Distribution patterns and influential factors of pathogenic bacteria in freshwater aquaculture sediments.

Pathogenic bacteria, the major causative agents of aquaculture diseases, are a serious impediment to the aquaculture industry. However, the bioinformatics of pathogenic bacteria and virulence factors (VFs) in sediments, an important component of freshwater aquaculture ecosystems, are not well characterized. In this study, 20 sediment samples were collected from fish pond sediments (FPS), shrimp field sediments (SFS), fish pond sediment control (FPSC), and shrimp field sediment control (SFSC). Molecular biological information was obtained on a total of 173 pathogenic bacteria, 1093 virulence factors (VFs), and 8475 mobile genetic elements (MGEs) from these samples. The results indicated that (1) aquaculture patterns and sediment characteristics can affect the distribution of pathogenic bacteria. According to the results of the Kruskal-Wallis H test, except for Mycobacterium gilvum, there were significant differences (P < 0.05) among the four sediment types in the average abundance of major pathogenic bacteria (top 30 in abundance), and the average abundance of major pathogenic bacteria in the four sediment types followed the following pattern: FPS > SFS > FPSC > SFSC. (2) Pathogenic bacteria are able to implement a variety of complex pathogenic mechanisms such as adhesion, invasion, immune evasion, and metabolic regulation in the host because they carry a variety of VFs such as type IV pili, HSI-I, Alginate, Colibactin, and Capsule. According to the primary classification of the Virulence Factor Database (VFDB), the abundance of VFs in all four types of sediments showed the following pattern: offensive VFs > non-specific VFs > defensive VFs > regulation of virulence-related genes. (3) Total organic carbon (TOC), total phosphorus (TP), available phosphorus (AP), nitrite, and nitrate were mostly only weakly positively correlated with the major pathogenic bacteria and could promote the growth of pathogenic bacteria to some extent, whereas ammonia was significantly positively correlated with most of the major pathogenic bacteria and could play an important role in promoting the growth and reproduction of pathogenic bacteria. (4) Meanwhile, there was also a significant positive correlation between CAZyme genes and major pathogenic bacteria (0.62 ≤ R ≤ 0.89, P < 0.05). This suggests that these pathogenic bacteria could be the main carriers of CAZyme genes and, to some extent, gained a higher level of metabolic activity by degrading organic matter in the sediments to maintain their competitive advantage. (5) Worryingly, the results of correlation analyses indicated that MGEs in aquaculture sediments could play an important role in the spread of VFs (R = 0.82, P < 0.01), and in particular, plasmids (R = 0.75, P < 0.01) and integrative and conjugative elements (ICEs, R = 0.65, P < 0.05) could be these major vectors of VFs. The results of this study contribute to a comprehensive understanding of the health of freshwater aquaculture sediments and provide a scientific basis for aquaculture management and conservation.

Diversity and biogenesis contribution of sulfate-reducing bacteria in arsenic-contaminated soils from realgar deposits.

Microbial sulfate reduction, a vital mechanism for microorganisms living in anaerobic, sulfate-rich environments, is an essential aspect of the sulfur biogeochemical cycle. However, there has been no detailed investigation of the diversity and biogenesis contribution of sulfate-reducing bacteria in arsenic-contaminated soils from realgar deposits. To elucidate this issue, soil samples from representative abandoned realgar deposits were collected. Microcosm assays illustrated that all three samples (2-1, 2-2, and 2-3) displayed efficient sulfate and As(V)-respiring activities. Furthermore, a total of 28 novel sequence variants of dissimilatory sulfite reductase genes and 2 new families of dsrAB genes were successfully identified. A novel dissimilatory sulfate-reducing bacterium, Desulfotomaculum sp. JL1, was also isolated from soils, and can efficiently respiratory reduce As(V) and sulfate in 4 and 5 days, respectively. JL1 can promote the generation of yellow precipitates in the presence of multiple electron acceptors (both contain sulfate and As(V) in the cultures), which indicated the biogenesis contribution of sulfate-reducing bacteria to the realgar mine. Moreover, this area had unique microbial communities; the most abundant populations belonged to the phyla Proteobacteria, Chloroflexi, and Acidobacteriota, which were attributed to the unique geochemistry characteristics, such as total organic carbon, total As, NO3-, and SO42-. The results of this study provide new insight into the diversity and biogenesis contributions of sulfate-reducing bacteria in arsenic-contaminated soils from realgar deposits.

Combination of polyurethane and polymethyl methacrylate thin films as a microextraction sorbent for rapid adsorption and sensitive determination of neonicotinoid insecticides in fruit juice and tea by ultra high performance liquid chromatography with tandem mass spectrometry.

An economical and effective thin film microextraction (TFME) for simultaneous analysis of ten neonicotinoid insecticides and metabolites in fruit juice and tea, was developed based on the combination of polyurethane (PU) and polymethyl methacrylate (PMMA) films as the sorbent followed by ultra high performance liquid chromatography with tandem mass spectrometry. The PU/PMMA composite was evidenced to possess rapid adsorption and strong accumulation towards neonicotinoids compared with the films used alone. A series of parameters were optimized, and the agitation mode, film size, ionic strength, desorption solvent and sample pH were found to dominate the microextraction process rather than the extraction temperature, agitation time and sample volume. The thin films are cost effective and efficient for single use analysis, but still can be reused at least 8 times with no significant loss in performance. The ten neonicotinoids were measured with good recoveries (81.1-107.9%), high enrichment factors (up to 135), low limits of detection (0.001-0.1 µg L-1), and wide linearity range (1-500 µg L-1, r2>0.9981) in fruit juice (apple, lemon, and pomegranate) and tea (green tea and black tea) samples. The proposed method was successfully applied to commercial fruit and tea drinks, and no samples were tested positive on target neonicotinoids. The PU/PMMA based TFME has shown great potential as an alternative to exhaustive extraction techniques for routine screening of trace neonicotinoids in fruit juice and tea by simplifying the analytical procedure, shortening the operation time, and lowering the material expense.

Using silicone rubber and polyvinylchloride as equilibrium passive samplers for rapid and sensitive monitoring of pyrethroid insecticides in aquatic environments.

Passive sampling to regularly identify the occurrence of pyrethroid insecticides in urban streams is a crucial work of risk management with respect to intrinsic toxicity of pyrethroids to aquatic organisms. Polymeric films, based on an equilibrium sampling principle, have found increasing use as passive samplers for hydrophobic contaminants. Herein, we investigated two thin-film samplers, namely silicone rubber (SR) and polyvinylchloride (PVC), compatible with a suite of 8 pyrethroids, for measuring freely dissolved concentrations (Cfree) in water. The characteristics of SR and PVC samplers were estimated in terms of equilibrium partitioning coefficients (Kf) with log units of 3.90-4.67 and sampling rates (Rs) of 0.011-0.016 L/h. The parameters were correlated positively with octanol-water partition coefficients of the compounds, whereas independent on water solubility. A strong agreement between Cfree obtained from the two samplers was observed in a range of 0.1-10 µg/L for pyrethroids under laboratory simulated conditions. Both of SR and PVC were confirmed as equilibrium samplers with faster sampling rates of pyrethroids that equilibrated on films within only one week, and higher accumulation at factors of 5.3-12.5 and 1.5-2.4 compared to a performance reference compound (PRC)-preload sampler. Additionally, the comparable results of the two passive sampling methods in multiple field applications indicated that the direct deployment of the two samplers without PRCs calibration can provide reliable assessment of trace concentrations. This study demonstrated the routine utilization of SR and PVC as promising tools for rapid and sensitive in-situ monitoring of pyrethroids, and indicators for the bioavailability against total chemical concentrations in variable aquatic environments.

Unique diversity and functions of the arsenic-methylating microorganisms from the tailings of Shimen Realgar Mine.

Microbial arsenic (As) methylation plays important roles in the As biogeochemical cycle. However, little is known about the diversity and functions of As-methylating microorganisms from the tailings of a Realgar Mine, which is characterized as containing extremely high concentrations of As. To address this issue, we collected five samples (T1-T5) from the tailings of Shimen Realgar Mine. Microcosm assays without addition of exogenous As and carbon indicated that all the five samples possess significant As-methylating activities, producing 0.8-5.7 µg/L DMAsV, and 1.1-10.7 µg/L MMAsV with an exception of T3, from which MMAsV was not detectable after 14.0 days of incubation. In comparison, addition of 20.0 mM lactate to the microcosms significantly enhanced the activities of these samples; the produced DMAsV and MMAsV are 8.0-39.7 µg/L and 5.8-38.3 µg/L, respectively. The biogenic DMAsV shows significant positive correlations with the Fe concentrations and negative correlations with the total nitrogen concentrations in the environment. A total of 63 different arsM genes were identified from the five samples, which code for new or new-type ArsM proteins, suggesting that a unique diversity of As-methylating microbes are present in the environment. The microbial community structures of the samples were significantly shaped by the environmental total organic carbon, total As contents and NO3- contents. These data help to better understand the microorganisms-catalyzed As methylation occurred in the environment with extremely high contents of As.

Microbial reactions and environmental factors affecting the dissolution and release of arsenic in the severely contaminated soils under anaerobic or aerobic conditions.

The soils near the abandoned Shimen Realgar Mine are characterized by containing extremely high contents of total and soluble arsenic. To determine the microbial reactions and environmental factors affecting the mobilization and release of arsenic from soils phase into pore water, we collected 24 soil samples from the representative points around the abandoned Shimen Realgar Mine. They contained 8310.84 mg/kg total arsenic and 703.21 mg/kg soluble arsenic in average. The soluble arsenic in the soils shows significant positive and negative correlations with environmental SO42-/TOC/pH/PO43-, and Fe/Mn, respectively. We found that diverse dissimilatory As(V)-respiring prokaryotes (DARPs) and As(III)-oxidizing bacteria (AOB) exist in all the examined soil samples. The activities of DARPs led to 65-1275% increase of soluble As(III) in the examined soils after 21.0 days of anaerobic incubation, and the microbial dissolution and releases of arsenic show significant positive and negative correlations with the environmental pH/TN and NH4+/PO43-, respectively. In comparison, the activities of AOB led to 24-346% inhibition of the dissolved oxygen-mediated dissolution of arsenic in the soils, and the AOB-mediated releases of As(V) show significant positive and negative correlations with the environmental SO42- and pH/NH4+, respectively. The microbial communities of 24 samples contain 54 phyla of bacteria that show extremely high diversities. Total arsenic, TOC, NO3- and pH are the key environmental factors that indirectly controlled the mobilization and release of arsenic via influencing the structures of the microbial communities in the soils. This work gained new insights into the mechanism for how microbial communities catalyze the dissolution and releases of arsenic from the soils with extremely high contents of arsenic.

A polyurethane-based thin film for solid phase microextraction of pyrethroid insecticides.

This work introduces polyurethane (PU) as an efficient and economic sorbent for thin film solid phase microextraction of pyrethroid insecticides, specifically of bifenthrin, fenpropathrin, lambda-cyhalothrin, permethrin, cypermethrin, flucythrinate, fenvalerate and deltamethrin. The PU film is immersed into chrysanthemum tea under ultrasonication for the adsorption of the analytes, and the analytes are desorbed by a mixture of hexane and ethyl acetate and then quantified by gas chromatography with electron capture detection. The film type, adsorption temperature, extraction time, sample condition, and desorption procedure were optimized. The adsorption capacity and robustness of the PU film is found to be excellent for analysis of pyrethroids in chrysanthemum tea. The limits of detection and method limits of detection range from 0.05-0.5 µg L-1 and 0.0003-0.003 µg L-1, respectively. The relative recoveries from spiked samples are between 84.5 and 104.1%, and enrichment factors up to 188. The method was validated through blind split analyses of chrysanthemum tea infusion and ready-to-drink samples with liquid-liquid extraction. Good agreement between the two approaches shows the method to have an accuracy that is similar to that of the conventional technique. Compared with other reported approaches, the PU-based method exhibites a higher sensitivity, easier operation, lower costs and less matrix effects. Graphical abstract Schematic representation of the use of a polyurethane film as an efficient and economic sorbent for the microextraction of 8 pyrethroids by gas chromatography. This method exhibites excellent performance of accuracy, sensitivity, and robustness, demonstrating its potential of application in the analysis of complex matrix.

Self-acidity induced effervescence and manual shaking-assisted microextraction of neonicotinoid insecticides in orange juice.

A novel dispersive liquid-liquid microextraction that combines self-induced acid-base effervescent reaction and manual shaking, coupled with ultra high performance liquid chromatography with tandem mass spectrometry was developed for simultaneous determination of ten neonicotinoid insecticides and metabolites in orange juice. An innovative aspect of this method was the utilization of the acidity of the juice for a self-reaction between acidic components contained in the juice sample and added sodium carbonate which generated carbon dioxide bubbles in situ, accelerating the analytes transfer to the extractant of 1-undecanol. The total acid content of juice sample was measured to produce the maximum amount of bubbles with minimum usage of carbonate. Manual shaking was subsequently adopted and was proven to enhance the extraction efficiency. The factors affecting the performance, including the type and the amount of the carbon dioxide source and extractant, and ionic strength were optimized. Compared with conventional methods, this approach exhibited low limits of detection (0.001-0.1 µg/L), good recoveries (86.2-103.6%), high enrichment factors (25-50), and negligible matrix effects (-12.3-13.7%). The proposed method was demonstrated to provide a rapid, practical, and environmentally friendly procedure due to no acid reagent, toxic solvent, or external energy requirement, giving rise to potential application on other high acid-content matrices.

Inhibitory effect of nitrate/nitrite on the microbial reductive dissolution of arsenic and iron from soils into pore water.

It was well established that microbial communities are the major drive for the formation of arsenic-contaminated groundwater. However, it remains to be elucidated for how nitrate/nitrite affects the microorganisms-catalyzed dissolution and reduction of arsenic. To address this issue, we collected soil samples containing high-contents of arsenic from the Shimen Realgar Mine area. Microcosm assay indicated that addition of nitrate/nitrite significantly inhibited the dissolution, reduction and release of As and Fe caused by the biological catalysis of microbial communities in the soils, meanwhile nitrate/nitrite was reduced into N2. To further investigate the molecular mechanism of this finding, we used a representative dissimilatory arsenate-respiring strain Shewanella sp. GL90 from the soils to perform the arsenic release assay. GL90 can efficiently catalyze the reductive dissolution, and promote the release of As and Fe in soils. It is interesting to see that the addition of nitrate/nitrite to the soils led to marked decreases in the GL90-mediated dissolution of As and Fe in the soils. Moreover, we found that this finding was attributed to that nitrate/nitrite significantly inhibited the transcription of the gene of the respiratory arsenate reductase protein in GL90 cells. This work provided new insights into the mechanisms for the coupling of As, N and Fe geochemical cycles in arsenic-rich soils, and for how environmental factors affect As concentration in groundwater.

Dissimilatory arsenate-respiring prokaryotes catalyze the dissolution, reduction and release of arsenic from paddy soils into groundwater: implication for the effect of sulfate.

The paddy soils in some areas in Jianghan Plain were severely contaminated by arsenic. However, little is known about the activity and diversity of the dissimilatory arsenate-respiring prokaryotes (DARPs) in the paddy soils, and the effects of sulfate on the microbial mobilization and release of arsenic from soils into solution. To address this issue, we collected arsenic-rich soils from the depths of 1.6 and 4.6 m in a paddy region in the Xiantao city, Hubei Province, China. Microcosm assays indicated that all of the soils have significant arsenate-respiring activities using lactate, pyruvate or acetate as the sole electron donor. Functional gene cloning and analysis suggest that there are diverse DARPs in the indigenous microbial communities of the soils. They efficiently promoted the mobilization, reduction and release of arsenic and iron from soils under anaerobic conditions. Remarkably, when sulfate was amended into the microcosms, the microorganisms-catalyzed reduction and release of arsenic and iron were significantly increased. We further found that sulfate significantly enhanced the arsenate-respiring reductase gene abundances in the soils. Taken together, a diversity of DARPs in the paddy soils significantly catalyzed the dissolution, reduction and release of arsenic and iron from insoluble phase into solution, and the presence of sulfate significantly increased the microbial reactions.

Microbially Mediated Methylation of Arsenic in the Arsenic-Rich Soils and Sediments of Jianghan Plain.

Almost nothing is known about the activities and diversities of microbial communities involved in As methylation in arsenic-rich shallow and deep sediments; the correlations between As biomethylation and environmental parameters also remain to be elucidated. To address these issues, we collected 9 arsenic-rich soil/sediment samples from the depths of 1, 30, 65, 95, 114, 135, 175, 200, and 223 m in Jianghan Plain, China. We used microcosm assays to determine the As-methylating activities of the microbial communities in the samples. To exclude false negative results, we amended the microcosms with 0.2 mM As(III) and 20.0 mM lactate. The results indicated that the microbial communities in all of the samples significantly catalyzed arsenic methylation. The arsM genes were detectable from all the samples with the exception of 175 m, and 90 different arsM genes were identified. All of these genes code for new or new-type ArsM proteins, suggesting that new As-methylating microorganisms are widely distributed in the samples from shallow to deep sediments. To determine whether microbial biomethylation of As occurs in the sediments under natural geochemical conditions, we conducted microcosm assays without exogenous As and carbons. After 80.0 days of incubation, approximately 4.5-15.5 µg/L DMAsV were detected in all of the microcosms with the exception of that from 30 m, and 2.0-9.0 µg/L MMAsV were detected in the microcosms of 65, 114, 135, 175, 200, and 223 m; moreover, approximately 18.7-151.5 µg/L soluble As(V) were detected from the nine sediment samples. This suggests that approximately 5.3, 0, 8.1, 28.9, 18.0, 8.7, 13.8, 10.2, and 14.9% of total dissolved As were methylated by the microbial communities in the sediment samples from 1, 30, 65, 95, 114, 135, 175, 200, and 223 m, respectively. The concentrations of biogenic DMAsV show significant positive correlations with the depths of sediments, and negative correlations with the environmental NH4+ and NaCl concentrations, but show no significant correlations with other environmental parameters, such as NO3-, SO42+, TOC, TON, Fe, Sb, Cu, K, Ca, Mg, Mn, and Al. This work helps to better understand the biogeochemical cycles of arsenic in arsenic-rich shallow and deep sediments.

Sulfate enhances the dissimilatory arsenate-respiring prokaryotes-mediated mobilization, reduction and release of insoluble arsenic and iron from the arsenic-rich sediments into groundwater.

Dissimilatory arsenate-respiring prokaryotes (DARPs) play key roles in the mobilization and release of arsenic from mineral phase into groundwater; however, little is known about how environmental factors influence these processes. This study aimed to explore the effects of sulfate on the dissolution and release of insoluble arsenic. We collected high-arsenic sediment samples from different depths in Jianghan Plain. Microcosm assays indicated that the microbial communities from the samples significantly catalyzed the dissolution, reduction and release of arsenic and iron from the sediments. Remarkably, when sulfate was added into the microcosms, the microorganisms-mediated release of arsenic and iron was significantly increased. To further explore the mechanism of this finding, we isolated a novel DARP, Citrobacter sp. JH001, from the samples. Arsenic release assays showed that JH001 can catalyze the dissolution, reduction and release of arsenic and iron from the sediments, and the presence of sulfate in the microcosms also caused a significant increase in the JH001-mediated dissolution and release of arsenic and iron. Quantitative PCR analysis for the functional gene abundances showed that sulfate significantly increased the arsenate-respiring reductase gene abundances in the microcosms. Thus, it can be concluded that sulfate significantly enhances the arsenate-respiring bacteria-mediated arsenic contamination in groundwater.