Computational protocol: Multiple syntrophic interactions drive biohythane production from waste sludge in microbial electrolysis cells

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Protocol publication

[…] Genomic DNAs of the electrode biofilms and bulk solution samples in parallel MECs were extracted by PowerSoil DNA Isolation Kit (Mobio laboratories, CA) according to the manufacturer’s protocol. DNA concentration and purity were detected by NanoPhotometer P-Class (Implen). Prior to PCR amplification, DNA from two parallel reactors were mixed. The V4-V5 region (length of ~400 bp) of bacterial and archaeal 16S rRNA gene was amplified separately using a set of primers: 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R (5′-CCGTCAATTCCTTTR AGTTT-3′) for bacteria, 519F (5′-CAGCMGCCGCGGTAATWC-3′) and 915R (5′-GTGCTCCCCCGCCAATTCCT-3′) for archaea. After integrated with barcode, PCR amplification was implemented using ABI GeneAmp® 9700 PCR system. High-throughput sequencing was performed on Illumina Miseq platforms according to the standard protocols. Raw sequencing data were filtered and analyzed using the pipelines of Quantitative Insights Into Microbial Ecology (QIIME) software (http://www.microbio.me/qiime). Operational taxonomic units (OTUs) were determined based on the threshold of 97 % similarity using UPARSE software (http://drive5.com/uparse/). Species diversity was evaluated in the MOTHUR (http://www.mothur.org). A representative sequence of each OTU was aligned for taxonomic identification using the Silva database (http://www.arb-silva.de) and Ribosomal Database Project (RDP) classifier (version 2.2 http://sourceforge.net/projects/rdp-classifier/) with a minimum confidence of 70 % [, ].The DNA samples extracted from anaerobic digestion raw waste sludge (RS-OCMEC), anode and cathode biofilms of MEC without alkali-pretreatment [RS-MEC (A), RS-MEC (C)] and with alkali-pretreatment [AS-MEC (A), AS-MEC (C)] were used to quantify archaea copies. Archaeal universal primers 787F (5′-ATTAGATACCCSBGTAGTCC-3′) and 1059R (5′-GCCATGCACCWCCTCT-3′) were chose to amplify archaeal community []. The q-PCR reaction mixtures (25 μL) contained 1× SYBR Green qPCR Mix (Tiangen, China), 300 nM of each primer and 1 μL of template DNA. Amplifications were performed on an ABI 7500 Real-Time PCR System (Applied Biosystems). The protocol of PCR amplification consisted of two steps: initial denaturation for 2 min at 95 °C followed by 40 cycles of denaturation for 10 s at 95 °C, annealing for 15 s at 60 °C, elongation for 30 s at 68 °C. Standard curve was obtained using diluted DNA of RS-OCMEC sample and the efficiency value calculated was up to 1.06 with an R2 of 0.99. All relative q-PCR reactions were performed in triplicate. […]

Pipeline specifications

Software tools QIIME, UPARSE, mothur, RDP Classifier
Application 16S rRNA-seq analysis
Diseases Carcinoma, Renal Cell, Retinoschisis
Chemicals Hydrogen, Methane