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Antimicrobial resistance and harmful cyanobacterial bloom are two health concerns of freshwater ecosystems; a lot of studies have been conducted on these threats. However, few studies have been con...

This study aimed to examine the drivers in shaping the occurrence and distribution of total viruses and picoplankton in tropical freshwater ecosystems. Flow cytometry was used to quantify the concentrations of total viruses, picoheterotrophs, picophytoplankton, and picocyanobacteria. Three land use patterns (urban-, agriculture- and parkland-dominated) were evaluated using ArcGIS. Significant correlations were observed between water-borne microbial targets and water quality parameters (0.175 ≤ |r| ≤ 0.441), nutrients (0.250 ≤ r ≤ 0.570) and land use factors (0.200 ≤ |r| ≤ 0.460). In particular, the concentrations of total viruses and picoheterotrophic cells were higher in catchments whereas the abundances of picophytoplankton and picocyanobacteria were higher in reservoirs. Total viruses and picoplankton had higher concentrations in urban- and agriculture-dominated areas, probably due to anthropogenic inputs and agricultural inputs, respectively. Although surface water is a complex matrix influenced by niche-based (i.e., physicochemical properties, nutrients, land use impact etc.) and neutral-based factors (i.e., ecological drift, dispersal and species), land use patterns could help to elucidate the occurrence and distribution of the total microbial community at the macroscopic level. Meanwhile, inter-correlations among viruses, picoplankton and picoheterotrophs (0.715 ≤ r ≤ 0.990) also substantiates their mutual interactions in influencing the microbial community. Furthermore, the relationships between total microbial cells and bacterial and viral indicators were also investigated. Concentrations of total viruses, picoplankton and picoheterotrophs were positively correlated with bacterial indicators (0.427 ≤ r ≤ 0.590) and viral indicators (0.201 ≤ r ≤ 0.563). These results indicated that faecal and viral contamination could contribute to the numbers of total viruses and bacteria.

Monitoring toxigenic cyanobacteria in freshwaters is of great importance due to the adverse health impacts on humans and aquatic organisms. Here we studied cyanobacterial occurrence and biodiversity in a drinking water reservoir in Tehran province, Iran. In total, nine different species representing three orders of Synechococcales, Oscillatoriales and Nostocales were isolated and classified into six families and seven genera ranging from 92.3% to 99.0% similarities in their partial 16S rDNA with GenBank sequences. The cultures were analyzed for cyanotoxins production by the Artemia salina bioassay, ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and also screened for the presence of marker genes involved in toxins production. Ethyl acetate extracts of three strains showed more than 50% mortality on A. salina larvae after 24 h at a concentration of 500 µg/ml. Production of at least one of the cyanotoxins, microcystin (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a), was detected in 6 of the strains. Seven MC variants with a total concentration of 130.6 ng/mg of biomass dry weight were detected for the strain Phormidium sp. UTMC6001 and molecular screening of the mcyE gene also confirmed the presence of this biomarker in its genome. Our study also revealed the production of CYN in a novel picocyanobacterial strain Cyanobium sp. UTMC6007 at 1.0 ng/mg of biomass dry weight. Considering the limited information on freshwater toxic cyanobacteria taxonomy in the Middle East, these findings will expand our knowledge and consequently aid in development of new water management policies in future.

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. Distinct from the majority of cyanophage isolates, PA-SR01 has a tailless morphology. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles.IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

This study employed high-throughput sequencing (HTS) to understand the variations in microbial community in the largest estuary reservoir located at the Yangtze River for a year. Correlations between the heterotrophic bacterial and cyanobacterial communities in the estuarine ecosystem were also investigated. Significant spatial and temporal changes were observed in the microbial community composition at all sites. These differences were mainly reflected on the variations of bacterial relative abundance. The modularity analysis on the network indicate that bacterial community response to the variations of environmental factors in the form of co-occurrence/exclusion patterns. In warm season, Synechococcus spp. being the dominant Cyanobacteria taxa exhibited high relative abundance in the reservoir. Water temperature was the critical driver for the proliferation of Synechococcus . Moreover, heterotrophic bacteria belonging to Actinobacteria, Proteobacteria (α-, β-, and γ-Proteobacteria), Bacteroidetes and Chlorobi, exhibited positive correlations with Synechococcus . The co-occurrence of these bacterial OTUs suggests that specific taxa may benefit from the proliferation of Synechococcus . In cold season, bacterial OTUs belonging to Actinobacteria and Bacteroidetes shown co-occurrence pattern with salt ions (including K + , Na + , Mg 2+ , Ca 2+ , Cl − and SO 4 2− ) inside the reservoir. In conclusion, further research is required to investigate the ecological functions of these taxa in estuarine ecosystems.

This study seeks to understand the general distribution of virome abundance and diversity in tropical freshwater ecosystems in Singapore and the geospatial distribution of the virome under different landuse patterns. Correlations between diversity, environmental parameters and land use patterns were analyzed and significant correlations were highlighted. Overall, the majority (65.5%) of the annotated virome belonged to bacteriophages. The percentage of Caudovirales was higher in reservoirs whereas the percentages of Dicistroviridae, Microviridae and Circoviridae were higher in tributaries. Reservoirs showed a higher Shannon-index virome diversity compared to upstream tributaries. Land use (urbanized, agriculture and parkland areas) influenced the characteristics of the virome distribution pattern. Dicistroviridae and Microviridae were enriched in urbanized tributaries while Mimiviridae, Phycodnaviridae, Siphoviridae and Podoviridae were enriched in parkland reservoirs. Several sequences closely related to the emerging zoonotic virus, cyclovirus, and the human-related virus (human picobirnavirus), were also detected. In addition, the relative abundance of PMMoV (pepper mild mottle virus) sequences was significantly correlated with RT-qPCR measurements (0.588, Graphical abstract Image 1, Highlights • Land use is the main driver of the viral community structure in surface waters. • 66 viral families were detected and 66% of the annotated virome was bacteriophages. • Reservoirs showed a higher Shannon diversity (10.4) than upstream tributaries (8.4). • Sequences closely related to human picobirnavirus and cyclovirus were detected. • The relative abundance of PMMoV sequences was correlated with RT-qPCR measurements.

In this study, Illumina MiSeq sequencing technique was employed to explore the characteristics and dynamics of cyanobacteria–heterotrophic bacteria between two estuarine reservoirs in sub-tropical (reservoir A in Shanghai) and tropical (reservoir B in Singapore) regions. The results indicated that significant differences in bacterial community composition were found between two estuarine reservoirs, which influenced by varied environmental variables. The environmental heterogeneity in reservoir A was much higher, which indicated that the composition of bacterial community in reservoir A was more complex. In contrast, reservoir B provided a suitable and temperate water environment conditions for bacterial growth, which resulted in higher community diversity and less co-exclusion correlations. The molecular ecological network indicated that the presence of dominant bacterial community in each of the reservoir were significant different. These differences mainly reflected the responses of bacterial community to the variations of environmental variables. Although Synechococcus was the dominant cyanobacterial species in both reservoirs, it exhibited co-occurrence patterns with different heterotrophic bacteria between reservoirs. In addition, the cyanobacteria–heterotrophic bacteria interaction exhibited highly dynamic variations, which was affected by nutrition and survive space. Also, the co-occurrence of Microcystis and Pseudanabaena found in reservoir B implied that the non-N-fixing Microcystis accompanied with N-fixing Pseudanabeana occurrence in freshwater lakes, so as to better meet the demand for nitrogen source.

Picocyanobacteria are small cyanobacteria, being about 0.8–1.5 µm in size. They are present in freshwater environments all over the world and are known to cause harmful algal blooms, although their effects are not well understood. Algal blooms are important to manage because they threaten freshwater resources, with potentially severe effects on ecological and human health. There is also increased urgency due to urbanization and climate change trends which are expected to exacerbate these bloom dynamics. These changes are expected to especially favour picocyanobacteria groups, emphasizing the need for better characterization of their effects in the environment. In this study, we report the discovery that Synechococcus sp. could produce cylindrospermopsin (CYN) and anatoxin-a (ATX). This ability had never been previously reported for this species. Their toxin genes were also partial compared to other major producers such as Raphidiopsis sp. and Anabaena sp., demonstrating potentially unique synthesis pathways that provides insight into the various mechanisms of genetic variation that drives toxin synthesis. The Synechococcus sp. strains were found to produce about 9.0 × 10−5–6.8 × 10−4 fg CYN cell−1 and 4.7 × 10−4–1.5 × 10−2 fg ATX cell−1. The potential for Synechococcus sp. to be toxic highlights a global concern due to its widespread distribution, and through environmental trends that increasingly favour its productivity within freshwater systems around the world.

Although the use of Sulfluramid (N-ethyl perfluorooctane sulfonamide (N-EtFOSA)) has been restricted by the Stockholm Convention, it is still frequently detected in the environmental matrices and in use in some countries. Employing constructed wetlands as treatment systems requires understanding of the biodegradation process in the rhizosphere and the effect of contaminants on the microbes of wetlands. This study aimed to investigate the interactions between the microbial community and N-EtFOSA under aerobic and anaerobic conditions. Aerobic biotransformation of N-EtFOSA occurred with a half-life of 0.51 day and yielded 85.1 mol% PFOS of after 91 days. Kinetic modelling revealed that cleavage of the S N was the rate-limiting degradation step. Biotransformation was not observed under anaerobic and anoxic conditions, suggesting that N-EtFOSA is recalcitrant to biodegradation without dissolved oxygen. Under aerobic condition, the presence of N-EtFOSA and its biotransformation products decreased the microbial richness and diversity and exerted selective pressure on the microbial community. Enrichment of Methylocaldum was significant (49%) in the presence of N-EtFOSA compared to unexposed conditions (11%), suggesting that Methylocaldum is relatively tolerant to N-EtFOSA and potentially degrading N-EtFOSA. Under anaerobic conditions, the microbial richness and diversity were not significantly altered by the presence of N-EtFOSA. Only Methanomethylovorans increased significantly in the spiked microcosm (30% vs. 20%). These findings provide knowledge for comprehending the contribution of N-EtFOSA to other PFASs in various environmental conditions, information about microbial community changes in response to PFASs and robust microbial species which can degrade N-EtFOSA in the environment.

The feasibility of cyanobacteria removal from freshwater by a dielectric barrier discharge (DBD) process is investigated. Seven commercial and environmental cyanobacteria strains, as well as real algae-laden water, were tested. The removal of the cyanobacteria was evaluated by analyzing the changes in chlorophyll a content, total organic carbon (TOC) concentration, and cell morphology. Nearly total removal of chlorophyll a was achieved within 20 min, while the TOC analysis exhibited an increase-decrease-increase trend in 60 min of treatment, likely due to the oxidation of intracellular and intercellular materials. Observation under light microscopy revealed the disruption of intracellular and intercellular structures within 5 min of DBD treatment and thus supported the TOC analysis. Increasing the salinity of the medium from 0 to 5 parts per thousand (ppt) improved treatment efficiency, where similar level of chlorophyll a removal (around 93%) was achieved in only half the treatment time. Application of DBD on real algae-laden water from a fish farm yielded higher treatment efficiency than in synthetic medium, indicating the promising application of DBD as a means to control cyanobacteria bloom in fresh and estuary water bodies.

Planktothricoides is a free-living filamentous cyanobacterium belonging to the order Oscillatoriales and the family Phormidiaceae, capable of forming bloom in fresh and brackish waters. A unicyanobacterial non-axenic culture dominated by Planktothricoides sp. SR001 was obtained from a freshwater reservoir in Singapore. The draft genome presented here is the first tropical freshwater Planktothricoides sp. ever sequenced. The genome of 7.0Mbp contains 5,776 genes predicted using the JGI IMG pipeline. The whole genome sequence allows identification of genes encoding for nitrogen-fixation, accessory photosynthetic pigments and biosynthesis of an off-flavor compound, 2-methylisoborneol, which has been experimentally verified here based on metabolite detection. In addition, strain SR001 genome contains an operon putatively involved in the production of a linear tripeptide cyanobactin related to viridisamide A and aeruginosamide, with the later known to possess anti-microbial or cytotoxic effect.

The identification of phytoplankton species and microbial biodiversity is necessary to assess water ecosystem health and the quality of water resources. We investigated the short-term (2 days) vertical and diel variations in bacterial community structure and microbially derived secondary metabolites during a cyanobacterial bloom that emerged in a highly urbanized tropical reservoir. The waterbody was largely dominated by the cyanobacteria Planktothricoides spp., together with the Synechococcus, Pseudanabaena, Prochlorothrix, and Limnothrix. Spatial differences (i.e., water depth) rather than temporal differences (i.e., day versus night) better-explained the short-term variability in water quality parameters and bacterial community composition. Difference in bacterial structure suggested a resource-driven distribution pattern for the community. We found that the freshwater bacterial community associated with cyanobacterial blooms is largely conserved at the phylum level, with Proteobacteria (β-proteobateria), Bacteroidetes, and Actinobacteria as the main taxa despite the cyanobacterial species present and geographical (Asia, Europe, Australia, and North America) or climatic distinctions. Through multivariate statistical analyses of the bacterial community, environmental parameters, and secondary metabolite concentrations, we observed positive relationships between the occurrences of cyanobacterial groups and off-flavor compounds (2-methyisoborneol and β-ionone), suggesting a cyanobacterial origin. This study demonstrates the potential of 16S rRNA gene amplicon sequencing as a supporting tool in algal bloom monitoring or water-resource management.

Cylindrospermopsis is known to be one of the major bloom-forming cyanobacterial genera in many freshwater environments. We report here the draft genome sequence of a tropical Cylindrospermopsis sp. strain, CR12, which is capable of producing the hepatotoxic cylindrospermopsin.

A non-axenic unialgal culture containing a Subsection V (Stigonematales) cyanobacterium, Hapalosiphon strain MRB 220, was obtained from a benthic freshwater algal mat through multiple transfers following growth in sterile media. Physiological characterization demonstrated the culture was capable of nitrogen-fixation and production of the off flavor compound 2-methylisoborneol (2-MIB). Total DNA isolated from this culture was sequenced using Illumina HiSeq and de novo assembled into contigs. The genome of MRB 220 was separated from co-occurring heterotrophic bacteria using sequence homology and compositional approaches, and its purity was confirmed based on best BLAST hit classification and principle component analysis of the tetranucleotide frequencies of fragmented contigs. The genome of ~7.4 Mbp contains 6,345 protein coding genes with 4,320 of these having functional prediction including predicted pathways for biosynthesis of the secondary metabolite welwitindolinone. Analyses of 16S rRNA gene and whole genome sequence average nucleotide identity indicated close relatedness of MRB 220 to the genera Hapalosiphon and Fischerella within the order Stigonematales. Microscopic examination showed that MRB 220 formed heterocystous branched filaments, thereby supporting identification of strain MRB 220 as a morphospecies of Hapalosiphon. Availability of the draft genome of Hapalosiphon strain MRB 220 enables future work to elucidate the pathway and dynamics for biosynthesis of 2-MIB and other secondary metabolites and understand the ecology and physiology of Stigonematales cyanobacteria in tropical freshwaters., Singapore. National Research Foundation (under its Environmental & Water Technologies Strategic Research Programme and administered by the Environment & Water Industry Programme Office (EWI) of the PUB (Grant number: 1102-IRIS-14-02)), Singapore. National Research Foundation (Singapore MIT Alliance for Research and Technology’s (SMART) Center for Environmental Sensing and Modeling (CENSAM) research program)

The temporal variations in cyanobacteria and microcystin production in a tropical reservoir, Kranji Reservoir in Singapore, were studied from February 2008 to August 2009 by using molecular techniques and enzyme-linked immunosorbent assay (ELISA) respectively. Results from PCR and qPCR showed that Microcystis and Anabaena were present (mean concentrations 4.16 × 10 6 gene copies/mL and 4.47 × 10 4 gene copies/mL respectively) and were well correlated to each other ( P Microcystis was the primary microcystin producer. The average percentage of toxigenic Microcystis spp. was 55.92%, whereas no Anabaena -specific microcystin producing gene was detected. Microcystis -specific microcystin-producing genes mcyB and mcyD were detected in all the samples, together with dominant detection of mcyA and mcyE genes. However, a low positive detection rate was observed for mcyC . Due to the combined effects of high temperature, light and nutrient conditions in this tropical reservoir, the Microcystis cell concentrations were higher and more uniform compared to other Microcystis blooms reported in subtropical and temperate regions. Furthermore, the proportion of toxigenic Microcystis in the total Microcystis population was considerably higher. Principle component analysis and multiple linear regression analysis showed that total nitrogen and total phosphorus were positively correlated with the abundances of total Microcystis and toxigenic Microcystis . In addition, total nitrogen, pH and dissolved oxygen were positively correlated with the microcystin concentration.

In this study, we focused on the dynamics of bacterial community composition in a large reservoir in the Yangtze estuary during spring and summer seasons, especially the variations of functional mechanisms of microbial community during the seasonal alternation between spring and summer. Both 16S rRNA gene sequencing and shotgun metagenomic sequencing technology were used for these purposes. The results indicated that obvious variations of bacterial community structures were found at different sites. Particle-associated bacterial taxa exhibited higher abundance at the inlet site, which was closer to the Yangtze River with a high level of turbidity. In other sites, Synechococcus, as the most dominant cyanobacterial species, revealed high abundance driven by increased temperature. Moreover, some heterotrophic bacterial taxa revealed high abundance following the increased Synechococcus in summer, which indicated potential correlations about carbon source utilization between these microorganisms. In addition, the shotgun metagenomic data indicated during the period of seasonal alternation between spring and summer, the carbohydrate transport and metabolism, energy production and conversion, translation/ribosomal biogenesis, and cell wall/membrane/envelope biogenesis were significantly enhanced at the exit site. However, the course of cell cycle control/division was more active at the internal site.

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

Mass occurrences of toxic cyanobacterial blooms have been reported world-wide in eutrophic waters. Due to rapid urbanization, the reservoirs in Singapore will be exposed to pollution. Therefore, it is important to assess toxic cyanobacteria species in local reservoirs to ensure a safe drinking water supply. While publications on the occurrences and characterizations of toxigenic cyanobacteria are rapidly increasing, most of the studies were done in temperate or cold climate regions. To date, there are only limited reports on cyanobacterial blooms in South East Asian region. With high and uniform temperature throughout the year, cyanobacterial blooms in the tropics could have distinct behaviors compared to those in the temperate regions as responses to local climatic condition. The temporal variations in cyanobacteria and microcystin production in a tropical reservoir, Kranji Reservoir in Singapore, were studied using molecular techniques and enzyme-linked immunosorbent assay (ELISA) respectively. Water samples were collected monthly from February 2008 to August 2009. Results from PCR and qPCR showed that Microcystis was the major microcystin producer. Anabaena spp. were found to coexist with the Microcystis spp. with Spearman’s rho coefficient, rs = 0.498 (P < 0.001). The average concentrations of Microcystis and Anabaena (equivalent to 16S rRNA) were 4.16×106 gene copies/mL and 4.47×104 gene copies/mL respectively. The average percentage of toxigenic Microcystis spp. was 55.92 % (SD 21.1 %), whereas no Anabaena-specific microcystin producing gene was detected. Microcystin-producing genes mcyB and mcyD were detected in all samples, together with the detections of mcyA and mcyE genes in 98 % and 96 % of the samples, respectively. However, a low detection rate was observed for mcyC. Due to the combined effects of high temperature, light and nutrient conditions in the tropics, the Microcystis cell concentrations were higher and more uniform compared to other Microcystis blooms reported in subtropical and temperate regions. Furthermore, the proportion of toxigenic Microcystis was considerably high and more stable throughout the year. Principle component analysis and multiple linear regression analysis showed that total nitrogen and total phosphorus were positively correlated with the abundances of total Microcystis and toxigenic Microcystis. In addition, total nitrogen, pH and dissolved oxygen were positively correlated with the microcystin concentration. The total microcystin concentration measured using an ELISA kit ranged from below the detection limit to14.4 µg/L with a mean concentration of 2.66±2.80 µg/L, which is higher than those reported by local water agency, PUB, using LC-MSMS detection on 4 microcystin variants. This suggests a more thorough survey of natural toxin variants in local waters is needed. Doctor of Philosophy (CEE)

A proper assessment of microbial water quality is necessary to give early warning of disease outbreak and protect public health. Conventional methods of analysis typically require the culturing of indicator microbes, but inherent problems exist, such as lengthy time of analysis and the inability to detect nonculturable species. Alternative methods exist (e.g., molecular and optical methods), which are faster but require adequate sampling preparation techniques. This chapter discusses some of the more common techniques used including filtration, centrifugation, and immunomagnetic separation as preconcentration techniques, the use of propidium monoazide and ethidium monoazide for preparing samples for analysis with molecular tools (e.g., polymerase chain reaction), and sample preservation/treatment for long-term storage. These methods are applied to the analysis of pathogenic bacteria, viruses, protozoans, and toxic algae (including their toxins).