Laboratory studies to assess the oil degradation potential of endogenous micro-organisms from tropical mangrove and salt marsh habitats in north Queensland

Hydrocarbon-degrading micro-organisms were obtained from mangrove sediments adjacent to Cocoa Creek on Cape Cleveland, Queensland. The area is a National Park with no known history of oil contamination. Separate sediment samples were collected from among the roots of the mangroves, Rhizophora stylosa and Avicennia marina, and the Holosarcia salt marsh. In the laboratory, sediments were added to Bushnell-Haas (BH) growth medium (Difco) and Gippsland Crude oil was added to stimulate the growth of hydrocarbon degraders. The flasks were incubated for 72 hours and the concentration of bacteria in the flasks containing the three sediment types, were sampled and the total numbers of bacteria estimated using a Neubauer haemocytometer. In the first series of flask experiments a medium range, preweathered crude oil (Gippsland Crude from the Bass Strait Basin) and micro-organisms isolated from Rhizophora mangrove sediments were used. To test whether mangrove pore water affected the rate of degradation, the oil and micro-organisms were incubated in BH growth medium made up with pore water instead of seawater. A chemical method of adding oxygen was tested by adding (MgO2) to flasks containing oil and micro-organisms in BH growth medium made up with seawater. A series of controls were also run.The relative efficiency of the micro-organisms isolated from the tropical mangrove and salt marsh habitats, was tested using preweathered Gippsland Crude, Arabian Light Crude and Bunker C oils. Flask experiments were conducted with nitrogen bubbled controls and time zero controls. The following analyses were conducted: Total Extractable Organic Matter (EOM) determined gravimetrically; Total oil determined by UV Fluorescence analysis (UVF); Total Hydrocarbons determined by gas chromatography with flame ionisation detection (GC-FID); The percentage of unresolved hydrocarbons (% UCM); Concentrations of individual isoprenoid and n-alkanes (µg/g dry weight); Ratios of isoprenoid to n-alkanes as biodegradation indices; Quantification of 218 individual aromatic and alkyl substituted aromatic hydrocarbons (PAH) over the biphenyl/naphthalene, fluorene, dibenzothiophene, phenanthrene/anthracene, benzanthracene/chrysene and pyrene through benzopyrene series determined by selected ion monitoring-gas chromatography/mass spectroscopy (SIM-GC/MS); Ratios of specific alkyl phenanthrene isomers to illustrate selective biodegradation; Sum of triterpine biomarkers in the hopane series as determined by SIM-GC/MS using m/z 191; selected sterane and potential demethylated hopane biomarkers as determined by SIM-GC/MS using m/z 217 and m/z 177; and Ratios of specific biomarkers useful as biodegradation indices.
A series of experiments were designed to provide information to assist in the formulation of a bioremediation strategy to treat oiled sediments in mangrove and salt marsh habitats in Queensland. The possible presence of inhibitors in pore water and low levels of molecular oxygen have been suggested as potentially limiting factors in oil degradation in mangrove mud.The first experiments tested the effects of mangrove pore water on degradation rates and also compared the degradation of oil under oxic and anoxic conditions, in the presence of micro-organisms isolated from mangrove sediments. Another set of experiments were designed to determine the rate at which hydrocarbon-degrading microorganisms from different intertidal habitats were able to degrade oil from different sources.

本研究的烃降解微生物（hydrocarbon-degrading microorganisms）分离自澳大利亚昆士兰州克利夫兰角可可溪附近的红树林沉积物。该区域为国家公园，无已知石油污染历史。研究人员分别从秋茄（Rhizophora stylosa）、白骨壤（Avicennia marina）两种红树林的根系周围，以及Holosarcia盐沼中采集沉积物样本。
实验室阶段，将沉积物添加至Bushnell-Haas（BH）培养基（Difco品牌）中，并添加吉普斯兰原油（Gippsland Crude oil）以刺激烃降解微生物生长。将三角烧瓶置于恒温培养箱中孵育72小时，随后对三种沉积物对应的烧瓶内的细菌浓度进行采样，并通过纽鲍尔血细胞计数板（Neubauer haemocytometer）估算细菌总数量。
第一组烧瓶实验采用中范围预风化原油（巴斯海峡盆地产出的吉普斯兰原油），以及从秋茄红树林沉积物中分离得到的微生物。为验证红树林孔隙水对降解速率的影响，研究人员将原油与微生物置于以孔隙水替代海水配制的BH培养基中进行孵育。同时，通过向以海水配制的BH培养基中添加过氧化镁（MgO₂）的方式，测试化学补氧方法的效果。此外，本研究还设置了一系列对照组。
为对比从热带红树林与盐沼生境中分离得到的微生物的相对降解效率，研究人员采用预风化吉普斯兰原油、阿拉伯轻质原油（Arabian Light Crude）及C重油（Bunker C oil）开展实验。实验设置了氮气吹扫对照组与零时刻对照组。
本研究开展了以下分析：
1. 采用重量法测定总可萃取有机物（Total Extractable Organic Matter, EOM）；
2. 通过紫外荧光分析法（UV Fluorescence analysis, UVF）测定总油含量；
3. 采用气相色谱-火焰离子化检测法（gas chromatography with flame ionisation detection, GC-FID）测定总烃含量；
4. 计算未分辨烃类占比（% Unresolved Complex Mixture, % UCM）；
5. 测定单个类异戊二烯与正构烷烃的浓度（单位：µg/g干重）；
6. 以类异戊二烯与正构烷烃的比值作为生物降解指数；
7. 通过选择离子监测-气相色谱/质谱联用法（selected ion monitoring-gas chromatography/mass spectroscopy, SIM-GC/MS），对联苯/萘系列、芴系列、二苯并噻吩系列、菲/蒽系列、苯并蒽/䓛（chrysene）系列及芘至苯并芘系列中的218种单个芳香烃及烷基取代芳香烃（多环芳烃Polycyclic Aromatic Hydrocarbons, PAH）进行定量分析；
8. 以特定烷基菲异构体的比值表征选择性生物降解过程；
9. 采用m/z 191选择离子监测模式，通过SIM-GC/MS定量分析藿烷系列（hopane series）中的三萜类生物标志物总量；
10. 采用m/z 217与m/z 177选择离子监测模式，通过SIM-GC/MS测定特定甾烷（sterane）及可能的脱甲基藿烷（demethylated hopane）生物标志物；
11. 以特定生物标志物的比值作为生物降解指数。
本研究设计了一系列实验，旨在为制定昆士兰州红树林与盐沼生境石油污染沉积物的生物修复策略（bioremediation strategy）提供数据支持。此前有研究指出，孔隙水中可能存在的抑制剂与低浓度分子氧，是红树林沉积物中石油降解的潜在限制因素。
首批实验验证了红树林孔隙水对降解速率的影响，并对比了从红树林沉积物中分离得到的微生物在有氧与无氧条件下的原油降解效果。另一组实验则旨在明确不同潮间带生境（intertidal habitats）来源的烃降解微生物，对不同来源原油的降解速率。