The Lake Erie HABs Grab: A binational collaboration to characterize the western basin cyanobacterial harmful algal blooms at an unprecedented high-resolution spatial scale.

Monitoring of cyanobacterial bloom biomass in large lakes at high resolution is made possible by remote sensing. However, monitoring cyanobacterial toxins is only feasible with grab samples, which, with only sporadic sampling, results in uncertainties in the spatial distribution of toxins. To address this issue, we conducted two intensive "HABs Grabs" of microcystin (MC)-producing Microcystis blooms in the western basin of Lake Erie. These were one-day sampling events during August of 2018 and 2019 in which 100 and 172 grab samples were collected, respectively, within a six-hour window covering up to 2,270 km2 and analyzed using consistent methods to estimate the total mass of MC. The samples were analyzed for 57 parameters, including toxins, nutrients, chlorophyll, and genomics. There were an estimated 11,513 kg and 30,691 kg of MCs in the western basin during the 2018 and 2019 HABs Grabs, respectively. The bloom boundary poses substantial issues for spatial assessments because MC concentration varied by nearly two orders of magnitude over very short distances. The MC to chlorophyll ratio (MC:chl) varied by a factor up to 5.3 throughout the basin, which creates challenges for using MC:chl to predict MC concentrations. Many of the biomass metrics strongly correlated (r > 0.70) with each other except chlorophyll fluorescence and phycocyanin concentration. While MC and chlorophyll correlated well with total phosphorus and nitrogen concentrations, MC:chl correlated with dissolved inorganic nitrogen. More frequent MC data collection can overcome these issues, and models need to account for the MC:chl spatial heterogeneity when forecasting MCs.

Whole-Community Metagenomics in Two Different Anammox Configurations: Process Performance and Community Structure.

Anaerobic ammonia oxidation (anammox) combined with partial nitritation (PN) is an innovative treatment process for energy-efficient nitrogen removal from wastewater. In this study, we used genome-based metagenomics to investigate the overall community structure and anammox species enriched in suspended growth (SGR) and attached growth packed-bed (AGR) anammox reactors after 220 days of operation. Both reactors removed more than 85% of the total inorganic nitrogen. Metagenomic binning and phylogenetic analysis revealed that two anammox population genomes, affiliated with the genus Candidatus Brocadia, were differentially abundant between the SGR and AGR. Both of the genomes shared an average nucleotide identify of 83%, suggesting the presence of two different species enriched in both of the reactors. Metabolic reconstruction of both population genomes revealed key aspects of their metabolism in comparison to known anammox species. The community composition of both the reactors was also investigated to identify the presence of flanking community members. Metagenomics and 16S rRNA gene amplicon sequencing revealed the dominant flanking community members in both reactors were affiliated with the phyla Anaerolinea, Ignavibacteria, and Proteobacteria. Findings from this research adds two new species, Ca. Brocadia sp. 1 and Ca. Brocadia sp. 2, to the genus Ca. Brocadia and sheds light on their metabolism in engineered ecosystems.

High Biofilm Conductivity Maintained Despite Anode Potential Changes in a Geobacter-Enriched Biofilm.

This study systematically assessed intracellular electron transfer (IET) and extracellular electron transfer (EET) kinetics with respect to anode potential (Eanode ) in a mixed-culture biofilm anode enriched with Geobacter spp. High biofilm conductivity (0.96-1.24 mS cm-1 ) was maintained during Eanode changes from -0.2 to +0.2 V versus the standard hydrogen electrode (SHE), although the steady-state current density significantly decreased from 2.05 to 0.35 A m-2 in a microbial electrochemical cell. Substantial increase of the Treponema population was observed in the biofilm anode at Eanode =+0.2 V, which reduced intracellular electron-transfer kinetics associated with the maximum specific substrate-utilization rate by a factor of ten. This result suggests that fast EET kinetics can be maintained under dynamic Eanode conditions in a highly conductive biofilm anode as a result of shift of main EET players in the biofilm anode, although Eanode changes can influence IET kinetics.

Ohmic resistance affects microbial community and electrochemical kinetics in a multi-anode microbial electrochemical cell.

Multi-anode microbial electrochemical cells (MxCs) are considered as one of the most promising configurations for scale-up of MxCs, but understanding of anode kinetics in multiple anodes is limited in the MxCs. In this study we assessed microbial community and electrochemical kinetic parameters for biofilms on individual anodes in a multi-anode MxC to better comprehend anode fundamentals. Microbial community analysis targeting 16S rRNA Illumina sequencing showed that Geobacter genus was abundant (87%) only on the biofilm anode closest to a reference electrode (low ohmic energy loss) in which current density was the highest among three anodes. In comparison, Geobacter populations were less than 1% for biofilms on other two anodes distant from the reference electrode (high ohmic energy loss), generating small current density. Half-saturation anode potential (EKA) was the lowest at -0.251 to -0.242 V (vs. standard hydrogen electrode) for the closest biofilm anode to the reference electrode, while EKA was as high as -0.134 V for the farthest anode. Our study proves that electric potential of individual anodes changed by ohmic energy loss shifts biofilm communities on individual anodes and consequently influences electron transfer kinetics on each anode in the multi-anode MxC.

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers.

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.

Comparative fecal metagenomics unveils unique functional capacity of the swine gut.

BACKGROUND: Uncovering the taxonomic composition and functional capacity within the swine gut microbial consortia is of great importance to animal physiology and health as well as to food and water safety due to the presence of human pathogens in pig feces. Nonetheless, limited information on the functional diversity of the swine gut microbiome is available. RESULTS: Analysis of 637, 722 pyrosequencing reads (130 megabases) generated from Yorkshire pig fecal DNA extracts was performed to help better understand the microbial diversity and largely unknown functional capacity of the swine gut microbiome. Swine fecal metagenomic sequences were annotated using both MG-RAST and JGI IMG/M-ER pipelines. Taxonomic analysis of metagenomic reads indicated that swine fecal microbiomes were dominated by Firmicutes and Bacteroidetes phyla. At a finer phylogenetic resolution, Prevotella spp. dominated the swine fecal metagenome, while some genes associated with Treponema and Anareovibrio species were found to be exclusively within the pig fecal metagenomic sequences analyzed. Functional analysis revealed that carbohydrate metabolism was the most abundant SEED subsystem, representing 13% of the swine metagenome. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Virulence factors associated with antibiotic resistance genes with highest sequence homology to genes in Bacteroidetes, Clostridia, and Methanosarcina were numerous within the gene families unique to the swine fecal metagenomes. Other abundant proteins unique to the distal swine gut shared high sequence homology to putative carbohydrate membrane transporters. CONCLUSIONS: The results from this metagenomic survey demonstrated the presence of genes associated with resistance to antibiotics and carbohydrate metabolism suggesting that the swine gut microbiome may be shaped by husbandry practices.

Identification of bacterial DNA markers for the detection of human fecal pollution in water.

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene.

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.

Development and applications of microbial ecogenomic indicators for monitoring water quality: report of a workshop assessing the state of the science, research needs and future directions.

This article brings forth recommendations from a workshop sponsored by the U.S. Environmental Protection Agency's Science to Achieve Results (STAR) and Environmental Monitoring and Assessment (EMAP) Programs and by the Council of State Governments, held during May 2002 in Kansas City, Kansas. The workshop assembled microbial ecologists and environmental scientists to determine what research and science is needed to bring existing molecular biological approaches and newer technologies arising from microbial genomic research into environmental monitoring and water quality assessments. Development of genomics and proteomics technologies for environmental science is a very new area having potential to improve environmental water quality assessments. The workshop participants noted that microbial ecologists are already using molecular biological methods well suited for monitoring and water quality assessments and anticipate that genomics-enabled technologies could be made available for monitoring within a decade. Recommendations arising from the workshop include needs for (i) identification of informative microbial gene sequences, (ii) improved understandings of linkages between indicator taxa, gene expression and environmental condition, (iii) technological advancements towards field application, and (iv) development of the appropriate databases.