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.

烃降解微生物（hydrocarbon-degrading micro-organisms）采集自澳大利亚昆士兰州克利夫兰角可可溪毗邻的红树林沉积物中。该区域为国家公园，暂无已知石油污染历史。研究人员分别从红海榄（Rhizophora stylosa）、白骨壤（Avicennia marina）的红树林根系周边以及Holosarcia盐沼中采集了沉积物样本。
实验室内，将沉积物添加至Bushnell-Haas（BH）培养基（Bushnell-Haas (BH) growth medium，Difco品牌）中，并加入吉普斯兰原油（Gippsland Crude oil）以刺激烃降解微生物的生长。将三角烧瓶孵育72小时后，对搭载三种沉积物样本的烧瓶中的细菌浓度进行取样，并采用纽鲍尔血细胞计数板（Neubauer haemocytometer）估算细菌总数量。
首轮三角烧瓶实验采用中量程预风化原油（巴斯海峡盆地来源的吉普斯兰原油，Bass Strait Basin）以及从红海榄红树林沉积物中分离得到的微生物开展。为验证红树林孔隙水是否会影响降解速率，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，研究人员测试了一种化学补氧方法：向以海水配制的BH培养基中搭载原油与微生物的三角烧瓶中添加过氧化镁（MgO₂），并同步设置了一系列对照组实验。
针对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能，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）与C级燃料油（Bunker C oil）开展了测试。实验设置了氮气鼓泡对照组与零时刻对照组。
本研究开展了以下分析：1. 总可提取有机物（Total Extractable Organic Matter, EOM）采用重量法测定；2. 总油量采用紫外荧光分析法（UV Fluorescence analysis, UVF）测定；3. 总烃采用气相色谱-火焰离子化检测法（gas chromatography with flame ionisation detection, GC-FID）测定；4. 未分辨烃类占比（% UCM）；5. 单体类异戊二烯烃与正构烷烃的浓度（单位：µg/g 干重）；6. 以类异戊二烯烃与正构烷烃的比值作为生物降解指数；7. 采用选择离子监测-气相色谱/质谱联用法（selected ion monitoring-gas chromatography/mass spectroscopy, SIM-GC/MS），对联苯/萘、芴、二苯并噻吩、菲/蒽、苯并蒽/屈以及芘至苯并芘系列中的218种单体芳香烃与烷基取代芳香烃（多环芳烃，PAH）进行定量分析；8. 采用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SIM-GC/MS（监测质荷比m/z 191）测定藿烷系列中三萜类生物标志物的总含量；10. 采用SIM-GC/MS（分别监测质荷比m/z 217与m/z 177）测定甾烷与潜在脱甲基藿烷类生物标志物的含量；11. 采用特定生物标志物的比值作为生物降解指数。
本系列实验旨在为制定针对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提供数据支撑。此前有研究指出，红树林沉积物孔隙水中可能存在抑制剂，且分子氧浓度偏低，这二者均可能成为石油降解过程的限制因素。首轮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情况。另一组实验则旨在明确不同潮间带生境来源的烃降解微生物对不同来源原油的降解速率。