Phosphate-solubilizing bacteria improve the antioxidant enzyme activity of Potamogeton crispus L. and enhance the remediation effect on Cd-contaminated sediment.

Phosphorus-solubilizing bacteria (PSB) assisted phytoremediation of cadmium (Cd) pollution is an effective method, but the mechanism of PSB-enhanced in-situ remediation of Cd contaminated sediment by submerged plants is still rare. In this study, PSB (Leclercia adecarboxylata L1-5) was inoculated in the rhizosphere of Potamogeton crispus L. (P. crispus) to explore the effect of PSB on phytoremediation. The results showed that the inoculation of PSB effectively improved the Cd extraction by P. crispus under different Cd pollution and the Cd content in the aboveground and underground parts of P. crispus all increased. The µ-XRF images showed that most of the Cd was enriched in the roots of P. crispus. PSB especially showed positive effects on root development and chlorophyll synthesis. The root length of P. crispus increased by 51.7 %, 80.5 % and 74.2 % under different Cd pollution, and the Ca/Cb increased by 38.9 %, 15.2 % and 8.6 %, respectively. Furthermore, PSB enhanced the tolerance of P. crispus to Cd. The contents of soluble protein, MDA and H2O2 in 5 mg·kg-1 and 7 mg·kg-1 Cd content groups were decreased and the activities of antioxidant enzymes were increased after adding PSB. The results showed that the application of PSB was beneficial to the in-situ remediation of submerged plants.

Analysis of diversity and function of epiphytic bacterial communities associated with macrophytes using a metagenomic approach.

Epiphytic bacteria constitute a vital component of aquatic ecosystems, pivotal in regulating elemental cycling. Despite their significance, the diversity and functions of epiphytic bacterial communities adhering to various submerged macrophytes remain largely unexplored. In this study, we employed a metagenomic approach to investigate the diversity and function of epiphytic bacterial communities associated with six submerged macrophytes: Ceratophyllum demersum, Hydrilla verticillata, Myriophyllum verticillatum, Potamogeton lucens, Stuckenia pectinata, and Najas marina. The results revealed that the predominant epiphytic bacterial species for each plant type included Pseudomonas spp., Microbacterium spp., and Stenotrophomonas rhizophila. Multiple comparisons and linear discriminant analysis effect size indicated a significant divergence in the community composition of epiphytic bacteria among the six submerged macrophytes, with 0.3-1% of species uniquely identified. Epiphytic bacterial richness associated with S. pectinata significantly differed from that of both C. demersum and H. verticillata, although no significant differences were observed in diversity and evenness. Functionally, notable variations were observed in the relative abundances of genes associated with carbon, nitrogen, and phosphorus cycling within epiphytic bacterial communities on the submerged macrophyte hosts. Among these communities, H. verticillata exhibited enrichment in genes related to the 3-hydroxypropionate bicycle and nitrogen assimilation, translocation, and denitrification. Conversely, M. verticillatum showcased enrichment in genes linked to the reductive citric acid cycle (Arnon-Buchanan cycle), reductive pentose phosphate cycle (Calvin cycle), polyphosphate degradation, and organic nitrogen metabolism. In summary, our findings offer valuable insights into the diversity and function of epiphytic bacteria on submerged macrophyte leaves, shedding light on their roles in lake ecosystems.

Ramichloridium endophyticum sp. nov., a novel species of endophytic fungus from Potamogeton pectinatus.

During a survey of endophytic fungi in aquatic plants collected from Tibet, PR China, a novel species, Ramichloridium endophyticum, was isolated from Potamogeton pectinatus. This novel species differs from other species of the genus Ramichloridium by its finely verrucose, obovoid, ellipsoidal-obovoid and occasionally subglobose conidia. Phylogenetic analysis of the combined sequences of the internal transcribed spacers (ITS) and the translation elongation factor 1-alpha gene (tef1-α) confirmed that the isolated strain represents a member of the genus Ramichloridium. A full description, illustrations and a phylogenetic tree showing the position of R. endophyticum are provided.

The abundance of nirS-type denitrifiers and anammox bacteria in rhizospheres was affected by the organic acids secreted from roots of submerged macrophytes.

Excessive nitrogen has been a global concern to cause lake eutrophication. The denitrification and anammox processes are considered to be effective biological pathways for nitrogen removal. Submerged macrophytes also play a key role in the nitrogen cycle of lakes. However, the mechanism of submerged macrophytes on regulating biological nitrogen removal pathways has not been well quantified. Therefore, this study investigated the impacts of submerged macrophytes on the community structures and abundance of the nirS-type denitrifiers and anammox bacteria in the rhizospheres. The qPCR results indicated that the abundance of two bacteria in the near-rhizospheres of submerged macrophytes was significantly lower than the root compartments and non-rhizospheres, while the concentrations of organic acids in the near-rhizospheres were higher than those of the root compartments and non-rhizospheres. Redundancy analysis results illustrated that concentrations of NO3--N, NO2--N, citric acid and oxalic acid were the key environmental indicators which had the significant impact on the microbial community. The concentrations of citric acid and oxalic acid were negatively correlated with the nirS-type denitrifiers abundance, and the oxalic acid concentrations were negatively correlated with the anammox bacteria abundance. These results indicated that submerged macrophytes could reduce the abundance of nirS-type denitrifiers and anammox bacteria by releasing organic acids. In addition, the highest diversity of denitrifier community were found in the rhizosphere of the Hydrilla verticillata, while the highest diversity of anammox community were found in the Potamogeton maackianus rhizosphere. These results indicate that the impacts of submerged macrophytes on the biological nitrogen removal pathways were species-dependent.

Nitrogen loading affects microbes, nitrifiers and denitrifiers attached to submerged macrophyte in constructed wetlands.

Submerged macrophytes and biofilms are important components of wetlands. However, little is known about the changes of microbes in biofilms attached to submerged macrophytes upon nitrogen loading. This study investigated the changes of microbes, algae, nitrifiers and denitrifiers in biofilms attached to the leaves of artificial plants (AP), Potamogeton malaianus (PM), Vallisneria natans (VN) and Hydrilla verticillata (HV) under varied initial concentrations of total nitrogen (TN). Nitrogen addition increased biofilm biomass and changed dissolved oxygen concentrations and pH values in overlaying water. Epiphytic algal densities showed the same trend at the same N level:AP>PM>VN>HV. As revealed by cluster analysis at phylum level, algae compositions in biofilm from four plants showed some host-specific at 2 and 12mgL-1 TN, but was clustered in the same group at 22mgL-1 TN regardless of plant species. Submerged macrophytes had better performance in total N removal than AP. In general, N application significantly increased the abundance of amoA, nirK, nirS, napA and cnorB in biofilm. The abundance of the denitrification genes (nirK, nirS, napA, narG and cnorB) was positively correlated with nitrogen application, while amoA was correlated with concentration of dissolved oxygen. These results indicate that N loadings stimulated the growth of biofilms attached to submerged macrophyte and the removal of total N can be partially ascribed to the synergistic interactions of submerged macrophyte and biofilms in wetlands. These results highlight the ecological role of submerged macrophyte-biofilm system in nitrogen removal in wetlands.

Submerged macrophytes shape the abundance and diversity of bacterial denitrifiers in bacterioplankton and epiphyton in the Shallow Fresh Lake Taihu, China.

nirK and nirS genes are important functional genes involved in the denitrification pathway. Recent studies about these two denitrifying genes are focusing on sediment and wastewater microbe. In this study, we conducted a comparative analysis of the abundance and diversity of denitrifiers in the epiphyton of submerged macrophytes Potamogeton malaianus and Ceratophyllum demersum as well as in bacterioplankton in the shallow fresh lake Taihu, China. Results showed that nirK and nirS genes had significant different niches in epiphyton and bacterioplankton. Bacterioplankton showed greater abundance of nirK gene in terms of copy numbers and lower abundance of nirS gene. Significant difference in the abundance of nirK and nirS genes also existed between the epiphyton from different submerged macrophytes. Similar community diversity yet different community abundance was observed between epiphytic bacteria and bacterioplankton. No apparent seasonal variation was found either in epiphytic bacteria or bacterioplankton; however, environmental parameters seemed to have direct relevancy with nirK and nirS genes. Our study suggested that submerged macrophytes have greater influence than seasonal parameters in shaping the presence and abundance of bacterial denitrifiers. Further investigation needs to focus on the potential contact and relative contribution between denitrifiers and environmental factors.

Remedial effects of Potamogeton crispus L. on PAH-contaminated sediments.

In this study, the remedial effects of submerged macrophyte Potamogeton crispus L. on polycyclic aromatic hydrocarbon (PAH)-contaminated sediments were investigated. After a 54-day experiment, the dissipation ratios of phenanthrene and pyrene were 84.8-88.3 and 72.4-78.5% in rhizosphere sediments, which were significantly higher than those in non-rhizosphere sediments (54.2-66.6 and 54.7-58.5%). The dissipation increment increased not only with increasing spiked concentration, but also over time, while plant uptake accounted for only a small portion (<6%) of the dissipation increment. Moreover, bioavailable fraction tests revealed that biodegradation was not controlled by the amount of bioavailable PAHs. For better understanding of the microbial mechanism involved, phospholipid fatty acid (PLFA) profiles were analyzed. Biomass of microorganisms indicated by the total PLFA content was higher in rhizosphere sediments than in non-rhizosphere sediments and was related well to the dissipation ratios of the two PAHs. Cluster analysis showed that community structure significantly changed in rhizosphere sediments. Moreover, the increments of PAH dissipation in rhizosphere sediments had a strong positive correlation with those of polyphenol oxidase activities in the same media. It can be concluded that the enhanced remediation of PAHs by P. crispus was mainly due to the increase of microbial biomass and activity as well as changes of microbial community structure in sediments as a result of plant growth stimulation.

Effects of Potamogeton crispus L.-bacteria interactions on the removal of phthalate acid esters from surface water.

To investigate the mechanism of submerged macrophyte-bacteria interactions on the removal of phthalic acid esters from surface water, experiments with and without Potamogeton crispus L. were performed. A two-compartment (i.e., water and plant) kinetic model was developed. The model adequately described the variation of dibutyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) in the plant-water system by providing the first-order rate constants of plant uptake (k1) and release (k2), microbial degradation in water (k3) and plant degradation (k4). During 10-d incubation, the presence of P. crispus enhanced the removal of DBP and DEHP from water by 6.3% and 22.4%. Compared with the experiment without P. crispus, biodegradation of DBP in water with P. crispus decreased by 8.3% because of plant uptake even though k3 increased by 30%. 21.4% of DBP transferred from water to plants, of which only small amount (5.1%) retained in the plant and the rest (94.9%) was degraded. Different from DBP, biodegradation of DEHP in water with P. crispus was a slightly higher than that without P. crispus. 25.5% of DEHP transferred from water to plants, of which a large portion (73.3%) retained in the plant and the rest (26.7%) was degraded. This finding reveals that the enhancement of DBP removal from surface water is mainly related to faster degradation in the plant, whereas it is mainly related to higher plant accumulation for DEHP.

Contrasting diversity of epibiotic bacteria and surrounding bacterioplankton of a common submerged macrophyte, Potamogeton crispus, in freshwater lakes.

Epibiotic bacteria on surfaces of submerged macrophytes play important roles in the ecological processes of shallow lakes. However, their community ecology and dynamics are far from understood in comparison with those of bacterioplankton. Here, we conducted a comparative study of the species diversity and composition of epibiotic bacterial and the surrounding bacterioplankton communities of a common submerged macrophyte, Potamogeton crispus, in 12 lakes at a regional scale in China. We found that in different freshwater lakes, epibiotic bacteria possessed higher taxonomic richness than bacterioplankton did. There existed a marked divergence in the community structure between epibiotic bacteria and bacterioplankton. Alphaproteobacteria was the most dominant group for epibiotic bacteria, whereas Actinobacteria dominated bacterioplankton. Although variations in both bacterioplankton and epibiotic bacterial community compositions in different lakes were better explained by environmental than spatial factors, both environment and space had more intensified effects on epibiotic bacteria. This implied more complex and diverse 'microhabitats' for epibiotic bacteria on surfaces of submerged macrophytes, which may lead to higher variations of epibiotic bacteria than bacterioplankton. Our study suggested that epibiotic bacteria exhibited higher diversity and distinct community composition than the surrounding bacterioplankton. More attention should be focused on the productive and diverse microbial habitats on submerged macrophytes.

An ultrasonic method for separation of epiphytic microbes from freshwater submerged macrophytes.

Epiphytic microbes are common inhabitants of freshwater submerged macrophytes, which play an important role in aquatic ecosystems. An important precondition for studying the epiphytic microbes is having an effective method of separating the attached microbes from the host macrophytes. We developed an ultrasound-based method for separating epiphytic microbes from freshwater submerged macrophytes, optimized the conditions of ultrasonic separation with an orthogonal experimental design, and compared the optimized ultrasonic method with manual separation. This method can be particularly useful for freshwater submerged macrophytes having a complex morphology.

Quantitative assessment of ammonia-oxidizing bacterial communities in the epiphyton of submerged macrophytes in shallow lakes.

In addition to the benthic and pelagic habitats, the epiphytic compartment of submerged macrophytes in shallow freshwater lakes offers a niche to bacterial ammonia-oxidizing communities. However, the diversity, numbers, and activity of epiphytic ammonia-oxidizing bacteria have long been overlooked. In the present study, we analyzed quantitatively the epiphytic communities of three shallow lakes by a potential nitrification assay and by quantitative PCR of 16S rRNA genes. On the basis of the m(2) of the lake surface, the gene copy numbers of epiphytic ammonia oxidizers were not significantly different from those in the benthic and pelagic compartments. The potential ammonia-oxidizing activities measured in the epiphytic compartment were also not significantly different from the activities determined in the benthic compartment. No potential ammonia-oxidizing activities were observed in the pelagic compartment. No activity was detected in the epiphyton of Chara aspera, the dominant submerged macrophyte in Lake Nuldernauw in The Netherlands. The presence of ammonia-oxidizing bacterial cells in the epiphyton of Potamogeton pectinatus was also demonstrated by fluorescent in situ hybridization microscopy images. By comparing the community composition as assessed by the 16S rRNA gene PCR-denaturing gradient gel electrophoresis approach, it was concluded that the epiphytic ammonia-oxidizing communities consisted of cells that were also present in the benthic and pelagic compartments. Of the environmental parameters examined, only the water retention time, the Kjeldahl nitrogen content, and the total phosphorus content correlated with potential ammonia-oxidizing activities. None of these parameters correlated with the numbers of gene copies related to ammonia-oxidizing betaproteobacteria.

[Ammonia-oxidizing bacteria community composition at the root zones of aquatic plants after ecological restoration].

To investigate the effects of aquatic plants on ammonia-oxidizing bacteria (AOB) at their root zones, four species of aquatic plants were selected, Phragmites communis, Typha angustifolia L., Potamogeton crispus L., and Limnanthemun nymphoides, which were widely used in ecological restorations. AOB in the samples were enumerated by most-probable-number (MPN) method. Nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) procedures were performed with ammonia oxidizer-selective primers. Main DGGE bands were excised from the gel and sequenced for phylogenetic affiliation. Results indicate that AOB densities are always higher at the root zones of emergent plants (Phragmites communis 2.8 x 10(5) cells/g and Typha angustifolia L.4.3 x 10(5) cells/g) than those of submerged and floating-leaved plant (Potamogeton crispus L. 9.3 x 10(4) cells/g and Limnanthemun nymphoides 7.7 x 10(4) cells/g). At the root zones, the oxidation-reduction potential is above zero and NH4+ concentration is lower than it in the bare surface sediment. Fourteen major bands were recovered from the DGGE gel, re-amplified and sequenced. Although the identified bands have their respective similar sequences in GenBank, most of them are related to Nitrosomonas-like. This type of bacteria would play an important role of nitrogen cycle in lake sediment after ecological restoration.

[Role of the clasping-leaved pondweed (Potamogeton perfoliatus) polysaccharides in formation of its bacterial environment].

The effect of the polysaccharides of clasping-leaved pondweed (Potamogeton perfioliatus) on the formation of a bacteriocenosis of this plant was demonstrated by research on chemoreception, relative surface hydrophobicity, and the growth characteristics of the members of five bacterial genera abundant in this microbiocenosis. The plant heteropolysaccharides of anionic and cationic nature were found to participate in selective stimulation or inhibition of growth of some microbial groups in surrounding water. These findings improve our understanding of the spectrum of physiological activity of glycopolymers of diverse origin.