1. INTRODUCTION
2.2. Materials & methods
2.2.3. Analytical methods
2.2.3.1. Physicochemical analyses
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Chemical oxygen demand (COD, based on standard dichromate chemistry) and sulfide concentrations were measured colorimetrically using a HS-COD-MR kit (HUMAS) and a HS-S kit (HUMAS), respectively. Solids were measured following the procedures in Standards Methods (APHA-AWWA-WEF., 2005). Major anions and cations were measured simultaneously using two ion chromatographs (Dionex ICS-1100, Thermo Scientific) respectively equipped with an IonPac AS14 column and an IonPac CS12A column. The C, H, O, N, and S contents of Ulva biomass were measured on a dry weight basis using an organic elemental analyzer (Flash 2000, Thermo Scientific). A gas chromatograph (7820A, Agilent) equipped with a flame ionization detector and an Innowax column (Agilent) was used to measure volatile fatty acids (VFAs, C2-C7). Biogas composition (CH4, CO2, and H2) was analyzed using the same gas chromatograph equipped with a thermal conductivity detector and a ShinCarbon ST column (Restek). The hydrogen sulfide content in biogas was determined using a 7890A gas chromatograph equipped with a flame photometric detector and an HP-1 column (Agilent). Samples for measuring soluble COD, VFAs, and ions were prepared by filtration through a 0.45-m-pore filter. All the above analyses were carried out at least duplicate. Biogas production from each reactor was periodically measured by water displacement and corrected to standard temperature and pressure (0 oC and 1 bar).
2.2.3.2. DNA extraction
Total DNA was extracted from the inoculum sludge, Ulva substrate, and reactor samples in batch operation, from the initial reactor sample of cycle 1, and final reactor samples of all cycles in repeated batch operation, and from the steady-state reactor sample using an automated nucleic acid extractor (Exiprogen, Bioneer) according to the manufacturer’s instructions. One milliliter of a sample was pelleted at 13,000 g for 5 min and repeatedly washed by resuspending in distilled water (up to 1 mL), supernatant decanting (900 L), and centrifuging (13,000 g for 1 min). The resulting pellet was resuspended in 1 mL of distilled water, and a 200-μL aliquot of the final resuspension was loaded onto the extractor with the ExiProgen Bacteria Genomic DNA kit (Bioneer). The purified DNA was recovered in 200 μL of elution buffer and stored at –20 °C until use.
2.2.3.3. Denaturing gradient gel electrophoresis (DGGE)
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Bacterial and archaeal 16S rRNA genes were amplified from total DNA samples by touch- down polymerase chain reaction (PCR) using BAC338F/509R and ARC787F/1059R primer pairs, respectively (Yu et al., 2005), as previously described (Kim & Lee, 2015b). Amplicons were electrophoresed in 8% (w/v) polyacrylamide gels with denaturant gradients of 25–60% for bacteria and 35–65% for archaea (100% denaturant corresponds to 7 M urea and 40% (v/v) formamide) at 80 V for 16 h in a D-code system (Bio-Rad). After electrophoresis, the gels were stained with SYBR Safe Dye (Molecular Probes) and scanned under blue light to visualize the band patterns.
Selected bands were cut from the gels and eluted in 40 μL of sterile water.
2.2.3.4. Real-time PCR
Real-time PCR was used to estimate the abundance of target microbial groups, i.e., bacteria, methanogens, and SRBs. The 16S rRNA gene concentrations of total bacteria and methanogens were determined as previously described (Kim & Lee, 2016). Six primers/probe sets specific for the domain Bacteria and five methanogen groups at the order or family level (i.e., Methanobacteriales, Methanomicrobiales, Methanococcales, Methanosarcinaceae, and Methanosaetaceae) were used to detect the corresponding microbial groups (Yu et al., 2005). For the quantification of SRBs, real- time PCR was carried out using two primer sets targeting the functional genes aprA and dsrA (Ben- Dov et al., 2007). Oligonucleotides used in this study are listed in Table S2. Reactions (20 L) were prepared using the THUNDERBIRD Probe qPCR Mix (TOYOBO) for the runs targeting bacteria or methanogens: 10 L of premix, 2 L of TaqMan probe (final concentration, 200 nM), 1 L of each primer (final concentration, 500 nM), 4 L of PCR-grade water, and 2 L of template DNA. For SRBs, reactions (20 L) were prepared using the THUNDERBIRD SYBR qPCR Mix (TOYOBO):
10 L of premix, 1 L of each primer (final concentration, 500 nM), 7 L of PCR-grade water, and 1 L of template DNA. All reactions were run on a QuantStudio 12K Flex system (Life Technologies) in a two-step thermal cycle procedure consisting of predenaturation (10 min at 95 °C) followed by 40 cycles of amplification (15 s at 95 °C and 1 min at 60 °C).
A standard curve was prepared as previously described for each primers/probe set used for the quantification of bacteria or methanogens using an equimolar mixture of the corresponding standard plasmids (Kim et al., 2013b). A 10-fold serial dilution series was constructed for each standard plasmid mixture and analyzed by real-time PCR with its corresponding primers/probe set. The crossing point values determined from the standard dilutions were plotted versus the logarithm of
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their template concentrations to generate a standard curve. For the quantification of SRBs, a standard curve was constructed for each target sequence in the same manner using standard plasmids. Three standard plasmids were generated for aprA and dsrA each by cloning the real-time PCR amplicons from reactor samples with different melting profiles (i.e., different sequences) into pGEM-T Easy vector (Promega). The concentration of a target sequence in an unknown sample was calculated from the corresponding standard curve within the linear range. Each sample was analyzed in duplicate.
2.2.3.5. High-resolution melting (HRM) analysis
PCR amplification and HRM analysis of aprA and dsrA were conducted on the QuantStudio 12K Flex system as previously described (Kim & Lee, 2014). The target genes were amplified using the same primers and thermal cycling protocol as those used for real-time PCR. Reactions (20 L) were prepared using the MeltDoctor HRM Master Mix (Applied Biosystems): 10 L of master mix, 1 L of each primer (final concentration, 500 nM), 7 L of PCR-grade water, and 1 L of template DNA. The PCR fragments were fully denatured at 95 °C for 10 s and renatured at 60 °C for 1 min, and then, their melting profiles were characterized by measuring the changes in fluorescence level with increasing temperature from 60 °C to 95 °C at a ramping rate of 0.015 °C/s. Melting peak plots were generated based on the melting curves with QuantStudio 12K Flex Software ver. 1.2 (Life Technologies), as previously described (Kim & Lee, 2014). Each sample was analyzed in duplicate.
2.2.3.6. Statistical analyses of microbial community data
DGGE gel images for bacteria and archaea were transformed to intensity matrices based on the relative contributions of individual bands to total band intensity in a lane. Band detection, alignment, and intensity measurement were carried out using the TotalLab 1D software. A matrix was constructed for aprA and dsrA based on the HRM peak profiles as described (Kim & Lee, 2014), with minor modifications. In each HRM peak plot, dissociation rates (–dRn/dT, the rate of decrease in the fluorescence level with temperature) were normalized to their sum within the analyzed temperature range (83.3–90.6 °C for aprA and 82.3–98.3 °C for dsrA). The normalized dissociation rates were taken at intervals of 0.2 °C (temperature resolution) to establish a relative abundance matrix.
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The intensity and relative abundance matrices were analyzed by clustering with the unweighted pair group method with arithmetic means (UPGMA) algorithm and/or nonmetric multidimensional scaling (NMS) to visualize shifts in microbial community structure during the experiment.
Clustering and NMS ordination calculation were conducted with the Sorensen (Bray–Curtis) distance measure, which is the most recommended measure for ecological community data (McCune et al., 2002), using PAST 3.14 (http://folk.uio.no/ohammer/past/) and PC-ORD 6 software (MjM software), respectively.