Project Overview

Summary:

Overview

Dr. Antonietta Quigg at Texas A&M University at Galveston was awarded an RFP-IV grant at $7,245,432.00 to lead the GoMRI Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers (ADDOMEx) Consortium which consisted of 6 collaborative institutions and 120 research team members (including students). The goals of ADDOMEx were to understand how the presence of hydrocarbons triggers production of exopolymeric substances or extracellular polymeric substances (EPS) that may protect organisms from the oil, emulsify the oil, or both, therefore altering its degradation, as well as to develop a process-based understanding of the role that EPS, micro-gels, and transparent exopolymer particles (TEP) play in the fate of oil (e.g., degradation, dispersion, or sedimentation) and, in addition, how the dispersant Corexit affects these processes.

Outreach

Over its award period (3 years, plus a 12-month no-cost extension), ADDOMEx organized approximately 86 outreach activities or products, including:

• Booth/display set up in the Sea Life Center at Texas A&M University at Galveston with hands-on activities for K-99 which has ~1200 visitors per year each year.

• Teacher training activities/workshops/community events across the country – topics include oil spills, marine snow, ocean circulation, phytoplankton and primary productivity, role of dispersants, and more

• Talks for K-99 audiences across the country including but not limited to federal and state agencies, non-profits, first responders, and many others

Research Highlights

As of January 31, 2019, ADDOMEx research, which entailed 3 research cruises/expeditions, resulted in 25 peer-reviewed publications, more than 85 scientific presentations and 74 datasets being submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are/will be available to the public. ADDOMEx engaged 14 Masters and PhD students over its award period. Significant outcomes of ADDOMEx research according to GoMRI Research Theme are highlighted below. ADDOMEx set out to test the following central hypothesis: Microbes (bacteria, phytoplankton) respond to oil and dispersant (Corexit), by producing exopolymeric substances (EPS) which interact with minerals, organic particles and organisms, and therewith determine the fate, distribution and potential effects of these hydrocarbon pollutants.

EPS which serves as a major source of dissolved organic carbon in the ocean are composed mainly of polysaccharides and proteins (Quigg et al. 2016; Santschi 2018). Despite its ubiquity, very little was known about the mechanism by which EPS, microgels, and transparent exopolymer particles (TEP) influence the fate of oil (e.g., degradation, emulsification, dispersion or sedimentation) and, in addition, how the dispersant Corexit affected these processes prior to the start of ADDOMEx. In order to do so, the research covered areas o the following themes:

Theme Two:

The vertical transport of sinking marine oil snow (MOS) during the DwH oil spill contributed appreciably to the unexpected, and exceptional accumulation of oil on the seafloor (Passow and Ziervogel 2016; Overton et al. 2016). It is now well established (see review of Passow and Hetland, 2016) that sedimentation of oil following the Deepwater Horizon (DwH) oil spill occurred largely in association with marine oil snow (MOS), a term that became accepted as describing marine snow that incorporates oil (Passow and Ziervogel 2016). A significant amount of the spilled oil made its way to the seafloor as MOS, appreciably affecting the distribution of oil within the ocean (Overton et al. 2016; Xu et al. 2018a,b).

MOS consists of a microbially colonized matrix of extracellular polymeric substances (EPS), to which cells, and both ionic (e.g., trace nutrients, Ca2+) and non-ionic (e.g., toxic oil) substances can attach. The secretion of EPS is one of the microbial defense strategies against harmful or stressful environmental situations. Bretherton et al. (2018) found that phytoplankton were either robust such that they were largely unaffected by any of the treatments (Synechococcus elongatus, Dunaliella tertiolecta, Phaeodactylum tricornutum and Navicula sp., and Skeletonema grethae CCMP775) or sensitive (reduced growth rates or increased lag time in response to oil and/or dispersant exposure). Kamalanathan et al. (2018b) revealed a robust multivariate model that can be used to identify phytoplankton exposed to oil with dispersant in field conditions. Collectively these studies have significant implications on how oil spills might impact phytoplankton community structure and bloom dynamics in the Gulf of Mexico; and future abilities to detect them rapidly.

Doyle et al. (2018) observed the formation of micron-scale aggregates of microbial cells around droplets of oil and dispersant and found that their rate of formation was directly related to the concentration of oil within the water column; these micro-aggregates may be precursors to the formation of MOS. Corexit significantly enhanced their formation compared to WAF – with further work to determine if this was because of higher oil concentrations (indirect effect) or because the of the Corexit proper. Microbial communities in marine surface waters respond much more rapidly than previously measured, with major shifts in community composition occurring within only a few hours. In WAF, this manifested as an increase in community diversity due to the outgrowth of several putative aliphatic- and aromatic-hydrocarbon degrading genera, including phytoplankton-associated taxa. In contrast, CEWAF microbial community diversity was reduced. Kamalanathan et al. (2018a) found higher bacterial extracellular enzyme activities, EPS production, bacterial and micro-aggregate counts in CEWAF compared to controls. Order Alteromonadaceae (most abundant bacterial amplicons) have alkaline phosphatase and leucine aminopeptidase; Alteromonadaceae and Pseudomonadaceae more lipase; while Piscirickettsiaceae has greater leucine aminopeptidase. Bacosa et al. (2018) isolated 100 strains, nine which produced remarkable amounts of EPS. Members of Alteromonas, Thalassospira, Aestuariibacter and Escherichia preferably degraded alkanes over polycyclic aromatic hydrocarbons. Our results reveal that Alteromonas and Thalassospira, among the commonly reported bacteria following the DwH spill, produce protein rich EPS that could have crucial roles in oil degradation and marine snow formation. These studies highlighted the link between EPS production and bacterial oil-degrading capacity that should not be overlooked during spilled oil clearance.

The chemicals effect the biology; the biology effects the chemistry. Wirth et al. (2018) incubated phytoplankton in roller tanks in the presence or absence of Macondo crude oil and Corexit and examined the partitioning of n-alkanes and polyaromatic hydrocarbon (PAH)s between MOS (> 1 mm), the dissolved phase and particles (<1 mm). Oil incorporation into MOS depended largely on the physiochemical properties of the respective oil compounds: insoluble compounds (n-alkanes and high molecular weight (HMW) PAHs), were integrated into MOS within entire oil droplets that were scavenged by phytoplankton while the water-soluble fraction (low molecular weight PAHs) was sorbed by cells in MOS (Wirth et al. 2018). Schwehr et al. (2018) investigated mechanisms governing the self-assembly and phase separation for protein- polysaccharide-oil-dispersant interactions. Treatments with oil and/or Corexit had EPS with enhanced protein:polysaccharide carbon-based ratios and lower surface tension (SFT), suggesting the effective bioemulsifying effects of proteins. Results from Schwehr et al. (2018) suggest that EPS are more efficient than Corexit at forming micelles which lead to the formation of emulsions and/or aggregates.

However, the role of the dispersant Corexit in mediating oil-sedimentation is still controversial. Using a series of roller table and mesocosm experiments, we investigated this phenomenon. Passow et al. (2017) demonstrated that the formation of diatom MOS in roller table experiments is enhanced by chemically undispersed oil, but inhibited by Corexit-dispersed oil. Chiu et al. (2017) reported that Corexit alone can significantly inhibit assembly of dissolved organic matter (DOM) microgel formation and reduce the stability of pre-existing microgels while chemically enhanced water accommodated fraction (CEWAF) could effectively facilitate DOM microgel formation. Nevertheless, the sedimentation rate of oil may at times be enhanced by Corexit application, because of an elevated oil content per aggregate when Corexit is used (Passow et al. 2017). Wirth et al. (2018) also found that Corexit significantly increased the amount of oil trapped in MOS, and caused MOS to be enriched in HMW oil compounds, of which many act as toxins. This was also consistent with findings by Xu et al (2018a,b) who, based on radiocarbon and ¹³C NMR results from mes0cosms, showed that the presence of Corexit enhanced the amount of petro-carbon being incorporated into the sinking oil-carrying aggregates (MOS). Xu et al. (2018a) found most of the chemically-dispersed oil preferentially partitioned into the colloidal and suspended particulate fractions rather than into the rapidly forming MOS. Thus the oil and non-petro-carbon sedimentation efficiency in treatments with a dispersant was much lower, compared to those in the Control and water accommodated fraction (WAF) treatments (Xu et al. 2018a).

Corexit promoted the association of oil and proteins, assisting to emulsify the oil in colloids and suspended particulate matter and delay the settling of EPS and MOS (Xu et al. 2018b). Formation of MOS and its subsequent sinking sequestered the oil in two stages: first via terrestrial-derived detritus containing humic compounds, and subsequently via freshly produced material, such as EPS produced by phytoplankton and bacteria (Xu et al. 2018). Quantitative solid-state ¹³C NMR readily distinguishes this oil from naturally formed marine snow and reveals that adding the dispersant Corexit enhances the amount of oil associated with the MOS, thus contributing to rapid removal from the water column (Hatcher et al (2018). In addition, results reveal that the oil associated with EPS is subjected to rapid transformation, in a matter of days, presumably by bacteria and fungi associated with EPS (Hatcher et al (2018). Microbially-mediated EPS are the key component that anchors the mineral ballast until the aggregates become dense enough and overcome the buoyancy added to the aggregates as a result of their association with oil/Corexit (Xu et al. 2018b). The interactions between Corexit and EPS components regulate petroleum hydrocarbon distribution between the water column and sinking MOS (Xu et al. 2018b). A conceptual framework developed by Passow et al. (2017) was used to explain the seemingly contradictory effects of Corexit application on the sedimentation of oil and marine particles.

Theme Three:

To test the effects of sunlight aggregation can occur in the marine environment, we conducted irradiation experiments on a well-characterized protein-containing EPS from the bacterium Sagitulla stellata. Our results show that after 1 h sunlight irradiation, the EPS was 60% higher (larger size particles with greater mass) than in the dark control and that reactive oxygen species hydroxyl radical and peroxide played critical roles in the photo-oxidation process (Sun et al. 2017). A non-protein containing phytoplankton EPS behaved similarly in the dark and light in terms of aggregation (Sun et al. 2017). Both high protein-to-carbohydrate ratio and high protein content of S. stellata promoted aggregation (Sun et al. 2017). We found that in the presence of oil as a WAF, natural sunlight stimulated polysaccharide secretion, coinciding with increased reactive oxygen species production and the formation of larger sized aggregates (>10 μm) (Sun et al.2018).

Changes to the microbial community in bioassays in which oil treatments were amended with the nutrients nitrate (N) and phosphate (P) showed that heterotrophic abundance was increased by oil regardless of nutrient concentrations while autotrophic abundance was inhibited by oil, but this reaction was less severe when nutrient concentrations were higher (Williams et al. 2016). Several PAH compounds were reduced in nutrient amended treatments relative to controls suggesting nutrient enhanced microbial PAH processing (see also Wirth et al. 2018 findings above). These findings provide a first-look at nutrient limitation during microbial oil processing (Williams et al. 2016). After this study, complex relationships among functionally different microbial groups were performed in either a WAF of oil or a chemically enhanced (CEWAF made by adding Corexit to WAF, 1:20) using a new method to produce large volumes of these mixtures (Wade et al. 2017).

 Proposal Abstract - ADDOMEx-1