PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions

Evaluation of Microfluidic Biosensor Development Using Microscopic Analysis of Molecular Beacon Hybridization

Abstract. Molecular beacons, oligonucleotide probes that fluoresce upon hybridization to a target nucleic acid, can be used in microfluidic devices to detect and quantify nucleic acids in solution as well as inside bacterial cells. Three essential steps towards the development of such devices as integrated microfluidic biosensors using molecular beacons were investigated in the present study. First, experiments using real-time confocal microscopy indicated that diffusion of DNA molecular beacons across a 100-µm diameter microfluidic channel took less than one minute after the flow of reagents was stopped. Second, experiments to evaluate hybridization kinetics of DNA molecular beacons with target nucleic acids in solution showed that DNA molecular beacons can be used to characterize hybridization kinetics in real time in microfluidic channels and that hybridization signals approached their maximum in approximately three minutes. Finally, it was demonstrated that peptide nucleic acid molecular beacons can be used to detect bacterial cells in microfluidic devices. These results suggest that the use of microfluidic devices to detect nucleic acids in solution and in bacterial cells is promising and that further development of an integrated microfluidic biosensor for bacterial detection based on this concept is warranted

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries. Supplemental materials (2 figures) are included

Probabilistic models to describe the dynamics of migrating microbial communities

In all but the most sterile environments bacteria will reside in fluid being transported through conduits and some of these will attach and grow as biofilms on the conduit walls. The concentration and diversity of bacteria in the fluid at the point of delivery will be a mix of those when it entered the conduit and those that have become entrained into the flow due to seeding from biofilms. Examples include fluids through conduits such as drinking water pipe networks, endotracheal tubes, catheters and ventilation systems. Here we present two probabilistic models to describe changes in the composition of bulk fluid microbial communities as they are transported through a conduit whilst exposed to biofilm communities. The first (discrete) model simulates absolute numbers of individual cells, whereas the other (continuous) model simulates the relative abundance of taxa in the bulk fluid. The discrete model is founded on a birth-death process whereby the community changes one individual at a time and the numbers of cells in the system can vary. The continuous model is a stochastic differential equation derived from the discrete model and can also accommodate changes in the carrying capacity of the bulk fluid. These models provide a novel Lagrangian framework to investigate and predict the dynamics of migrating microbial communities. In this paper we compare the two models, discuss their merits, possible applications and present simulation results in the context of drinking water distribution systems. Our results provide novel insight into the effects of stochastic dynamics on the composition of non-stationary microbial communities that are exposed to biofilms and provides a new avenue for modelling microbial dynamics in systems where fluids are being transported

Separation of the bacterial species, Escherichia coli, from mixed-species microbial communities for transcriptome analysis

Abstract Background The study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. However, current applications of microarrays are mostly limited to single species studies. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis. Results E. coli cells were separated from a dual-species (E. coli and Stenotrophomonas maltophilia) community using immuno-magnetic separation (IMS). High recovery rates of E. coli were achieved. The purity of E. coli cells was as high as 95.0% separated from suspended mixtures consisting of 1.1 - 71.3% E. coli, and as high as 96.0% separated from biofilms with 8.1% E. coli cells. Biofilms were pre-dispersed into single-cell suspensions. The reagent RNAlater (Ambion, Austin, TX) was used during biofilm dispersion and IMS to preserve the transcriptome of E. coli. A microarray study and quantitative PCR confirmed that very few E. coli genes (only about eight out of 4,289 ORFs) exhibited a significant change in expression during dispersion and separation, indicating that transcriptional profiles of E. coli were well preserved. Conclusions A method based on immuno-magnetic separation (IMS) and application of RNAlater was developed to separate a bacterial species, E. coli as an example, from mixed-species communities while preserving its transcriptome. The method combined with cDNA microarray analysis should be very useful to study species interactions in mixed-species communities.http://deepblue.lib.umich.edu/bitstream/2027.42/112838/1/12866_2011_Article_1346.pd

Effect of backwashing on perchlorate removal in fixed bed biofilm reactors

The influence of backwashing on biological perchlorate reduction was evaluated in two laboratory scale fixed bed biofilm reactors using 1- or 3-mm glass beads as support media. Influent perchlorate concentrations were 50 μg/L and acetate was added as the electron donor at a concentration of 2 mg C/L. Perchlorate removal was evaluated at various influent dissolved oxygen (DO) concentrations. Complete perchlorate removal was achieved with an influent DO concentration of 1 mg/L resulting in bulk phase DO concentrations below the detection limit of 0.01 mg/L. The influence of increasing influent DO concentrations for 12 h periods was evaluated before and after individual backwash events. Partial perchlorate removal was achieved with an influent DO concentration of 3.5 mg/L before a strong backwash (bulk phase DO concentrations of approximately 0.2 mg/L), while no perchlorate removal was observed after the strong backwash at the same influent DO level (bulk phase DO concentrations of approximately 0.8 mg/L). The immediate effect of backwashing depended on influent DO concentrations. With influent DO concentrations of 1 mg/L, strong backwashing resulted in a brief (\u3c12 h) increase of effluent perchlorate concentrations up to 20 μg/L; more pronounced effects were observed with influent DO concentrations of 3 mg/L. Daily weak backwashing had a small and, over time, decreasing negative influence on perchlorate reduction, while daily strong backwashing ultimately resulted in the breakdown of perchlorate removal with influent DO concentrations of 3 mg/L

Nitrogen and Sulfur Concentrations and Flow Rates of Corn Wet‐Milling Streams

Nitrogen (N) and sulfur (S) concentrations can affect the market value of coproducts from corn wet‐milling. The composition of parent streams would be expected to affect composition of the resulting coproducts but there are few published data available to examine this relationship. Concentration and flow data are needed to determine which streams are important in modifying N and S coproduct concentrations. The objective was to measure concentrations and flows of N and S in corn wet‐milling streams. Samples were taken from 21 process streams from 3 wet‐milling plants during two periods of three weeks each; N and S concentrations of each sample were determined. There were large differences in N and S concentrations among processing streams; within most streams, N and S concentrations were similar among plants. Concentrations of N and S were related inversely to flow rates. Steepwater and gluten streams contained most of the N and S flow and provide an opportunity for modification. The process water stream carried large quantities of N and S and represents another opportunity for improving process efficiency and coproduct value.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141537/1/cche0260.pd

Impact of Ammonium on Syntrophic Organohalide-Respiring and Fermenting Microbial Communities

Citation: Delgado, A. G., Fajardo-Williams, D., Kegerreis, K. L., Parameswaran, P., & Krajmalnik-Brown, R. (2016). Impact of Ammonium on Syntrophic Organohalide-Respiring and Fermenting Microbial Communities. Msphere, 1(2), 10. doi:10.1128/mSphere.00053-16Syntrophic interactions between organohalide-respiring and fermentative microorganisms are critical for effective bioremediation of halogenated compounds. This work investigated the effect of ammonium concentration (up to 4 g liter(-1) NH4+-N) on trichloroethene-reducing Dehalococcoides mccartyi and Geobacteraceae in microbial communities fed lactate and methanol. We found that production of ethene by D. mccartyi occurred in mineral medium containing = 1 g liter(-1) NH4+-N, organohalide-respiring dynamics shifted from D. mccartyi and Geobacteraceae to mainly D. mccartyi. An increasing concentration of ammonium was coupled to lower metabolic rates, longer lag times, and lower gene abundances for all microbial processes studied. The methanol fermentation pathway to acetate and H-2 was conserved, regardless of the ammonium concentration provided. However, lactate fermentation shifted from propionic to acetogenic at concentrations of >= 2 g liter(-1) NH4+-N. Our study findings strongly support a tolerance of D. mccartyi to high ammonium concentrations, highlighting the feasibility of organohalide respiration in ammonium-contaminated subsurface environments. IMPORTANCE Contamination with ammonium and chlorinated solvents has been reported in numerous subsurface environments, and these chemicals bring significant challenges for in situ bioremediation. Dehalococcoides mccartyi is able to reduce the chlorinated solvent trichloroethene to the nontoxic end product ethene. Fermentative bacteria are of central importance for organohalide respiration and bioremediation to provide D. mccartyi with H2, their electron donor, acetate, their carbon source, and other micronutrients. In this study, we found that high concentrations of ammonium negatively correlated with rates of trichloroethene reductive dehalogenation and fermentation. However, detoxification of trichloroethene to nontoxic ethene occurred even at ammonium concentrations typical of those found in animal waste (up to >= 2 g liter(-1) NH4+-N). To date, hundreds of subsurface environments have been bioremediated through the unique metabolic capability of D. mccartyi. These findings extend our knowledge of D. mccartyi and provide insight for bioremediation of sites contaminated with chlorinated solvents and ammonium