The introduction of influent sulfamethoxazole loads induces changes in the removal pathways of sulfamethoxazole in vertical flow constructed wetlands featuring hematite substrate.

High frequent detection of sulfamethoxazole (SMX) in wastewater cannot be effectively removed by constructed wetlands (CWs) with a traditional river sand substrate. The role of emerging substrate of hematite in promoting SMX removal and the effect of influent SMX loads remain unclear. The removal efficiency of SMX in hematite CWs was significantly higher than that in river sand CWs by 12.7-13.8% by improving substrate adsorption capacity, plant uptake and microbial degradation. With increasing influent SMX load, the removal efficiency of SMX in hematite CWs slightly increased, and the removal pathways varied significantly. The contribution of plant uptake was relatively small (< 0.1%) under different influent SMX loads. Substrate adsorption (37.8%) primarily contributed to SMX removal in hematite CWs treated with low-influent SMX. Higher influent SMX loads decreased the contribution of substrate adsorption, and microbial degradation (67.0%) became the main removal pathway. Metagenomic analyses revealed that the rising influent load increased the abundance of SMX-degrading relative bacteria and the activity of key enzymes. Moreover, the abundance of high-risk ARGs and sulfonamide resistance genes in hematite CWs did not increase with the increasing influent load. This study elucidates the potential improvements in CWs with hematite introduction under different influent SMX loads.

[Effects of Foliar Application of Silicon Fertilizers on Phyllosphere Bacterial Community and Functional Genes of Paddy Irrigated with Reclaimed Water].

Agricultural utilization of reclaimed water is considered to be an effective way to solve water shortage and reduce water environmental pollution. Silicon fertilizer can improve crop yield and quality and enhance crop resistance. The effect of foliar spray with silicon fertilizer on phyllosphere microbial communities remains lacking. In this study, a pot experiment was conducted to explore the effects of different types of silicon fertilizer on the composition and diversity of a phyllosphere bacterial community and the abundances of related functional genes in rice irrigated with reclaimed water. The results showed that Firmicutes, Proteobacteria, Actinobacteriota, Bacteroidota, and Verrucomicrobiota dominated the phyllosphere bacteria of rice. The relative abundance of Bacillus was higher than that of other treatments in RIS3. Reclaimed water irrigation significantly increased the relative abundances of the potential pathogens Pantoea and Enterobacter. The unclassified bacteria were also an important part of the bacterial community in the rice phyllosphere. Bacillus, Exiguobacterium, Aeromonas, and Citrobacter were significantly enriched by silicon fertilizer treatments. Functional prediction analysis showed that indicator species were mainly involved in metabolism and degradation functions, and the predicted functional groups of phyllosphere bacteria were attributed to chemoheterotrophy, aerobic chemoheterotrophy, nitrate reduction, and fermentation. Quantitative PCR results showed that AOA, AOB, and nifH genes were at low abundance levels in all treatments, and nirK genes was not significantly different among treatments. These results contribute to the in-depth understanding of the effects of foliar spray silicon fertilizer on the bacterial community structure and diversity of rice phyllosphere and provide a theoretical basis for the application of silicon fertilizer in reclaimed water irrigation agriculture.

Roles of substrates in removing antibiotics and antibiotic resistance genes in constructed wetlands: A review.

Antibiotics and corresponding antibiotic resistance genes (ARGs) are emerging pollutants in wastewater that pose a significant threat to the environment and human health. Constructed wetlands (CWs) are a cost-effective technology for eliminating these pollutants through substrates, plants, and microorganisms. Detailed reviews of the roles of CW substrates on antibiotic and ARG removal and recent progress in the field are lacking. This paper reviews the mechanisms influencing antibiotic and ARG (intracellular and extracellular) removal in CWs, and natural, biomass, chemical, modified, industrial, novel, and combined substrates on their removal efficiencies. Generally, substrates remove antibiotics and ARGs mainly through adsorption, biodegradation, chemical oxidation, and filtration. Other mechanisms, such as photolysis, may also contribute to removal. Natural substrates (e.g., gravel, zeolite) are more frequently employed than other types of substrates. The removal performance of antibiotics and intracellular ARGs by zeolite was better than that of gravel through enhanced substrate adsorption, filtration, and biodegradation processes. Moreover, Mn ore showed promising high capability to remove high concentration of antibiotics through various removal pathways. In addition, combined substrates of soil/sand/gravel and other substrates further facilitate antibiotic removal. Future research is suggested to explore the mechanisms of competitive adsorption and redox-controlled biodegradation, investigate the effect of Fe/Mn oxides on the removal of antibiotics and ARGs via chemical oxidation, evaluate the removal of extracellular ARGs by CWs with different substrates, and investigate the effect of substrates on removal of antibiotics and ARGs in full-scale CWs.

[Effects of Soil Amendments on the Bacterial Diversity and Abundances of Pathogens and Antibiotic Resistance Genes in Rhizosphere Soil Under Drip Irrigation with Reclaimed Water].

Due to reclaimed water, irrigation can cause human health and environmental risks. Soil amendments are applied to reveal the abundance of pathogens and antibiotic resistance genes in rhizosphere soil irrigated by reclaimed water and to better understand the effects of environmental factors on the rhizosphere soil bacterial composition, which has guiding significance for the reasonable use of soil amendments. In this study, the effects of biochar, bioorganic fertilizer, humic acid, loosening soil essence, and corn vinasse on bacterial community diversity and certain gene abundances in rhizosphere soil under drip irrigation with reclaimed water were studied using high-throughput assays and quantitative PCR. The results showed that biochar significantly increased pH, organic matter, and total nitrogen contents in the rhizosphere soil. The corn vinasse significantly decreased soil pH and increased the contents of total nitrogen and total phosphorus but significantly increased the soil EC value (P<0.05). The effects of the five soil amendments on the α-diversity of rhizosphere bacteria were not significantly different. The bacterial community structure and diversity of rhizosphere bacteria were similar at different taxonomic levels, but their relative abundance was different. α-Proteobacteria, γ-Proteobacteria, Bacteroidia, Actinobacteria, Acidimicrobiia, and Anaerolineae were the dominant bacteria in all treatments. The dominant genera consisted of Pseudomonas, Sphingobium, Sphingomonas, Cellvibrio, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Flavobacterium, and Algoriphagus (relative abundance>1%). Correlation analysis of environmental factors showed that the composition of the rhizosphere bacterial community was strongly correlated with pH, EC, total nitrogen, and total phosphorus content. The abundances of pathogenic bacteria and antibiotic resistance genes were 103-107 copies·g-1 and 104-108 copies·g-1, respectively. There were significant differences in the detection levels of pathogens and antibiotic resistance genes. Bioorganic fertilizer, loosening soil essence, and corn vinasse significantly increased the abundances of some antibiotic resistance genes, whereas humic acid and corn vinasse significantly decreased the abundances of Pseudomonas syringae, Ralstonia solanacearum, and total coliforms (P<0.05). A significant correlation was found between pathogens (Arcobacter, Bacillus cereus, Pantoea agglomerans, and Fecal bacteroidetes) and antibiotic resistance genes (tetA, tetB, tetO, tetQ, sul1, ermB, and ermC). In conclusion, while monitoring pathogens and antibiotic resistance genes in the agricultural environment under reclaimed water irrigation, attention should be paid to the rational application of soil amendments to avoid exacerbating the spread of biological contamination.

Reduction effect of individual N, P, K fertilization on antibiotic resistance genes in reclaimed water irrigated soil.

The transfer of antibiotic resistance genes (ARGs) in soil under reclaimed water irrigation poses a potential environmental risk. Regulation of NPK fertilizer could influence the behavior of bacterial communities, mobile genetic elements (MGEs), and soil properties, which determine the fate of ARGs. To identify the key element in NPK fertilizer and realize efficient regulation, we explored the effect of individual N, P, K fertilization on ARG variation in tomato rhizosphere and bulk soils. Compared with an unfertilized treatment, N fertilization resulted in greater decreases in the abundance of ARGs (decreases of 24.06%-73.09%) than did either P fertilization (increases of up to 35.84%, decreases of up to 58.80%) or K fertilization (decreases of 13.47%-72.47%). The influence of different forms of N (CO(NH2)2, NaNO3, and NH4HCO3), P (Ca(H2PO4)2 and CaMgO4P+), and K (KCl and K2(SO4)) fertilizers was also investigated in this study, and showed the influence of NaNO3, CaMgO4P+, and K2(SO4) on reducing ARGs abundance was greater in different types of N, P, K fertilizers. Bacterial communities showed the strongest response to N fertilization. The reduced bacterial diversity and abundance of ARG-host and non-host organisms explained the decline of total ARG abundance in soil. In soils fertilized with either P or K, the effect of soil properties, especially total nitrogen and pH, on ARG variation was greater than that of bacterial community and MGEs. These results suggest that N regulation of in NPK fertilizer may be an effective way to reduce the risks of ARGs in soil associated with reclaimed water irrigation.

Ryegrass (Lolium multiflorum L.) and Indian mustard (Brassica juncea L.) intercropping can improve the phytoremediation of antibiotics and antibiotic resistance genes but not heavy metals.

Lolium multiflorum and Brassica juncea display phytoremediation potential for heavy metals and antibiotics pollution. However, there is limited understanding of their function in removing combined pollutants (heavy metals, antibiotics and antibiotic resistance genes (ARGs)) under different cropping patterns. Sole cropping had little effect on heavy metals, but reduced antibiotics by 2.46%-84.88% and increased ARGs by 15.96%-33.82%. Intercropping was more beneficial to soil remediation and plant accumulation of L. multiflorum, and further increased the remediation of antibiotics by 2.38%-54.40%. Members of phyla (Actinobacteria, Bacteroidetes, and Proteobacteria) were mainly responsible for most antibiotics removal. Compared with sole cropping, intercropping reduced more ARGs abundance in rhizosphere soil for L. multiflorum (20.43%) and in bulk soil for B. juncea (23.22%). Mobile genetic elements (MGEs) played a significant role in the variation of ARGs. Further, sample type showed a higher indirect negative impact on ARGs by mainly affecting soil properties and bacterial community, and the co-occurrence between the bacterial community and ARGs in bulk soil was more complex than that in rhizosphere soil. Together these results suggest that phytoremediation of combined soil pollution was positive but limited, and intercropping resulted in enhanced removal efficiency when compared with sole cropping.

[Effects of Selected Biochars Application on the Microbial Community Structures and Diversities in the Rhizosphere of Water Spinach (Ipomoea aquatica Forssk.) Irrigated with Reclaimed Water].

The utilization of reclaimed water is one of the most important ways of alleviating the shortage of water resources for agricultural irrigation. As an effective disposal method for biomass waste, biochar has been widely used in the improvement and remediation of agricultural environments. However, few studies have been performed on the effects of biochar application on microbial community structures and pathogen abundances in rhizosphere soils irrigated with reclaimed water. Based on a pot experiment, high throughput sequencing technology and quantitative polymerase chain reaction (PCR) methods were used to investigate the effects of different biochars on the microbial community structure and diversity and pathogen abundance of rhizosphere soils irrigated with reclaimed water. The results showed that four different types of biochars had different effects on the soil nutrient status. Rice hull-derived biochar and rice straw-derived biochar resulted in significantly increased soil pH with reclaimed water irrigation, while peanut shell-derived biochar, rice straw-derived biochar, and wheat straw-derived biochar significantly increased EC values (P<0.05). The Sobs index, Shannon index, and Chao1 index of bacterial community in the rhizosphere soil significantly increased with rice straw-derived biochar treatment, while the Simpson index significantly decreased by adding peanut shell-derived biochar, rice hull-derived biochar, and wheat straw-derived biochar (P<0.05). There were differences in the relative abundances of bacterial communities in rhizosphere soils under different treatments. The dominant taxonomic groups at the phylum level were Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, and Acidobacteria. The dominant genera included Pseudomonas, Rheinheimera, Arthrobacter, Sphingomonas, and Aeromonas (relative abundance>5%). Redundancy (RDA) and heatmap analyses showed that the diversities and compositions of bacterial communities in rhizosphere soils in different treatments were closely related to soil EC values, organic matter, total nitrogen, and cadmium contents. Biochar application had no significant effect on the abundances of Aeromonas hydrophila and Bacillus cereus. Rice straw-derived biochar and peanut shell-derived biochar could significantly reduce the γ-Proteobacteria, while rice hull-derived biochar and wheat straw-derived biochar could significantly reduce the relative abundance of AOA (P<0.05). In conclusion, there were no obvious negative effects observed from reclaimed water irrigation on soil quality. Biochar application not only significantly improved the physicochemical properties of the soil, but also impacted the bacterial community structure and the abundance of the functional bacteria in the rhizosphere soil, which was closely related to the soil properties.

[Effect of Different Reclaimed Water Irrigation Methods on Bacterial Community Diversity and Pathogen Abundance in the Soil-Pepper Ecosystem].

Reclaimed water is considered to be a reasonable and sustainable alternative water resource to improve water resource layout and mitigate the shortage of traditional water resources. Its use in irrigation will cause changes in the microbial community structure and opportunistic pathogen abundance in soils and crops, but few studies have been conducted on this subject. Peppers were used as the research subjects, and the treatments were direct irrigation of reclaimed water, mixed irrigation with freshwater and reclaimed water, rotated irrigation with freshwater and reclaimed water, with potable water irrigation as the control. The effects of different irrigation methods of reclaimed water on the soil physicochemical properties were analyzed through a pot experiment. Furthermore, changes in bacterial community and opportunistic pathogen abundance in pepper fruit and the rhizosphere under reclaimed water irrigation conditions were investigated based on high-throughput sequencing technology and quantitative PCR methods. The results showed that direct irrigation with reclaimed water increased soil EC and decreased soil pH. 16S rDNA high-throughput sequencing showed that Proteobacteria, Bacteroides, Actinobacteria, and Firmicutes were present in both pepper fruit and the rhizosphere at phylum level, and the most dominant genera (Pantoea, Pseudomonas, Sphingomonas, Sphingopyxis, Luteimonas, and Mariniflexile) were greatly affected by reclaimed water irrigation methods. Quantitative PCR results indicated that the influence of reclaimed water irrigation on the distribution and abundance of pathogenic bacteria in the soil-pepper system was different, and the abundance of Legionella spp. in pepper fruit and Pseudomonas syringae in the rhizosphere increased with reclaimed water irrigation. Our results indicated that the reclaimed water was suitable for agricultural irrigation, but different reclaimed water irrigation methods may introduce different degrees of microbial contamination. In addition, attention must be given to some opportunistic pathogens and phytopathogens.

Reducing water use by alternate-furrow irrigation with livestock wastewater reduces antibiotic resistance gene abundance in the rhizosphere but not in the non-rhizosphere.

Livestock wastewater is rich in nutrients but may contain antibiotics and antibiotic resistance genes (ARGs). Their discharge to watercourses or soil may result in proliferation of ARGs. Irrigation with wastewater appears to be the most feasible option of disposing of it. One efficient irrigation technology used in arid regions is alternate-furrow irrigation (AFI) by alternately drying part of the plant roots for a prolonged period to physiologically reduce transpiration without compromising yield. However, the extent to which AFI with wastewater influences the concentration of antibiotics and spread of ARGs in soil is poorly understood. The purpose of this paper is to investigate how AFI using swine wastewater alters antibiotic kinetics and ARGs abundance under different irrigation rates, using pepper as the model plant. We examined three AFI treatments using 50%, 65% and 80% of the amount of water employed in sufficient conventional furrow irrigation. Each treatment had a groundwater irrigation control. The results showed that antibiotic concentrations and relative ARGs abundance in the top 20 cm of soil did not increase with the irrigation amount, although they were higher than those in the groundwater-irrigated soils. The relative ARGs abundance in the soil was modulated by irrigation amount and reducing the irrigation amount in AFI reduced ARGs dispersion only in rhizosphere. When the soil moisture was close to field capacity, ARGs were more abundant in rhizosphere than in non-rhizosphere, possibly because the rhizosphere is rich in microbes and increasing antibiotic concentrations due to an increase in irrigation rate favors antibiotic-resistant microbiome in competing for substrates. These, however, were not mirrored in the relative ARGs abundance in the roots. These results have important implications as it revealed that reducing the input of antibiotics and ARGs into soil with AFI does not necessarily reduce ARGs proliferation.

Amendment soil with biochar to control antibiotic resistance genes under unconventional water resources irrigation: Proceed with caution.

The spread of antibiotic resistance genes (ARGs) has become a cause for serious concern because of its potential risk to public health. The use of unconventional water resources (e.g., reclaimed water or piggery wastewater) in agriculture to relieve groundwater shortages may result in an accumulation of ARGs in soil. Biochar addition has been proven to be a beneficial method to alleviate the pollution of ARGs in manure-amended soil. However, the role of biochar on ARGs in soil-plant systems repeatedly irrigated with unconventional water resources is unknown. Under reclaimed water or piggery wastewater irrigation, rhizobox experiments using maize plants in soil amended with biochar were conducted to investigate the variation of typical ARGs (tet and sul genes) in soil-plant systems during a 60-day cultivation, and ARGs was characterized by high-throughput qPCR with a 48 (assays) × 108 (samples) array. Only piggery wastewater irrigation significantly increased the abundance of ARGs in rhizosphere and bulk soils and root endophytes. Following 30-day cultivation, the abundance of ARGs in soil was significantly lower due to biochar addition. However, by day 60, the abundance of ARGs in soil supplemented with biochar was significantly higher than in the control soils. Antibiotics, bio-available heavy metals, nutrients, bacterial community, and mobile gene elements (MGEs) were detected and analyzed to find factors shaping ARGs dynamics. The behavior of ARGs were associated with antibiotics but not with bio-available heavy metals. The correlation between ARGs and available phosphorus was stronger than that of ARGs with total phosphorus. MGEs had good relationship with ARGs, and MGEs shifts contributed most to ARGs variation in soil and root samples. In summary, this study provides insights into potential options for biochar use in agricultural activities.

Fate of antibiotic and metal resistance genes during two-phase anaerobic digestion of residue sludge revealed by metagenomic approach.

The prevalence and persistence of antibiotic resistance genes in wastewater treatment plants (WWTPs) is of growing interest, and residual sludge is among the main sources for the release of antibiotic resistance genes (ARGs). Moreover, heavy metals concentrated in dense microbial communities of sludge could potentially favor co-selection of ARGs and metal resistance genes (MRGs). Residual sludge treatment is needed to limit the spread of resistance from WWTPs into the environment. This study aimed to explore the fate of ARGs and MRGs during thermophilic two-phase (acidogenic/methanogenic phase) anaerobic digestion by metagenomic analysis. The occurrence and abundance of mobile genetic elements were also determined based on the SEED database. Among the 27 major ARG subtypes detected in feed sludge, large reductions (> 50%) in 6 ARG subtypes were achieved by acidogenic phase (AP), while 63.0% of the ARG subtypes proliferated in the following methanogenic phase (MP). In contrast, a 2.8-fold increase in total MRG abundance was found in AP, while the total abundance during MP decreased to the same order of magnitude as in feed sludge. The distinct dynamics of ARGs and MRGs during the two-phase anaerobic digestion are noteworthy, and more specific treatments are required to limit their proliferation in the environment.

The behavior of antibiotic resistance genes and arsenic influenced by biochar during different manure composting.

The effect of two different biochar types, rice straw biochar (RSB) and mushroom biochar (MB), on chicken manure composting was previously examined by monitoring the fate of antibiotic resistance genes (ARGs) and arsenic. The behavior of ARGs and arsenic in other kinds of manure composting with the same biochar types had not been examined. In this study, we added either RSB or MB to pig and duck manure composts to study the behavior of ARGs (tet genes, sul genes, and chloramphenicol resistance genes) and arsenic under the same experimental condition. The results showed that the average removal values of selected ARGs were respectively 2.56 and 2.09 log units in duck and pig manure compost without the addition of biochar. The effect of biochar addition on the average removal value of ARGs depended on the type of biochar and manure. For instance, in pig manure compost, MB addition increased the average removal value of ARGs, while RSB addition decreased. And both biochar additions had a negative influence on the average removal value of ARGs in duck manure compost. Analytical results also demonstrated that MB addition reduced total arsenic and the percentage of bioavailable arsenic more than RSB.

Influence of two-phase anaerobic digestion on fate of selected antibiotic resistance genes and class I integrons in municipal wastewater sludge.

The response of representative antibiotic resistance genes (ARGs) to lab-scale two-phase (acidogenic/methanogenic phase) anaerobic digestion processes under thermophilic and mesophilic conditions was explored. The associated microbial communities and bacterial pathogens were characterized by 16S rRNA gene sequencing. A two-phase thermophilic digestion reduced the presence of tetA, tetG, tetX, sul1, ermB, dfrA1, dfrA12 and intI1 exhibiting 0.1-0.72 log unit removal; in contrast, tetO, tetW, sul3, ermF and blaTEM even increased relative to the feed, and sul2 showed no significant decrease. The acidogenic phase of thermophilic digestion was primarily responsible for reducing the quantity of these genes, while the subsequent methanogenic phase caused a rebound in their quantity. In contrast, a two-phase mesophilic digestion process did not result in reducing the quantity of all ARGs and intI1 except for ermB and blaTEM. ARGs patterns were correlated with Proteobacteria and Actinobacteria during the two-phase anaerobic digestion.

Effect of different biochars on antibiotic resistance genes and bacterial community during chicken manure composting.

Rice straw biochar (RSB) and mushroom biochar (MB) were added to lab-scale chicken manure composting to evaluate their effects on the behaviors of antibiotic resistance genes (ARGs) and on total and bio-available heavy metals (Cu, Zn and As). The associated bacterial community was characterized by 16SrRNA high-throughput sequencing. The abundance of pathogenic bacteria was also calculated. At the end of the control composting experiment, the average removal rate of ARGs was 0.86 log units and the removal rate of pathogenic bacteria was 57.1%. MB addition resulted in a higher removal rate than that in the control composting experiment. However, RSB addition yielded opposite results, which may be due to the higher abundance of Erysipelotrichaceae, Lactobacillaceae, Family_XI_Incertae_Sedis (belonging to Firmicutes carrying and disseminating ARGs) and pathogenic bacteria carrying ARGs. Furthermore, the correlations between bio-available heavy metals and ARGs were more obvious than those between total heavy metals and ARGs.