1. INTRODUCTION
4.2. Materials and methods
4.2.3. Analytical methods
4.2.3.1. Physicochemical analysis
The reactor effluent samples were periodically collected and analyzed to monitor the reactor performances. COD was measured spectrophotometrically using an HS-COD-MR kit (HUMAS, Korea). The volatile fatty acids (VFAs, C2–C7) concentration was analyzed using a gas
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chromatograph (7820A, Agilent) equipped with a flame ionization detector and an Innowax column (Agilent). The concentration of total dissolved sulfide and Fe2+ concentration was measured spectrophotometrically using HS-S kit and HS-Fe2+ kit (HUMAS), respectively. Samples for soluble COD, VFAs, and Fe2+ measurements were prepared using filtration through a 0.45-m pore syringe filter. Biogas content (CH4, CO2, and H2) was determined by another 7820A gas chromatograph coupled with a thermal conductivity detector and a ShinCarbon ST column (Restek).
The H2S content in the biogas was monitored using both a 7890A gas chromatograph (Agilent) equipped with a flame photometric detector and an HP-1 column (Agilent), and gas detector tube systems comprising of gas sampling pump (GV-100S, GASTEC) and detector tubes (4H, GASTEC). Biogas production from each reactor was periodically measured through water displacement and corrected to standard temperature and pressure (0 °C and 1 bar). Concentration of anions and cations was analyzed by a Dionex ICS-1100 ion chromatograph (Thermo Scientific) installed with an IonPac AS14 and an IonPac CS12A column, respectively. Samples for ions were prepared by filtration through a 0.22 m pore size syringe filter. Solids were determined according to the procedures in Standards Methods (APHA-AWWA-WEF., 2005). Structural characteristics of digestate were identified by Raman spectroscopy using a micro Raman system (alpha 300S, WITec) and by X-ray diffraction (XRD) analysis using a D/MAX2500V X-ray diffractometer (RIGAKU, Japan) equipped with an ultra 18 kW cu-rotating anode X-ray source. Samples for both Raman and XRD analyses were prepared by centrifugation (at 3,000 rpm for 10 min), resuspension, drying (at 105 oC overnight), and crushing in a mortar. Iron sulfide (FeS) was measured using the HCl extraction method following the literature (Thamdrup et al., 1994). The Element contents of substrates (C, H, O, N, and S) were analyzed on a dry weight basis using an organic elemental analyzer (Flash 2000, Thermo Scientific). All analyses were performed at least in duplicate.
4.2.3.2. Magnetite quantification
A residual magnetite concentration was measured using oxalate extraction (Kostka & Luther, 1994). A 5-mL reactor effluent sample was collected, then centrifuged (3000 rpm for 5 min), and the supernatant was discarded. The resulting pellet was loosened using mild vortexing and subjected to magnetite extraction with 10 mL of oxalate solution (0.2 M sodium oxalate/oxalic acid, pH 2.5) in an orbital shaker (280 rpm) for 48 h. The extractant was filtered through a 0.22-mm-pore filter and analyzed for soluble Fe concentration using HS-Fe(T) kit (HUMAS).
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4.2.3.3. Extracellular S0 analysis
Each reactor sample was divided into two 20-mL aliquots in 50-mL centrifuge tubes: one for measuring intracellular S0 and one for total cell-associated S0. The aliquot for intracellular S0 measurement was washed through repeated pelleting, decanting, and resuspending (in 20 mL of phosphate-buffered saline, pH 7) at increasing centrifugal forces (2,000 g for 15 min and 5,000 g for 15 min). The final resuspension was then treated by ultrasonication (20 kHz, 480 W) for 10 min, followed by centrifugation at 20,000 g for 20 min and supernatant decanting (Kim et al., 2016). The aliquot for total cell-associated S0 measurement was pelleted by centrifugation at 2,000 g for 5 min without any pretreatment. The resulting pellets from both aliquots were loosened by mild vortexing and subjected to S0 extraction with 20 mL of perchloroethylene in an orbital shaker (280 rpm) for 16 h. The extractions were filtered through a 0.22-m-pore filter and analyzed for the S0 concentration using a 1200 series high performance liquid chromatograph (Agilent, Germany) equipped with a diode array detector and an Acclaim 120 C18 column (Dionex, USA). The extracellular S0 concentration was determined by subtracting the concentration of intracellular S0 from that of total cell-associated S0 (i.e., sum of intracellular and extracellular S0).
The S0 was visualized using a fluorescent probe to detect sulfur (SSP4, DOJINDO). A 1-mL reactor sample was washed through repeated pelleting, decanting, and resuspending (in 1 mL of serum free medium). A 1-mL sample of SSP4 working solution (10 mM SSP4 in DMSO) was added into the final resuspension and incubated (at 37 oC for 15 min). After incubation, the sample was washed through repeated pelleting, decanting, and resuspending (in 1 mL of serum free medium).
The S0 in the sample was observed under microscope (Nikon).
4.2.3.4. Cyclic voltammetry
Cyclic voltammetry (CV) was performed using a single-chamber cell with three electrodes;
namely, two graphite felt electrodes as anode and cathode (each with 1 cm × 1.2 cm × 4 cm dimensions) and one Ag/AgCl as reference electrode. The electrodes were cleaned by sonication in acetone for 1 h prior to use. Two cells with a working volume of 225 mL were inoculated with the RM and RC digestates taken on day 555 (i.e., Phase M5 and C2, respectively) at a seeding ratio of 33% (v/v). The remaining volume was filled with a medium containing sodium acetate (5 g COD/L),
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sodium sulfate (0.35 g/L), and phosphate buffer (78 mM KH2PO4/K2HPO4, pH 7). The CV cells for RM and RC digestates were added with magnetite (8 mM Fe) and FeCl2 (2 mM Fe), respectively, to create the same conditions as in the reactors (i.e., Phase M5 and C2). After being purged with N2 for 5 min, the CV cells were incubated at 37 °C. CV was measured after the first 24 and 48 h of incubation using a potentiostat (WMPG1000S, WonATech, Korea) within the potential range of – 0.8 to 0.8 V (vs. Ag/AgCl) at a scan rate of 50 mV/s.
4.2.3.5. Electron transport system activity
The electron transport system (ETS) activities of reactor effluent sample were measured using INT-ETS assay, which is a measure of microbial ability to reduce an indicator under defined conditions (Blenkinsopp & Lock, 1990). A 1-mL of reactor sample was collected in a centrifuge tube, mixed with 2-mL HCl-Tris (pH 8) and 1.5-mL of iodonitrotetrazolium (INT, 0.2%), and incubated for 30 min in dark conditions (200 rpm, 37 oC). After adding 1-mL of formaldehyde solution (37%) to terminate the reaction, the sample was centrifuged (5,000 g for 5 min), and the supernatant was discarded. The resulting pellet was treated with 5 mL of methanol to extract formazan, reduced products of INT, for 10 min in dark conditions (200 rpm, 37 oC). The formazan- extracted sample which was prepared by filtration through a 0.45-m pore syringe filter was spectrophotometrically measured at 485 nm (Equation 4-1).
𝑈 =𝐷485×𝑉𝑒
𝐾×𝑉×𝑡 (4-1)
where U is the ETS activity (g/mL·min), D485 is the absorbance at 485 nm, Ve is the volume of extractant (mL), K is the slope of the standard curve, V is the volume of sample (mL), and t is the incubation time (min).
4.2.3.6. Scanning electron microscopy (SEM)
The surface morphology of reactor digestate was characterized using SEM. A 20-mL of reactor effluent sample was centrifuged (2,000 g for 10 min), washed three times with 0.1 M phosphate buffer (pH 7.4). The resulting pellet was fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5%
glutaraldehyde for 4 h at 4 oC. The fixed pellet was washed three times with 0.1 M phosphate buffer
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(pH 7.4), which was followed by serial ethanol dehydration (50, 70, 90, and 100 %, v/v) for 15 min at each step. The resulting specimen was loaded on SEM stubs using carbon tape, sputter-coated with platinum, and characterized using a field-emission SEM system (NanoSEM 230, FEI, USA).
4.2.3.7. Preparation of nucleic acids
Biomass samples for microbial community analysis were collected from steady-state samples of RM and RC. A 1-mL aliquot of each biomass sample was prepared by repeated centrifugation (13000 g for 3 min), discarding supernatant (900 L), and resuspension in DW (up to 1-mL). A 200-L aliquot of the final suspension was loaded on ExiProgen Bacteria Genomic DNA Kit (Bioneer, Korea) and total DNA extraction was performed using an ExiProgen automated nucleic acid extractor (Bioneer, Korea) following the manufacturer’s instructions. The purified DNA eluted in 100 L of elution buffer was stored at –20 °C prior to use. Total RNA was isolated from 5-L aliquot of the final suspension and then cDNA was synthesized from the isolated RNA by reverse- transcription using the SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO, Japan) in accordance with the manufacturer’s instructions. Reverse transcription was operated by incubation at 37 oC for 15 min, 50 oC for 5 min, followed by heat inactivation at 98 oC for 5 min. The resulting cDNA was stored at –20 °C until later use.
4.2.3.8. High-throughput sequencing
To construct next-generation sequencing (NGS) libraries, the total DNA and cDNA samples were amplified by polymerase-chain reaction (PCR) with universal prokaryotic 16S rRNA gene primers (Rognes et al., 2016) of which an Illumina adapter sequence was tagged at the 5’ end. The PCR was performed following thermal cycling step of initial denaturation (at 94 oC for 10 min), amplification (at 94 oC for 30 s, at 55 oC for 30 s, and at 72 oC for 30 s), and final extension (at 72
oC for 7 min). The resulting PCR products were sent to Macrogen, Inc. (Korea) for sequencing on the Illumina Miseq platform. Readings with low quality scores, ambiguous bases, or potential chimeric sequences were excluded. The trimmed sequences were aligned and clustered using CD- HIT-OTU(http://weizhongli-lab.org/cd-hit-otu/) with an operational taxonomic unit (OTU) definition of ≥97% sequence similarity. Taxonomic classification was conducted via RDP Classifier.
The taxonomy of the OTUs was assigned against the RDP database using UCLUST tool (Edgar,
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2010) in QIIME (ver 1.8.0) pipeline (Caporaso et al., 2010). The sequences obtained in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the bioproject accession number (PRJNA579001).