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The operational conditions used in this study are summarized in Table 1. The composition of the medium was as follows (L−1, the values for x were adjusted at each operation phase; see Table 1): x g MnCl2·4H2O, x g (NH4)2SO4, x g KNO3, 0.04 g KH2PO4, 1.33 g CaCl2·4H2O, 3.4 g MgSO4·7H2O, 4.18 g MgCl2·6H2O, 0.33 g KCl, 24 g NaCl, 0.084 g NaHCO3, 1 mL trace mineral element solution, and 1 mL vitamin solution. The trace mineral element solution contained the following (L−1): 2.5 g EDTA·2Na, 5 g MnSO4·2H2O, 5 g CoSO4·7H2O, 1.8 g ZnSO4·7H2O, 0.1 g CuSO4·5H2O, 0.2 g KAl(SO4)2·12H2O, 0.1 g H3BO3, 0.01 g Na2MoO4·2H2O, 0.1 g SrCl2·6H2O, 0.1 g NaBr, 0.1 g KI, 1 g FeCl2·4H2O, 0.25 g NiCl2·6H2O, 0.0017 g Na2SeO3, and 0.0033 g Na2WO4·2H2O. The vitamin solution was composed of the following vitamins (L−1): 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine hydrochloride, 5 mg thiamine hydrochloride, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg dl-calcium pantothenate, 0.1 mg vitamin B12, 5 mg p-aminobenzoic acid, and 5 mg lipoic acid. The salinity of the medium showed 3.55%, which is the same level of that of natural seawater. Although we did not measure the salinity of the original sediment samples, the salinity level is probably very similar to that of natural seawater. The pH of the medium was adjusted to 7 or 7.5. A total of 10 L of medium was made at a time. The 10 L medium bottle was stored at 15 °C in the sample incubation chamber. The influent medium was supplied into the reactor column from the top inlet port by a peristaltic pump (Masterflex L/S tubing pump 7550-50, Cole-Parmer, Vernon Hills, IL, USA). The medium then flowed down, passing through the sponge cubes by gravity, and was finally pumped out of the PVC column. The sponge cubes were not submerged, and hung freely in the atmosphere. Air (for reactor A) or a gas mixture of air and CH4 (final 2% or 4% of CH4, for reactor M) was packed into an aluminum bag (AAK-10, ASONE, Osaka, Japan) and supplied to the lower part of the bioreactor column via a peristaltic pump. The medium and gases were supplied intermittently at 1 min/9 min (on/off) regulated by an automatic on/off timer (FT-011, Tokyo Glass Kikai Co. Ltd, Tokyo, Japan) connected to the peristaltic pump. The HRT and GRT were changed by altering the flow rate of the peristaltic pump. The bioreactors were operated under atmospheric pressure.

Table 1

Operational conditions for the DHS reactors

PhaseOperation time (days)Interval time (days)Mn2+ (μM)NH4+ (μM)NO3 (μM)pHCH4 (%)HRTa (hrs)GRTb (hrs)
Operation with NH4+ (reactor A) 
 1 0–77 77 118 363 – – 4.5 192 
 2 78–161 83 118 363 – – 4.5 192 
 3 162–168 118 363 – – 2.3 96 
 4 169–258 89 118 182 – – 2.3 96 
 5 259–370 111 118 182 – 7.5 – 2.3 96 
 6 371–532 161 27 182 – 7.5 – 2.3 96 
 7 533–636 103 27 182 – 7.5 – 4.5 96 
Operation with CH4 (reactor M) 
 1 0–71 71 118 363 297 4.5 22.5 
 2 72–196 124 118 36 297 4.5 22.5 
 3 197–210 13 118 36 297 4.5 11.3 
 4 211–231 20 118 36 297 4.5 11.3 
 5 232–258 26 118 36 4.5 11.3 
 6 259–341 82 118 36 7.5 4.5 11.3 
 7 342–370 28 118 36 7.5 4.5 5.6 
 8 371–559 188 27 36 297 7.5 4.5 2.8 
 9 560–587 27 27 297 7.5 4.5 2.8 
 10 588–746 158 27 36 297 7.5 4.5 2.8 
PhaseOperation time (days)Interval time (days)Mn2+ (μM)NH4+ (μM)NO3 (μM)pHCH4 (%)HRTa (hrs)GRTb (hrs)
Operation with NH4+ (reactor A) 
 1 0–77 77 118 363 – – 4.5 192 
 2 78–161 83 118 363 – – 4.5 192 
 3 162–168 118 363 – – 2.3 96 
 4 169–258 89 118 182 – – 2.3 96 
 5 259–370 111 118 182 – 7.5 – 2.3 96 
 6 371–532 161 27 182 – 7.5 – 2.3 96 
 7 533–636 103 27 182 – 7.5 – 4.5 96 
Operation with CH4 (reactor M) 
 1 0–71 71 118 363 297 4.5 22.5 
 2 72–196 124 118 36 297 4.5 22.5 
 3 197–210 13 118 36 297 4.5 11.3 
 4 211–231 20 118 36 297 4.5 11.3 
 5 232–258 26 118 36 4.5 11.3 
 6 259–341 82 118 36 7.5 4.5 11.3 
 7 342–370 28 118 36 7.5 4.5 5.6 
 8 371–559 188 27 36 297 7.5 4.5 2.8 
 9 560–587 27 27 297 7.5 4.5 2.8 
 10 588–746 158 27 36 297 7.5 4.5 2.8 

aHydraulic retention time.

bGas retention time.

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