OEX_16S_V3-V4

The compositions of artificial seawater medium are as follow: 30 g∙L-1 of NaCl, 0.14 g∙L-1 of K2HPO4, 0.14 g∙L-1 of CaCl2·2H2O, 0.25 g∙L-1 of NHCl, 0.25g∙L-1 of NaNO3, 4.18 g∙L-1 of MgCl2·6H2O, 0.33 g∙L-1 of KCl, 0.5g∙L-1 of MgSO4·7H2O. 1 g∙L-1 of Kraft lignin (471003-500G, Lot#MKCK3344, SIGMA-ALDRICH, St.Louis, USA) was added into the artificial seawater medium as the sole carbon and energy source. 1 g∙L-1 of resazurin as oxygen indicator was added into the medium. Before autoclaving, the mixed medium was sparged with N2 for 15 min to crowd out O2 and subsequently sealed with a butyl rubber stopper and plastic cap. After autoclaving, trace elements (SL-10 in DSMZ medium 320) and vitamins (DSMZ medium 141) were added to the medium. The final pH was 7.6. Around 1 % of original enrichment enriched with plant detritus in the deep sea of the South China Sea was added into the medium amended with lignin and incubated under the conditions of no light, 4 °C and 0.2 MPa. After cultivation for three months (label this incubation as G0), 1 ml of incubation liquid was added into two new vials of medium amended with lignin and incubated under the same conditions. After six months and one year, 10 ml liquid was absorbed from the incubations and immediately filtered with a 0.22-μm pore size polycarbonate membrane (Millipore, USA). Here, we labeled the incubated cultures for six months and one year as G1 and G2, respectively.