Sampling along a transect within the PARWQCP occurred on 12 November 2008. Upon temporary shutdown of the rotating distributor arm in one of the trickling filters, a surface PVC media module (0.9 m deep) was removed from the reactor, and 1-3 g biofilm samples were obtained with a sterile spatula, placed in sterile 1.5 ml tubes, weighed, and stored at -20oC. Concurrently, samples of influent, trickling filter effluent, and activated sludge were obtained. Activated sludge samples represented a 24-h composite from the combined outlet of all four aeration basins using a Sigma 900 refrigerated automatic sampler (Hach, Loveland, CO). Eight 1.5 ml activated sludge samples were centrifuged onsite for 5 min at 5000 g, decanted, and stored at -20oC. Raw influent and trickling filter effluent samples were transported on ice to the laboratory, vacuum filtered in 10 ml aliquots onto 0.22 μm Hydrophilic Durapore PVDF filters (25 mm diameter, Millipore, Billerica, MA), and archived at -20oC.
Growth protocol
Environmental Samples
Extracted molecule
genomic DNA
Extraction protocol
Activated sludge and trickling filter biofilm samples were washed using 1 ml Tris-EDTA buffer (1 mM, pH=7.0) and genomic DNA (gDNA) was extracted in triplicate using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) following the manufacturer’s protocol, except for the initial bead-beating step. A Vortex Adapter (MO BIO laboratories, Inc., Carlsbad, CA) with the Vortex Genie 2T (Scientific Industries, Inc., Bohemia, NY) was used to physically disrupt cells in lysing matrix at maximum speed for 15 min. gDNA extraction from raw influent and trickling filter effluent proceeded via incubation of filters in lysis solution (800 μl water, 100 μl 10% SDS (Invitrogen, Carlsbad, CA) and 100μl 10mg/ml Proteinase K (Invitrogen, Carlsbad, CA) at 55oC for 2.5 h. Filtrate from the lysis incubation was purified with the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) following the manufacturer’s protocol but without the initial bead-beating step.
Label
biotin
Label protocol
Bacterial and archaeal 16S rRNA genes (~1450-bp) were separately PCR amplified for application to the PhyloChip platform using universal primers 27F.1 (5’-AGRGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) for the Bacterial domain and primer pair 4Fa (5’-TCCGGTTGATCCTGCCRG-3’) and 1492R for Archaeal domain. Each PCR reaction mix contained 1X buffer with 0.8 mM dNTP mixture (Takara Bio Inc., Japan), 1.25U Ex Taq polymerase (Takara Bio Inc., Japan), 1 μg/μl BSA (Roche Applied Science, Indianapolis, IN), 300 nM of each primer, and 10 ng DNA. PCR conditions were set to 1 cycle of 3 min at 95°C, followed by 30 cycles of 95°C (30 s), annealing (30 s), 72°C (60 s), and a final extension at 72°C for 7 min. With the exception of the duplicate influent samples, DNA extracts were amplified using a range of eight different annealing temperatures between 48°C and 58°C. Due to low DNA concentrations, two DNA extracts from raw influent samples were amplified in duplicate at three different annealing temperatures (48oC, 54.2oC, and 58oC). All amplicons from different annealing temperatures associated with a single sample were combined, and pooled amplicons were purified with the NovaGen SpinPrep PCR Clean-up Kit (EMD4Biosciences, Gibbstown, NJ). Presence or absence of the expected amplicon was verified via agarose gel electrophoresis. Amplicons were further purified in selected samples via agarose gel electrophoresis with the Qiagen QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA). Pooled purified amplicons were quantified via agarose gel electrophoresis using the GelDoc XR System and Quantity One software (Biorad, Hercules, CA) with an E-Gel Low Range Quantitative DNA Ladder (Invitrogen, Carlsbad, CA). 500 ng of bacterial 16S rRNA gene amplicons and 100 ng of archaeal 16S rRNA gene amplicons were pooled for each sample for use in downstream microarray processing steps. Pooled bacterial and archaeal 16S rRNA gene amplicons from each sample were spiked with internal standards consisting of known concentrations of amplicons of yeast and bacterial metabolic genes (Brodie et al. 2006), and then fragmented to 50-200 bp using Dnase I (0.02 U/ mg DNA, Invitrogen, Carlsbad, CA) and One-Phor-All buffer (GE Healthcare, Piscataway, NJ), as per the standard Affymetrix protocol. Terminal 3’ labeling of fragmented amplicons via biotinylation was accomplished using the GeneChip DNA Labeling Reagent (Affymetrix, Santa Clara, CA) with recombinant Terminal Deoxynucleotitidyl Transferase (Promega, Madison, WI), as per the manufacturer’s instructions.
Hybridization protocol
The labeled DNA was denatured (99°C for 5 min) and hybridized to PhyloChip arrays at 48°C overnight (> 16 h) at 60 rpm in a rotisserie oven with prewarmed hybridization cocktail (biotin-labeled target, hybridization solution, and control oligonucleotides.) Microarray washing and staining occurred on a GeneChip Fluidics Station 400 (Affymetrix, Santa Clara, CA) following standard manufacturer’s protocols.
Scan protocol
Arrays were scanned using a GeneArray Scanner (Affymetrix, Santa Clara, CA) with a 488-nm argon-ion laser with emission detection at 520 nm.
Description
Affymetrix G2 PhyloChip
Data processing
The scan was captured as a pixel image using standard Affymetrix software (GeneChip Microarray Analysis Suite, version 5.1) that reduced the data to an individual signal value for each probe. Probes producing hybridization intensities in the lowest 2% of all measured intensities were considered background, and the average background intensity was subtracted from the fluorescence intensity of all remaining probes. To correct for variation associated with quantification of amplicon target (quantification variation) and downstream variation associated with target fragmentation, labeling, hybridization, washing, staining and scanning (microarray technical variation), a two-step PhyloChip data normalization procedure was performed in the R statistical programming environment (http://www.r-project.org/). First, for each PhyloChip experiment, a scaling factor best explaining the intensities of the spiked control probes under a multiplicative error model was estimated using a maximum-likelihood procedure (Ivanov et al. 2009). The intensities in each experiment were multiplied with its corresponding optimal scaling factor. Second, the intensities for each experiment were corrected for the variation in total array intensity by dividing the intensities with its corresponding total array intensity separately for Bacteria and Archaea. Scoring of probe pairs as positive or negative followed previously described protocols (DeSantis et al. 2007). For each set of probe pairs comprising an OTU, the positive fraction (PosFrac, or pf) was defined as the fraction of total probe pairs in a probe set that were scored as positive. An OTU was considered present when its PosFrac was greater than 0.9. Hierarchical taxonomic nodes (subfamily, family, order, class, phylum, domain) were considered present if at least one underlying OTU was present. OTUs that were not detected in at least one sample (pf>0.9) are not included in the matrix table and were not considered in downstream analyses.
