Microbial lipids from a nearshore sediment core from Bowling Green Bay, North Queensland

Sediment samples were collected from 2 sites in Bowling Green Bay, a shallow coastal embayment located between Townsville and the Burdekin River delta in the central Great Barrier Reef province. A sediment core was collected at Station U for use in a depth profile study. The core was collected using a plastic, 51 mm diameter, core tube. The core was returned to the laboratory within 3 hours of collection, subsectioned and transferred to centrifuge tubes for pore water isolation. A surface sediment sample, collected at nearby Station 11 was used for an intact lipid study. The surface sediment (0-1 cm depth) was collected using a frame supported grab sampler and immediately frozen.All sediment samples were freeze dried and ground to < 100 µm in a stainless steel ring mill. Total carbon was measured using a LECO CHN Analyser. Total phosphorus was determined by ICPES after nitric acid/perchloric acid digestion. Pore waters were analysed for ammonium, phosphate, nitrite and nitrate using an Autoanalyser system.The results of pore water analyses indicated that bacterial activity responsible for the mineralization of the nutrients was confined to the upper 15-20 cm of the core. On this basis, samples from five depths were selected for lipid analysis (0-1 cm, 3-4 cm, 7-9 cm, 11-13 cm and for comparison 30-35 cm). Sediment samples (1-5 g dry wt) were extracted (x2) with hexane:isopropanol:water (HIP) 30:20:1.25. An aliquot of the extract was saponified and the resultant fatty acids were isolated and esterified to methyl esters. Lipid phosphate was determined by P analysis of an aliquot of the TSE after combustion and acid hydrolysis.An aliquot of the lipid extract from the surface sediment sample was separated into fractions by silica column chromatography. Four fractions were obtained. Each fraction was evaporated just to dryness under nitrogen and then transesterified. Non-ionic compounds were extracted from the resultant mixture with toluene. The toluene extract was evaporated to a small volume, treated and analysed by capillary GC and GC-MS.
This research was undertaken to:1. examine the results of direct application of the total solvent extract (TSE) fatty acid based method to estimate bacterial biomass in a sediment core from an environment with low bacterial populations and significant relict sediment input2. examine methods which make the lipid phosphate and fatty acid procedures more reliable.
Sedimentary monoenoic fatty acids have been shown to be potentially able to indicate the microbial abundance and community structure in diverse marine sediments. This procedure has been extended to enable detailed examination of bacterial community structure changes in surficial mangrove-associated sediments based solely on total solvent extract (TSE) fatty acid compositions. However, in environments where relict organic material contributes to the extractable fatty acid pool, or where bacterial populations are relatively low, the TSE fatty acids will include free fatty acids derived from dead and for decomposing organic matter. In such circumstances, biomass estimates based on TSE fatty acids can potentially lead to gross overestimations.

研究于大堡礁中部区域、汤斯维尔与伯德金河三角洲之间的浅海海湾鲍林格林湾（Bowling Green Bay）的2处站位采集沉积物样品。在U站位采集沉积物岩芯以开展深度剖面研究，采样采用直径51 mm的塑料岩芯管。岩芯在采集后3小时内被运回实验室，进行分段切割后转移至离心管以分离孔隙水。于邻近的11站位采集表层沉积物样品，用于完整脂质分析。表层沉积物（深度0~1 cm）采用框架式抓斗采样器采集，并立即冷冻保存。
所有沉积物样品均经冷冻干燥处理，并使用不锈钢环磨研磨至粒径小于100 µm。总碳含量采用力可（LECO）碳氢氮分析仪进行测定。总磷含量则经硝酸-高氯酸消解后，通过电感耦合等离子体发射光谱仪（ICPES）进行定量。孔隙水样品采用自动分析仪系统检测铵态氮、磷酸盐、亚硝酸盐及硝酸盐含量。
孔隙水分析结果显示，负责营养盐矿化作用的细菌活性仅局限于岩芯上部15~20 cm区域。基于此，选取5个深度层位的样品开展脂质分析：0~1 cm、3~4 cm、7~9 cm、11~13 cm，以及作为对照的30~35 cm。称取1~5 g干重沉积物样品，采用正己烷:异丙醇:水（体积比30:20:1.25，简称HIP）进行两次萃取。取部分萃取液进行皂化反应，分离所得脂肪酸并酯化生成脂肪酸甲酯。脂质磷含量通过对总溶剂提取物（Total Solvent Extract，TSE）的等分试样进行燃烧及酸水解后的磷元素分析来测定。
取表层沉积物样品的脂质萃取液等分试样，通过硅胶柱色谱法分离为4个组分。将每个组分在氮气吹扫下蒸发至近干，随后进行酯交换反应。向反应混合物中加入甲苯以萃取非离子化合物，将甲苯萃取液浓缩至少量体积后进行前处理，再通过毛细管气相色谱（GC）及气相色谱-质谱联用（GC-MS）进行分析。
本研究旨在达成以下目标：1. 评估基于总溶剂提取物（TSE）脂肪酸的方法，在细菌种群较低且存在显著残余沉积物输入的环境中，直接应用于沉积物岩芯细菌生物量估算的效果；2. 优化使脂质磷与脂肪酸分析流程更可靠的方法。
沉积物中单不饱和脂肪酸已被证实可潜在反映多样海洋沉积物中的微生物丰度与群落结构。该方法已被拓展至仅基于总溶剂提取物（TSE）脂肪酸组成，详细解析与红树林相关的表层沉积物中细菌群落结构的变化。然而，在残余有机质贡献于可萃取脂肪酸库，或细菌种群相对较低的环境中，TSE脂肪酸会包含源自死亡且正在分解的有机质的游离脂肪酸。在此类场景下，基于TSE脂肪酸的生物量估算可能会出现严重的高估问题。