Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea@en

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. […]古尔法克斯油田（Gullfaks）是挪威四大主要油气田之一，坐落于北海高原东北缘。汤姆默利滕（Tommeliten）位于北海中部的埃科菲斯克（Ekofisk）大区域内。在HE 208与AL 267科考航次期间，研究人员对北海的多处海底天然气渗漏点位进行了考察。在古尔法克斯海域的海因克渗漏点（Heincke seep），研究人员于2004年5月随HE 208航次采用视频引导式多管采样器（MUC；基尔大学Octopus型）采集了沉积物样品。样品采自一处被细菌席密集覆盖且观测到气体沸腾逸出的区域。粗砂质沉积物将多管采样器的最大下入深度限制为30厘米，而高渗透性砂层因孔隙水流失，无法开展高分辨率垂直分层取样。对汤姆默利滕海域的气体喷溢成像与摄像观测显示，该区域存在气体逸出现象，分布有数块直径小于50厘米的小型细菌席斑块，绝大多数斑块处均有单股气泡流涌出。斑块间距为10至100米。仅可借助搭载于遥控水下机器人（ROV）“切罗基号”的3枚直径3.8厘米的小型推送式岩心管，采集细菌席覆盖下的沉积物样品。上述岩心以3厘米为间隔进行分层取样。从10至17克湿沉积物中提取脂质生物标志物的实验流程，详见此前研究（Elvert等，2003；DOI:10.1080/01490450303894）。简言之，在提取前，研究人员向沉积物样品中分别加入已知碳同位素δ¹³C值的定量胆甾烷（cholestane）、十九烷醇（nonadecanol）和十九烷酸（nonadecanolic acid），作为烃类、醇类和脂肪酸组分的内标物。通过使用极性依次降低的有机溶剂对沉积物进行超声萃取，获得总脂质提取物。采用氢氧化钾甲醇溶液进行皂化反应，将酯化脂肪酸（FAs）从甘油骨架上解离下来。从此混合体系中，使用正己烷萃取得到中性组分。经后续酸化处理后，再次使用正己烷萃取得到游离脂肪酸。为开展分析，采用甲醇中的三氟化硼（BF₃）对脂肪酸进行甲酯化反应，得到脂肪酸甲酯（FAMES）。总细胞计数与催化报道者沉积荧光原位杂交（CARD-FISH）的样品固定工作于采样后即刻在科考船上完成。两种分析方法均采用甲醛溶液对沉积物进行固定。固定两小时后，用于CARD-FISH染色的分样经1×PBS缓冲液（含10mmol/L磷酸钠、130mmol/L氯化钠，pH值调至7.2）洗涤后，最终以1:1体积比的PBS与乙醇混合溶液于-20℃下保存，以待后续处理。[……]