Aromatic Compounds

WEATHERING OF OIL AND

GAS IN THE COASTAL MARINE ENVIRONMENT: QUANTIFYING RATES OF MICROBIAL METABOLISM

Principal Investigator: David Valentine (UCSB)

Summary of Research

Large quantities of oil and gas are released into the Santa Barbara Channel by way of natural seepage with lesser amounts emitted during petroleum removal and recovery. These emissions greatly affect beach, air and water quality along the Southern California Coast. As a result, many studies and a substantial amount of resources have been devoted to developing a better understanding of the weathering processes occurring in hydrocarbon-rich environments. These studies have provided evidence for natural hydrocarbon-consuming communities thriving in heavily contaminated regions. It is assumed that native assemblages of microorganisms having the capability to consume a variety of hydrocarbons emitted from natural oil seeps are present in the Santa Barbara channel. Although microbial oxidation is known to occur, little is known about the distribution of relevant microbial communities, rates of oxidation and the extent to which various hydrocarbons are broken down or consumed.

This MMS-UC CMI funded research focuses on the microbial weathering of aromatic compounds released into marine environments. The objectives of this research include: (1) determing the intermediates and end products arising from microbial decomposition of these most persistant and harmful hydrocarbons and (2) the development of techniques to quantify rates for microbial consumption and decomposition of aromatic and polycyclic aromatic compounds in marine environments. Specifically, we intend to assay the weathering patterns and microbial activity in anoxic, sulfidic sediments. The general approach is to incubate hydrocarbon-contaminated seep sediments under controlled conditions while assaying CO₂/H₂S/CH₄ production, sulfate consumption, and changes in petroleum composition.

We are using two distinct approaches to study the intermediates and end products of hydrocarbon weathering. The first approach is the use of radio-labeled substrates to assess major products of microbial metabolism. The second technique involves chromatographic technology designed to completely resolve the undefined complex mixture typical of weathered petroleum, so-called two-dimensional gas chromatography. We are collaborating with Chris Reddy at Woods Hole Oceanographic Institution, who has helped develop the GC×GC technology.

We have also performed our first experiments designed to assess the rates of hydrocarbon weathering. One experiment involved collecting a time series of surface slick samples at Shane Seep, using the slick sampler developed by other MMS-funded scientists (Liefer et al). Samples were collected at the seep and ‘down-slick’ and represent a rough time series.

A second experiment collected 5 L of sediment from anoxic regions of Shane’s seep to be used in a long-term incubation experiment. The sediment was transported back to the lab, while being kept under environmental conditions.

Analytical procedures:

Gases (CO₂/CH₄/H₂S) are quantified in the headspace using an agilent Micro GC/TCD at UCSB shown in Figure 1. TCO2 in the aqueous phase will also be quantified using the agilent Micro GC/TCD. Aqueous sulfur (~ sulfate) is quantified by spectrophotometry and ICP-OES from the liquid phase overlying the sediment at UCSB. Organic Acids are to be measured by HPLC at UCSB. DIC will be measured at UCSB. Hydrocarbon composition will be monitored using GC×GC at WHOI.

Figure 1. Left panel shows an image of the agilent Micro GC/TCD.

Figure 2. Preliminary results are shown in the right panel for CO₂ produced in the experimental, blank and kill control bottles over the first 120 days of the experiment.

Selected Results:

During the first 120 days of incubation kill controls (autoclaved petroleum, sediment and seawater), experimental blanks (sediment and seawater w/out petroleum) and experimental bottles (petroleum, sediment and seawater) were analyzed periodically for various markers/indicators of microbial growth. These experiments have focused on quantifying CO₂ in the head-space of the incubation bottles, determining the δ¹³C of the CO₂ in the head-space and an assay for the production of sulfide. The isotope ratio mass spectrometer used for this experiment, along with our initial results, are shown in Figure 2. Quantifying CO₂, the end-product of petroleum consumption by microbes, and analyzing the δ¹³C of that CO₂ will aid in determining the extent of microbial activity. The production of sulfide should indicate both that conditions are sufficient for microbial growth and the presence of sulfate reducers which has been postulated to be a necessary condition for microbial hydrocarbon oxidation.

Figure 3. Shows an image of the IR-MS used to determine isotopic values for CO₂ produced during the experiment.

Figure 4. Right panel shows initial results for the experimental (E#1), blanks and kill controls during the first 120 days of the experiment.

Analyses performed on the first 4 time series samples show a steady increase in the quantity of the CO₂ in the experimental bottles. Results from experiments using the isotope ratio mass spectrometer (IR-MS) on the CO₂ in the head-space show a decrease in the δ¹³C. Analyses performed on December 18, 2003 show a δ¹³C value of approximately +3‰ whereas values from tests performed on experimental bottles on March 24, 2004 showed a decrease in the δ¹³C to approximately -12.5‰. The δ¹³C value determined in the kill controls has been static at approximately - 10‰. Sulfide assays show an increase in sulfide produced in the experimental bottles from T=0 to T=4. Aqueous sulfide concentrations have increased from below detection to slightly less than 300 µM in the first 120 days of incubation. Sulfide concentrations in the kill controls have remained below detection throughout the experiment.

The surface slick samples collected for the ‘down slick’ time series did not contain enough oil to quantify and will be repeated in the coming year.

Future plans:

We are in the process of analyzing samples from the long-term incubation experiment, designed to broadly assay hydrocarbon weathering patterns, including aromatics. The planned duration of the experiment is 12-18 months. Samples will be sacrificed on a monthly basis and assayed using the methods described above, including the new GC×GC techniques developed at Woods Hole Oceanographic Institution. We have acquired a Spectronic 20 spectrophotometer for use in quantifying aqueous sulfate and to calculate sulfate reduction rates in the experimental bottles. Since microbial activity has been indicated, the first 5 time series will be shipped to WHOI for GC×GC analyses.