 and 0.49, p < 0.001). According to illumina sequencing results of 16S rRNA and pmoA genes, α-proteobacteria (type II) and γ-proteobacteria (type I) were the dominant methanotroph classes in all epiphytic biofilms. The ratio of type I/type II methanotrophs and pmoA gene abundance in epiphytic biofilm was considerably lower in treatment with 16 mg/L NOR than without it (p < 0.05). pmoA gene abundance was negatively correlated with methane fluxes (p < 0.05). Additionally, the assembly of epiphytic bacterial community was mainly governed by deterministic processes, while stochastic dispersal limitation was the primary assembly process in the epiphytic methanotrophic community under NOR stress. The deterministic process gained more importance with time both in bacterial and methanotrophic community assembly. Network analysis revealed that relationships among bacteria in epiphytic biofilms weakened with time but associations among methanotrophic members were enhanced under NOR stress over time. It could be concluded that submerged macrophytes-epiphytic biofilms symbiotic system exhibited potential prospects to reduce methane emissions from wetlands under reasonable management.

Responses of a Submerged Macrophyte Potamogeton crispus and Epiphytic Biofilm to Humic-Substance Enrichment Coupled with Brownification in Freshwater Habitats

Brownification denotes increasing water color, partly caused by increasing dissolved organic matter of terrestrial origin in freshwater. Brownification has become a wide-spread environmental problem because water color alters the physicochemical environment and biological communities in aquatic ecosystems. However, our understanding of its ecological effects on aquatic macrophytes is limited. Here, an indoor mesocosm experiment with a common submerged macrophyte, Potamogeton crispus, along an increasing gradient of brownification was conducted over a period of 42 days. Results showed that P. crispus was able to overcome low degrees of brownification owing to the plasticity in morphological and physiological traits and P. crispus growth even benefitted from the concomitant nutrients along with brownification. However, the macrophyte growth was negatively affected by a 10-fold increase in water color beyond its current level. Additionally, collapse in antioxidant systems and potent photosynthesis inhibition implied that P. crispus could not adapt to the low-light stress generated under the high degree of brownification. Epiphytic bacteria are more sensitive to brownification than their hosts. Any degree of brownification initially caused a decrease in microbial diversity of epiphytic biofilm, whereafter the concomitant nutrients under brownification favored the growth of epiphytic microorganisms. The shading effect of a large number of epiphytic biofilms under brownification may further aggravate the low-light stress on macrophytes. Overall, the study provides new insights into the comprehensive effects and underlying mechanisms of brownification on macrophytes.

Temporal characteristics of algae-denitrifying bacteria co-occurrence patterns and denitrifier assembly in epiphytic biofilms on submerged macrophytes in Caohai Lake, SW China

Temporal characteristics of algae-denitrifying bacteria co-occurrence patterns and denitrifier assembly in epiphytic biofilms on submerged macrophytes in Caohai Lake, SW China

The response of structure and nitrogen removal function of the biofilm on submerged macrophytes to high ammonium in constructed wetlands

The ammonium exceedance discharge from sewage treatment plants has a great risk to the stable operation of subsequent constructed wetlands (CWs). The effects of high ammonium shocks on submerged macrophytes and epiphytic biofilms on the leaves of submerged macrophytes in CWs were rarely mentioned in previous studies. In this paper, the 16S rRNA sequencing method was used to investigate the variation of the microbial communities in biofilms on the leaves of Vallisneria natans plants while the growth characteristics of V. natans plants were measured at different initial ammonium concentrations. The results demonstrated that the total chlorophyll and soluble sugar synthesis of V. natans plants decreased by 51.45% and 57.16%, respectively, and malondialdehyde content increased threefold after 8 days if the initial NH4+-N concentration was more than 5 mg/L. Algal density, bacterial quantity, dissolved oxygen, and pH increased with high ammonium shocks. The average removal efficiencies of total nitrogen and NH4+-N reached 73.26% and 83.94%, respectively. The heat map and relative abundance analysis represented that the relative abundances of phyla Proteobacteria, Cyanobacteria, and Bacteroidetes increased. The numbers of autotrophic nitrifiers and heterotrophic nitrification aerobic denitrification (HNAD) bacteria expanded in biofilms. In particular, HNAD bacteria of Flavobacterium, Hydrogenophaga, Acidovorax, Acinetobacter, Pseudomonas, Aeromonas, and Azospira had higher abundances than autotrophic nitrifiers because there were organic matters secreted from declining leaves of V. natans plants. The analysis of the nitrogen metabolic pathway showed aerobic denitrification was the main nitrogen removal pathway. Thus, the nitrification and denitrification bacterial communities increased in epiphytic biofilms on submerged macrophytes in constructed wetlands while submerged macrophytes declined under ammonium shock loading.

Effects of eutrophic water with ammonium chloride, urea, potassium dihydrogen phosphate and sodium-β-glycerophosphate on Myriophyllum verticillatum and epiphytic bacteria

With the rapid development of industry and agriculture, excessive nitrogen and phosphorus released into natural surface water have caused eutrophication. Applying submerged plants to manage eutrophic water has attracted widespread attention. However, there are limited studies on the effects of different nitrogen and phosphorus in the water environment on submerged plants and their epiphytic biofilm. Therefore, this paper investigated the effects of eutrophic water with ammonium chloride (IN), urea (ON), potassium dihydrogen phosphate (IP), and sodium-β-glycerophosphate (OP) on Myriophyllum verticillatum and epiphytic biofilms. The results showed that Myriophyllum verticillatum exhibited a good purification effect on the eutrophic water with inorganic phosphorus, the removal rates of IP were 68.0%, and the plants grew best in this condition. The fresh weight of the IN group and ON group increased by 12.24% and 7.12%, and the shoot length of the IN group and the ON group increased by 17.71% and 8.33%; the fresh weight of the IP group and OP group increased by 19.19% and 10.83%, the shoot length of the IP group and the OP group increased by 21.09% and 18.23%. In addition, the enzyme activities of superoxide dismutase, catalase, nitrate reductase, and acid phosphatase in plant leaves were significantly changed in eutrophic water with different forms of nitrogen and phosphorus. Finally, the analysis of the epiphytic bacteria showed that different forms of nitrogen and phosphorus nutrients could significantly alter the abundance and structure of microorganisms and microbial metabolism also had significant changes. This study provides a new theoretical basis for evaluating the removal of different forms of nitrogen and phosphorus by Myriophyllum verticillatum, and it also provides new insights for the subsequent engineering of epiphytic microorganisms to improve the capability of submerged plants to treat eutrophic water.

Vallisneria natans decreased CH4 fluxes in wetlands: Interactions among plant physiological status, nutrients and epiphytic bacterial community

Submerged macrophytes provide niches for epiphytic microbes (including aerobic methanotrophs) growth. However, little is known about the impacts of submerged macrophytes growth status and nutrients loadings on methanotroph community and methane release in wetlands. In the present study, methane fluxes, bacterial and methanotroph community in epiphytic biofilm, and environmental parameters were investigated during Vallisneria natans senescence in wetlands under low (VnL) and high (VnH) nutrients for seven weeks. Relative conductivity and concentration of H2O2, total chlorophyll and malondialdehyde were higher in leaves of V. natans in VnH than VnL at the same sampling time. Nutrients loading increased methane fluxes in treatments with or without (Control) macrophytes, while healthy V. natans plants reduced the methane flux and nutrients concentration in water columns. CH4 fluxes were positively correlated to temperature and COD (p < 0.05). Methane oxidation rates were 3.04–31.68 μmol methane mg−1 fresh weight of V. natans leaves - epiphytic biofilm within 1 h. Proteobacteria, Cyanobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, Actinobacteria and Acidobacteria were dominant phylum in all epiphytic biofilms. The mean abundances of pmoA/16S rRNA were higher in VnL than VnH. According to Illumina sequencing results of pmoA gene, γ-proteobacteria and α-proteobacteria were the dominant methanotroph class in epiphytic biofilm from VnH and VnL, respectively. Among seven detected methanotrophic genera, Methylomonas was significantly higher in VnH than VnL. Network analysis revealed that there were much closer relationships between the environmental parameters and epiphytic bacterial community in VnH than in VnL. COD and MDA were negatively correlated with Methyloglobulus, Methylosarcina, Methylobacter and Methylocystis, but positively correlated with Methylomonas and Methylosinus. This study highlights that methanotrophs in epiphytic biofilm play important roles in methane-oxidizing, which can be affected by plant physiological status and environmental parameters.

Effect of erythromycin on epiphytic bacterial communities and water quality in wetlands

The occurrence of antibiotics such as erythromycin (ERY) under macrolide group, has long been acknowledged for negatively affecting ecosystems in freshwater environments. However, the effects of ERY on water quality and microbial communities in epiphytic biofilms are poorly understood. Here, Scanning Electron Microscopy (SEM), High-throughput sequencing, and physicochemical analytical methods were employed to unravel the impact of ERY on the water quality and bacterial morphology, biodiversity, composition, interaction, and ecological function in epiphytic biofilms attached to Vallisneria natans and artificial plants in mesocosmic wetlands. The study showed that ERY exposure significantly impaired the nutrient removal capacity (TN, TP, and COD) and altered the epiphytic bacterial morphology of V. natans and artificial plants. ERY did not affect the bacterial α-diversity. Notwithstanding ERY decreased the bacterial composition, but the relative abundance of Proteobacteria and Patescibacteria spiked by 62.2 % and 54 %, respectively, in V. natans, while Desulfobacteria and Chloroflexi increased by 8.9 % and 11.2 %, respectively, in artificial plants. Notably, ERY disturbed the food web structure and metabolic pathways such as carbohydrate metabolism, amino acid metabolism, energy metabolism, cofactor and vitamin metabolism, membrane transport, and signal transduction. This study revealed that ERY exposure disrupted the bacterial morphology, composition, interaction or food web structure, and metabolic functions in epiphytic biofilm. These data underlined that ERY negatively impacts epiphytic bacterial communities and nutrient removal in wetlands.

Combined toxic effects of enrofloxacin and microplastics on submerged plants and epiphytic biofilms in high nitrogen and phosphorus waters

With the wide application of plastic products, microplastic pollution has become a major environmental issue of global concern. Microplastics in aquatic environments can interact with organic pollutants, causing a combined effect on submerged macrophytes. This study investigated the response mechanisms of the submerged plant Myriophyllum verticillatum and epiphytic biofilm to the antibiotic enrofloxacin, microplastics, and their combined exposure in a high nitrogen and phosphorus environment. The results indicated that Myriophyllum verticillatum was not sensitive to enrofloxacin of 1 mg L−1, while 10 and 50 mg L−1 enrofloxacin inhibited the uptake of nitrogen and phosphorus by the plants, as well as triggered oxidative stress in the plant leaves, causing irreversible damage to the plant cells. In addition, enrofloxacin altered the structure of the leaf epiphytic biofilm community. Interestingly, 1, 5, and 20 mg L−1 microplastics had no significant effect on the plant, while they facilitated the aggregation of microorganisms, increasing the abundance of the leaf epiphyte biofilm. The combination of enrofloxacin and microplastics induced a synergistic effect on Myriophyllum verticillatum. Specifically, the rate of nitrogen and phosphorus uptake by the plant was reduced, the content of photosynthetic pigments decreased, and antioxidant enzyme activity was further increased. In addition, the diversity of the leaf epiphytic biofilm community was similar to the single enrofloxacin exposure. These results demonstrated the differences between single and combined exposures and provided a new theoretical basis to evaluate the harmful effects of enrofloxacin and microplastics on submerged macrophytes.

Unraveling the ecological mechanisms of bacterial succession in epiphytic biofilms on Vallisneria natans and Hydrilla verticillata during bioremediation of phenanthrene and pyrene polluted wetland

In wetland ecosystem, the microbial succession in epiphytic biofilms of submerged macrophytes remains to be fully elucidated, especially submerged macrophytes used to remediate organic pollutants contaminated sediment. Herein, 16 S rRNA gene sequencing was used to investigate the bacterial dynamics and ecological processes in the biofilms of two typical submerged macrophytes (Vallisneria natans and Hydrilla verticillata) settled in sediment polluted by polycyclic aromatic hydrocarbons (PAHs) at two growth periods. The results presented that the variations of bacterial community in the biofilms were influenced by attached surfaces (explanation ratio: 17.30%), incubation time (32.30%) and environmental factors (39.10%). Bacterial community assembly was mainly driven by dispersal limitation which triggered more positive co-occurrence associations in microbial networks, maintaining ecological stability in the process of bioremediation of PAHs. Additionally, the functional redundancy strength of bacterial community was more affected by attached surface than incubation time. The structural equation model illustrated that community assembly drove β-diversity and explained a part of ecological functions. Environmental factors, community assembly, and β-diversity jointly affected microbial networks. Overall, our study offers new insights into the microbial ecology in biofilms attached on the submerged macrophytes settled in PAH-polluted sediment, providing important information for deeply understanding submerged macrophyte-biofilm complex and promoting sustainable phytoremediation in shallow lacustrine and marshy ecosystems.