Effect of target gene sequence evenness and dominance on real-time PCR quantification of artificial sulfate-reducing microbial communities.

Quantitative real-time PCR of phylogenetic and functional marker genes is among the most commonly used techniques to quantify the abundance of microbial taxa in environmental samples. However, in most environmental applications, the approach is a rough assessment of population abundance rather than an exact absolute quantification method because of PCR-based estimation biases caused by multiple factors. Previous studies on these technical issues have focused on primer or template sequence features or PCR reaction conditions. However, how target gene sequence characteristics (e.g., evenness and dominance) in environmental samples affect qPCR quantifications has not been well studied. Here, we compared three primer sets targeting the beta subunit of the dissimilatory sulfite reductase (dsrB) to investigate qPCR quantification performance under different target gene sequence evenness and dominance conditions using artificial gBlock template mixtures designed accordingly. Our results suggested that the qPCR quantification performance of all tested primer sets was determined by the comprehensive effect of the target gene sequence evenness and dominance in environmental samples. Generally, highly degenerate primer sets have equivalent or better qPCR quantification results than a more target-specific primer set. Low template concentration in this study (~105 copies/L) will exaggerate the qPCR quantification results difference among tested primer sets. Improvements to the accuracy and reproducibility of qPCR assays for gene copy number quantification in environmental microbiology and microbial ecology studies should be based on prior knowledge of target gene sequence information acquired by metagenomic analysis or other approaches, careful selection of primer sets, and proper reaction conditions optimization.

Tracking de novo protein synthesis in the activated sludge microbiome using BONCAT-FACS.

In order to ensure stable performance of engineered biotechnologies that rely on mixed microbial community systems, it is important to identify process-specific microbial traits and study their in-situ activity and responses to changing environmental conditions and system operational parameters. We used BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) in combination with Fluorescence-Activated Cell Sorting (FACS) and 16S rRNA gene amplicon sequencing to identify translationally active cells in activated sludge. We found that only a subset of the activated sludge microbiome is translationally active during the aerobic treatment phase of a full-scale sequencing batch reactor designed to enhance biological phosphorus removal from municipal wastewater. Relative abundance of amplicon sequence variants was not a reliable predictor of species activity. BONCAT-positive and -negative cells revealed a broad range of population-wide and taxa-specific translational heterogeneity. BONCAT-FACS in combination with amplicon sequencing can provide new insights into the ecophysiology of highly dynamic microbiomes in activated sludge systems.

Different Engineering Designs Have Profoundly Different Impacts on the Microbiome and Nitrifying Bacterial Populations in Municipal Wastewater Treatment Bioreactors.

Numerous wastewater treatment processes are designed by engineers to achieve specific treatment goals. However, the impact of these different process designs on bacterial community composition is poorly understood. In this study, 24 different municipal wastewater treatment facilities (37 bioreactors) with various system designs were analyzed by sequencing of PCR-amplified 16S rRNA gene fragments. Although a core microbiome was observed in all of the bioreactors, the overall microbial community composition (analysis of molecular variance; P = 0.001) as well as that of a specific population of Nitrosomonas spp. (P = 0.04) was significantly different between A/O (anaerobic/aerobic) systems and conventional activated sludge (CAS) systems. Community α-diversity (number of observed operational taxonomic units [OTUs] and Shannon diversity index) was also significantly higher in A/O systems than in CAS systems (Wilcoxon; P < 2 × 10-16). In addition, wastewater bioreactors with short mean cell residence time (<2 days) had very low community α-diversity and fewer nitrifying bacteria compared to those of other system designs. Nitrospira spp. (0.71%) and Nitrotoga spp. (0.41%) were the most prominent nitrite-oxidizing bacteria (NOB); because these two genera were rarely prominent at the same time, these populations appeared to be functionally redundant. Weak evidence (AOB:NOB « 2; substantial quantities of Nitrospira sublineage II) was also obtained suggesting that complete ammonia oxidation by a single organism was occurring in system designs known to impose stringent nutrient limitation. This research demonstrates that design decisions made by wastewater treatment engineers significantly affect the microbiome of wastewater treatment bioreactors. IMPORTANCE Municipal wastewater treatment facilities rely on the application of numerous "activated sludge" process designs to achieve site-specific treatment goals. A plethora of microbiome studies on municipal wastewater treatment bioreactors have been performed previously; however, the role of process design on the municipal wastewater treatment microbiome is poorly understood. In fact, wastewater treatment engineers have attempted to control the microbiome of wastewater bioreactors for decades without sufficient empirical evidence to support their design paradigms. Our research demonstrates that engineering decisions with respect to system design have a significant impact on the microbiome of wastewater treatment bioreactors.

Localized plasticity in the streamlined genomes of vinyl chloride respiring Dehalococcoides.

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism.

Unusual codon bias in vinyl chloride reductase genes of Dehalococcoides species.

Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes vcrA and bvcA showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available Dehalococcoides genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes.