High-throughput diagnosis of human pathogens and fecal contamination in marine recreational water.

Waterborne pathogens and their associated diseases are major threats to public health, and surveillance of pathogens and identification of the sources of pollution are imperative for preventing infections. However, simultaneously quantitative detection of multiple pathogens and pollution sources in water environments is the major challenge. In this study, we developed and validated a highly sensitive (mostly >80%) and highly specific (>99%) high-throughput quantitative PCR (HT-qPCR) approach, which could simultaneously quantify 68 marker genes of 33 human pathogens and 23 fecal markers of 10 hosts. The HT-qPCR approach was then successfully used to investigate pathogens and fecal pollution in marine recreational water samples of Xiamen, China. Totally, seven pathogenic marker genes were found in 13 beach bathing waters, which targeted Acanthamoeba spp., Clostridium perfringens, enteropathogenic Escherichia coli, Klebsiella pneumoniae, Vibrio cholera/V. parahaemolyticus and Legionella spp.. Fecal markers from human and dog were the most frequently detected, indicating human and dog feces were the main contamination in the recreational waters. Nanopore sequencing of full-length 16S rRNA gene revealed that 28 potential human pathogens were detected and electrical conductivity, salinity, oxidation-reduction potential and dissolved oxygen were significantly correlated with the variation in bacterial community. Our results demonstrated that HT-qPCR approach had the potential rapid quantification of microbial contamination, providing useful data for assessment of microbial pathogen associated health risk and development of management practices to protect human health.

Enhanced nitrogen removal in filled-and-drained vertical flow constructed wetlands: microbial responses to aeration mode and carbon source.

For the purpose of enhancing the removal rate of nitrogen (N) and organic matters, intermittent aeration and carbon source were used in filled-and-drained vertical flow constructed wetlands (VFCWs). The results showed that the best removal of COD (74.16%), NH4+-N (93.56%), TN (86.88%), and NO3--N (79.65%) was achieved in VFCW1 (aerated with carbon source system). Illumina MiSeq300 high-throughput sequencing showed that carbon source aerated system increases the diversity and richness of the microbial community. The copy numbers of nitrification functional genes (nxrA, amoA), denitrification functional genes (nirS, nirK, nosZ), and anammox functional gene (anammox 16S rRNA) displayed various changes when applied different aeration modes and additional carbon source to each system. An increase of the DO concentration and carbon source facilitated the absolute abundance of microbial nitrification and denitrification functional genes, respectively. All in all, these results demonstrate that carbon source combined with intermittent aeration is valid to improve the pollutant treatment performance in these systems.

The simultaneous antibiotics and nitrogen removal in vertical flow constructed wetlands: Effects of substrates and responses of microbial functions.

A vertical flow constructed wetland (VFCW) packed with the different substrates was designed to remediate the antibiotics in the wastewater. Zeolite (CW-Z) paralleled with Manganese (Mn) ore (CW-M) and biochar (CW-C) were used to enhance the synchronous removal of ciprofloxacin hydrochloride (CIPH), sulfamethazine (SMZ) and nitrogen (N) from the wastewater. The result indicated that CW-M had a significant potential to remove CIPH (93%), SMZ (69%), TN (71%), NH4+-N (94%) and NO3--N (94%) across all the treatments. The abundance of amoA, nirK and nirS genes are dramatically higher in CW-M and CW-C, while CW-C inhibited the production of quinolone resistance genes. Results showed that different substrates could affect the microbial diversity and structure. The addition of Mn ore to the water led to an improved abundance of nitrogen-related phyla. Overall, Mn ore has a considerable potential to simultaneously remove antibiotics and N in VFCWs.

Enhanced optimal removal of nitrogen and organics from intermittently aerated vertical flow constructed wetlands: Relative COD/N ratios and microbial responses.

Carbon source and dissolved oxygen are the critical factors which sustain the stable redox environment for the microbes to implement the removal of nitrogen and organics in vertical flow constructed wetlands (VFCWs). The effect mechanisms of the COD/N ratios in intermittently aerated VFCWs are needed to be investigated in order to increase the synchronous removal efficiency of pollutants. In this study, the combined effects of COD/N ratios (3, 6, 12) and intermittent aeration in VFCWs on pollutant removal, microbial communities and related function genes were studied. The results showed the increase of COD/N ratios from 3 to 12 enhanced the removal efficiency of TN, NO3--N and COD. The removals of NH4+-N decreased as the COD/N ratio increased. The optimal removals of TN (87.65%), NH4+-N (93.20%), NO3--N (80.80%) and COD (73.93%) were obtained in VFCW2 (COD/N ratios was 6). Illumina Miseq High-throughput sequencing analysis showed that high COD/N ratios increased the richness and diversity of microbial communities. The absolute abundance of nirK, nosZ, nirS, amoA, nxrA, and anammox bacterial 16S rRNA presented various changes under the different ratios of COD/N. The increase of COD/N ratios enhanced the copy numbers of nirS, nirK and nosZ, which participate in denitrification process. High COD/N ratios (6 and 12) were in favor of Actinobacteria, Firmicutes and Chloroflexi, which mainly play important roles in the process of denitrification. This paper implies that the combination of carbon source and aeration is necessary to sustain high microbial activities during pollutant removal in VFCWs.

Microbial utilization of rice straw and its derived biochar in a paddy soil.

The application of straw and biochar to soil has received great attention because of their potential benefits such as fertility improvement and carbon (C) sequestration. The abiotic effects of these materials on C and nitrogen (N) cycling in the soil ecosystem have been previously investigated, however, the effects of straw or its derived biochar on the soil microbial community structure and function are not well understood. For this purpose, a short-term incubation experiment was conducted using (13)C-labeled rice straw and its derived biochar ((13)C-labeled biochar) to deepen our understanding about soil microbial community dynamics and function in C sequestration and greenhouse gas emission in the acidic paddy soil amended with these materials. Regarding microbial function, biochar and straw applications increased CO2 emission in the initial stage of incubation and reached the highest level (0.52 and 3.96mgCkg(-1)soilh(-1)) at 1d and 3d after incubation, respectively. Straw amendment significantly (p<0.01) increased respiration rate, total phospholipid fatty acids (PLFAs) and (13)C-PLFA as compared to biochar amendment and the control. The amount and percent of Gram positive bacteria, fungi and actinomycetes were also significantly (p<0.05) higher in (13)C-labeled straw amended soil than the (13)C-labeled biochar amended soil. According to the (13)C data, 23 different PLFAs were derived from straw amended paddy soil, while only 17 PLFAs were derived from biochar amendments. The profile of (13)C-PLFAs derived from straw amendment was significantly (p<0.01) different from biochar amendment. The PLFAs18:1ω7c and cy17:0 (indicators of Gram negative bacteria) showed high relative abundances in the biochar amendment, while 10Me18:0, i17:0 and 18:2ω6,9c (indicators of actinomycetes, Gram positive bacteria and fungi, respectively) showed high relative abundance in the straw amendments. Our results suggest that the function, size and structure of the microbial community were strongly influenced by the substrate composition and availability.

Effect of rice straw application on microbial community and activity in paddy soil under different water status.

Rice straw application and flooding are common practices in rice production, both of which can induce changes in the microbial community. This study used soil microcosms to investigate the impact of water status (saturated and nonsaturated) and straw application (10 g kg(-1) soil) on soil microbial composition (phospholipid fatty acid analysis) and activity (MicroResp(™) method). Straw application significantly increased total PLFA amount and individual PLFA components independent of soil moisture level. The amount of soil fungal PLFA was less than Gram-negative, Gram-positive, and actinomycete PLFA, except the drained treatment with rice straw application, which had higher fungal PLFA than actinomycete PLFA at the initial incubation stage. Straw amendment and waterlogging had different effects on microbial community structure and substrate-induced pattern. PLFA profiles were primarily influenced by straw application, whereas soil water status had the greater influence on microbial respiration. Of the variation in PLFA and respiration data, straw accounted for 30.1 and 16.7 %, while soil water status explained 7.5 and 29.1 %, respectively. Our results suggest that (1) the size of microbial communities in paddy soil is more limited by carbon substrate availability rather than by the anaerobic conditions due to waterlogging and (2) that soil water status is more important as a control of fungal growth and microbial community activity.