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

Brief description

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.

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Maintenance and Update Frequency: notPlanned

Statement: Statement: Bacterial culture:Sediments (10 g) were added to 100 ml of Bushnell-Haas (BH) medium (Difco) made with fresh seawater in 250 ml Erlenmeyer flasks. Bushnell-Haas media has been specifically designed to provide the nutrients necessary for microbial decomposition of oil (Brown and Braddock, 1990; Swannell et al., 1995) Gippsland Crude oil was added (1.0 g) to the media to stimulate the growth of hydrocarbon degraders. The flasks were incubated for 72 hours. The presence of hydrocarbon degraders was confirmed by increases in turbidity, by microbial colonisation of oil droplets and by microscopic examination of the dominant micro-organisms present. 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.Brown EJ and Braddock JF (1990) Sheen screen: a miniaturized most probable number for enumeration of oil-degrading micro-organisms. App. Environ. Microbiol. 56: 3895-3896.Swannell RPJ, Croft BC, Grant AL and Lee K (1995) Evaluation of bioremediation agents in beach microcosms. Spill Science and Technology Bulletin 2(2/3): 151-154.Preweathering of oil:The crude oils used in experiments (Gippsland Crude, Arabian Light Crude and Bunker C) were pre-weathered by addition of a known amount to an aluminium dish that was left in a fume hood for 48 hours. This procedure was followed to allow some of the most volatile components to escape as would happen in an oil spill. Weathered oil was mixed and 0.5 g added to each flask using a glass pipette. All the flasks were placed on an orbital shaker table set at 85 rpm. The shaker was located in a darkened room where temperature was a constant 25 ± 1°C over the 14 days of the incubations. This temperature could be expected to represent a median between night and day temperatures over much of year in tropical Queensland. Daily visual checks were made for temperature, gas flows and fluid volumes. After 7 days, Super-Q water was added to flasks to restore their initial volumes. Each flask was rotated by hand twice a day to resuspend oil which had adhered to the sides of the flasks.Addition of oxygen:The oxygen release compound (ORC) contained magnesium peroxide, which decomposes in water to magnesium hydroxide and oxygen as follows: MgO2 + H2O -> Mg(OH)2 + O2Magnesium peroxide is a white powder, which according to technical bulletins (Regenesis, USA) has been useful for treating oil contaminated ground waters. No trial in marine sediments had been undertaken prior to these experiments.Experimental procedure:The experiments were conducted in sterile 250 ml Erlenmeyer flasks. Each treatment was run in triplicate. Growth media was mixed thoroughly using a magnetic stirrer and 100 ml was added to each flask. Flasks were inoculated with ~ 4 x 10^7 bacterial cells. Compressed air and N2 were bubbled into the media through narrow glass tubing (i.d. 4 mm). The N2 sparged flasks (used to determine evaporative losses and anaerobic degradation) were initially sparged with nitrogen gas for at least 10 min to remove traces of dissolved oxygen. The nitrogen flow rate into the N2 flasks was set to maintain a dissolved oxygen level of less than 0.1 mg/l. A visually equivalent flow rate of air was supplied to the aerated flasks, and this was found to maintain oxygen levels of 4.5 mg/l in media containing an active hydrocarbon-degrading microbial community. Oxygen levels were confirmed using a portable YSI dissolved oxygen meter (Yellow Spring Instruments, USA). The flasks were sealed with plugs of cotton wool pushed firmly around the glass tubes delivering N2 or air. The gases were high purity grade and the gas lines to the flasks were filtered through 0.45 µm in-line filters. The plastic pipes and glass tubes, which supplied air to the flasks, were steam sterilised at 121°C and 15 psi.Oil analysis:The contents of one incubation flask were poured into a 500 ml glass separatory funnel. Fifty ml of distilled dichloromethane (CH2Cl2) were added to the flask to rinse out oil adhered to the flask. The CH2Cl2 rinse was then added to the separatory funnel that was then sealed with a glass stopper and shaken vigorously. After separation of the phases, the CH2Cl2 extract was dried and vacuum-filtered through a 1 cm diameter X 10 cm high glass column filled with 5 cm of precombusted Na2SO4 into a pre-weighed Erlenmeyer flask. The procedure was repeated with a second aliquot of 25 ml CH2Cl2 which was combined with the first extraction. Samples from aeration treatments formed an emulsion, which did not easily separate. These solvent emulsions were poured into 250 ml polypropylene bottles and centrifuged until the phases separated. The mixture was returned to the separatory funnel and the CH2Cl2 was then drained. Each sample extract was weighed to determine the exact volume of CH2Cl2 using a density of 1.327 g/ml. The weight of extractable organic matter (EOM) recovered in each sample was determined by gentle evaporation of 10 µl aliquots of the extracts onto the pan of an electronic microbalance, which reads to 0.1 µg. Twenty ml of each extract were transferred to a glass vial, sealed with an aluminium foil lined screw cap, and stored in the refrigerator until sub-sampled for analysis by methods described in detail by UNEP/IOC/IAEA (1992) and briefly described here. Ultraviolet fluorescence (UVF) analysis was done using a Hitachi F-4000 scanning fluorescence spectrophotometer. The Arabian Light Crude and the Bunker C oils gave maximum fluorescence at 310 nm excitation and 360 nm emission wavelengths. The Gippsland Crude had its maximum at 280 nm excitation, 330 nm emission. Calibration curves were constructed using the prepared (To) oils dissolved in hexane. Samples were measured under the same conditions as the calibration standard oils and were diluted to provide readings within the linear calibrated range for each oil (0 to ~10 µg/ml). Synchronous excitation / emission spectra were taken of a hexane dilution of each sample to characterise the types of aromatic hydrocarbons contained in each sample. Since only the aromatics fluoresce, and not the alkanes, this technique provides an estimate of the fate of the aromatic components of the oil as an average, non compound-specific mixture. For each extract, a concentration of ~ 3 µg/µl (based on EOM) was analysed by gas chromatography. One-n-eicosene (C20:1) was used as an internal standard. The Fisons gas chromatograph was equipped with on-column injector, autosampler and flame ionisation detector. The column used was 30 m DB-5 fused silica (J and W Scientific). High purity nitrogen was used as carrier gas and detector make-up gas. The oven program was as follows: 50°C for 1 min; then 50° to 100°C at 6°/min, 100° to 300° at 4°/min, then hold for 13 minutes. This facilitated baseline resolution of pristane/C17 and phytane/C18 peaks for most samples. Individual alkanes were quantified using response factors generated from external alkane standards over the C10 to C36 elution range. Peak areas were integrated using Fisons Chromcard software. Unresolved hydrocarbons (UCM) were calculated as the total oil signal over specific elution windows minus the amount of total resolved hydrocarbons determined by peak integration and an average response factor.For Selected Ion Monitoring Gas Chromatography Mass Spectrometry (SIM GC/MS), a concentration of 1.0 µg/µl of each extract was analysed from the second set of experiments on an Hewlett Packard 6890 Series GC/MS equipped with a DB5-MS column (J and W Scientific). Three deuterated aromatic hydrocarbons (D10-biphenyl, D10-phenanthrene, and D12-perylene) were added as internal standards. The GC/MS oven program was similar to the program used for GC/FID analysis. The SIM program quantified 218 individual aromatic and alkyl substituted aromatic hydrocarbons (PAH) over the biphenyl/naphthalene, fluorene, dibenzothiophene, phenanthrene/anthracene, benzanthracene/chrysene and pyrene through benzopyrene series. The triterpane and sterane biomarkers were monitored using the m/z 191 and 217 ions plus appropriate parent ions. The areas of the 8 most predominant peaks in the hopane biomarker series common in each oil, were used to normalize the concentrations of individual aromatic hydrocarbons determined in the same GC/MS analysis. These were Ts or C27 trisnorneohopane, Tm or C27 trisnorhopane, C29 H norhopane, C30H hopane, and the C31 and C32 R and S hopanes. Burns and Codi (1998) showed this normalization method to be useful in reducing analytical variation in oil degradation studies.Burns KA and Codi S (1998) Contrasting impacts of localised versus catastrophic oil spills in mangrove sediments. Mangroves and Salt Marshes 2: 63-74.UNEP/IOC/IAEA (1992). Determination of hydrocarbons in sediments. Reference Methods for Marine Pollution Monitoring No. 20. (Prepared by K.A. Burns). International Laboratory of Marine Radioactivity, Monaco MC98000. 97 pp.

Notes

Credit
Burns, Kathryn A, Dr (Principal Investigator)