Project Overview

Summary:

In January 2018, Dr. Antonietta Quigg at Texas A&M University at Galveston, was awarded an RFP-VI grant totaling $2,540,646 to lead the GoMRI project entitled “Aggregation and Degradation of Dispersants and Oil by Microbial Exopolymers 2 (ADDOMEx-2): Towards a synthesis of process and pathways of marine oil snow formation” which consisted of 6 collaborative institutions and approximately 40 research team members (including students).

This project was proposed to perform the next steps following research done by ADDOMEx-2 by integrating ADDOMEx-2 derived insights into a comprehensive conceptual model framework. Key experiments generated measurements needed to improve numerical model (in conjunction with FOMOSA and others) which will enhance prediction capabilities in order to guide the decision process of first responders. The primary experimental goal of ADDOMEx-2 was to perform a series of “wrap-up” experiments intended to fill current knowledge gaps. These experiments centered around two main hypotheses:

       i.          Particle formation and fragmentation is governed mainly by stickiness.

      ii.         The fate of oil (chemically undispersed or Corexit dispersed) trapped within marine oil snow (MOS) is dictated by both chemical and microbial oxidation. Both processes lead to rapid (half-life of days) oxidative alteration of the oil. This affects the sinking and dispersion of MOS and the associated oxidized oil.

This project is investigated the detailed steps of water-column processes that lead to sinkable MOS formation and oxidative loss terms for oil through controlled laboratory experiments. Sub-hypotheses addressed the mechanisms of the growth of nano- to micro- to macrogels and their role in dispersing oil, the factors that control MOS sedimentation, and the role of light versus microbially produced ROS in oxidation and crosslinking of MOS aggregates. The project was able to provide input into oil tracking prediction models, that is, realistic measures of (i) sinking velocities, (ii) stickiness, and (iii) disaggregation kinetics of MOS. Towards meeting the goals of a GOMRI legacy, the team has participated in numerous synthesis activities, most notably, the MOSSFA synthesis working group.

Outreach Highlights

As of June 30, 2020, ADDOMEX-2 research team members have participated in more than 20 outreach related activities including: school presentations, invited talks, social media engagement, news articles and more. Here are a few of our key outreach products and activities:

• Over 20 undergraduate students participated in various aspects of the ADDOMEx-2 project resulting in poster presentations and co-authorship on several papers. Of these students, many have continued into MS or PhD programs.
• Numerous presentations were given to K-12 students across all institutions. Sharing this information with the next generation has contributed to broadening public education and engagement as well as training our own teams in science communication.
• The ADDOMEx facebook page continues to be a point for outreach of our work with the world. We continue to post both our findings and other relevant information on this page.

Research Highlights

As of June 30, 2020, this project’s research resulted in 25 peer-reviewed publications, 32 scientific presentations, and 59 datasets being submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are/will be made available to the public. The project also engaged 7 PhD and 2 Masters student’s over its award period. Further, the project trained 11 postdoctoral fellows and 6 research scientists. Significant outcomes of this project’s research are highlighted below.

ADDOMEx- and ADDOMEx-2 has shown that the marine microbial community actively responds to oil/dispersant (Corexit) challenges by regulating its exopolymeric substances (EPS) release accordingly, and that the EPS released interacts with the oil/dispersant. These results enhance the fundamental understanding of the how marine microbes (especially phytoplankton and bacteria) and their exudates physically determine the fate and transport of oil and dispersants in the ocean. Although a variety of environmental factors and microbial contribution have been demonstrated to influence oil/dispersant fate and transport, the relationship whereby these contributing factors influence each other are still not fully understood. The most important and unique impact of the research is it established the critical factors that determine the fate of oil pollutants and the associated ecological impact. This information will serve as the basis in establishing improved predictive models for risk assessment and to develop potential interventions to reduce the environmental impact and to formulate better response/management plans for future oil spill incidents.

MOS formed rapidly in most ADDOMEx replicate controlled mesocosms experiments with both the WAF and the CEWAF treatments. The mesocosms mimicked environmental conditions and concentrations of found during the Deepwater Horizon (DwH) oil spill or other oil spills. ADDOMEx mesocosms predicted half-lives of water column hydrocarbons that agreed with those predicted during the DwH, other oil spills and by other micro/mesocosum studies. ADDOMEx large (100L) mesocosms provided sufficient MOS to do detailed biological, microbiological and chemical studies.

Exopolymeric substances (EPS) with higher protein ratios was released under higher cellular stress levels induced by CEWAF, suggesting that higher protein ratio EPS, which is more hydrophobic, is secreted to physically or chemically ameliorate the hazardous agents. Our findings provide evidence that microbes can actively modify their EPS release and composition in responses to various adverse stress. Collectively, the results support the hypothesis that higher stress not only can trigger more EPS release, but also induce changes in its composition (protein/carbohydrate - P/C), thereby affecting environmental processes such as marine oil snow formation and the characteristic of marine organic matter.

The P/C ratio of EPS can be “conditionally” used as a proxy for their ‘stickiness’ and aggregation propensity, e.g., relative hydrophobicity, surface activity and surface tension, attachment efficiency, light-induced chemical crosslinking, and sedimentation efficiency of marine snow in marine environments. Not only the P/C ratio of EPS but also their physical properties, e.g., molecular size, work cooperatively to regulate the MOS-crude oil droplet aggregation. A high-throughput microplate-based protocol was established to assess P/C ratio of EPS. We found that under oil or Corexit challenges, phytoplankton and bacteria will release EPS with higher P/C ratio. These higher P/C ratio EPS can result in more aggregation and more stable aggregates.

With hydrophobic particles (surrogate for oil droplets), we were able to demonstrate that hydrophobic surfaces can promote aggregation and stability of formed aggregates. We have established a magnetic tweezer-based platform to assess the stickiness of EPS under various environmental conditions, such as for different salinities, temperatures, pHs and ionic conditions. We successfully demonstrated that P/C ratio (hydrophobicity) of EPS plays a determining role for the stickiness. Oil spills and Corexit administration not only can directly promote aggregation through direct physico-chemical (hydrophobic) interactions, but also can facilitate aggregation through indirect biological mechanisms that regulate the P/C ratio of the released EPS.

Phytoplankton are impacted and mediate the fate of oil in the marine environment, including producing EPS with various P/C ratios. Using metatranscriptomic data we developed a phytoplankton functional group oil sensitivity index and showed in a Gulf of Mexico mesocosm experiment that dinoflagellates were the most sensitive to exposure to oil and oil-dispersant mixtures while Chrysophytes benefit under oil exposure relative to other groups. More generally RNA expression analyses suggest that oil exposure causes cell membrane interference, stress to the photosynthetic apparatus and stimulates genes associated with ketogenesis in microbial eukaryotes. We hypothesize that under mild oil stress marine microbial eukaryotes may mediate the resultant oil stress with ketogenesis.

Further laboratory experiments to understand the differences in phytoplankton response on a functional group and species level suggested that anti-oxidant ability of a given species can act as a selective factor in deciding the fate of phytoplankton composition during oil exposure. In addition, we studied both direct and indirect interaction between phytoplankton-bacteria during oil exposure. We found that polysaccharide molecules secreted as EPS by diatoms are crucial for the surrounding bacteria and that the exposure to oil significantly affected the composition of bacteria in a way that the abundance of oil degrading bacteria were enhanced. In addition, exposure to oil negatively affected the bacterial secretion of EPS and therefore the uptake of bacterial EPS by phytoplankton was impacted. On the other hand, the impact of oil exposure was significantly lower on phytoplankton EPS secretion and therefore the uptake of phytoplankton EPS by bacteria was not affected.

To study indirect phytoplankton-bacteria interaction, we measured activities of extracellular enzymes like α- and β-glucosidase, lipase, alkaline-phosphatase, leucine-amino-peptidase along with organic carbon content and metagenomic sequencing. This was a mesocosm study conducted using seawater collected from the Gulf of Mexico with oil mixed with the chemical dispersant, Corexit. Overall, we found that addition of oil and dispersant Corexit enhanced extracellular enzyme activity and EPS production with microbial community mostly dominated by hydrocarbon degraders. Further monitoring of enzyme activities over a longer mesocosm experiment showed that the microbial utilization of dissolved organic matter during oil exposure is dynamic in nature, with a switch from oil to EPS-polysaccharide with the near depletion of oil. As phytoplankton-bacteria interaction is a critical process in marine snow formation, the above findings are likely to have significant implication in MOSSFA dynamics during an oil spill.

The discovery of MOSSFA events led to a series of experiments that investigated the formation of different types of marine oil snow, and examined especially the mechanisms behind marine oil snow formation. Details of marine oil snow formation mechanisms are essential for the development of predictive models that can be used for response planning and damage assessment. As the importance of marine oil snow was discovered due to the (co-)sedimentation of a large fraction of the spilled DwH oil, flux and sinking velocities were important parameters to characterize within this research focus. Mesocosm and smaller scale experiments lead to the discovery of the importance of the protein to carbohydrate (P/C) ratio in the aggregation process for particle and colloid formation. Novel mechanisms that lead to particle aggregation and ultimately, to sediment flux, such as radical oxygen species (ROS) mediated chemical crosslinking of proteins, as well as hydrophobic interactions, were demonstrated. Sedimentation and deposition onto the seafloor is however, only one of the potential fates of material (including oil) incorporated into marine snow.

Marine snow is also known as hotspots of bacterial activity and microbial activity in aggregates is significantly elevated compared to the surrounding seawater. This begs the hypothesis that oil incorporated into marine oil snow may also be degraded at a faster rate than freely suspended oil droplets. First indications of such rapid oxidation of oil within marine snow came from analysis by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), two-dimensional GCxGC-MS, and solid-state ¹³C NMR. Mesocosm experiments during ADDOMEx2 were designed to collect samples with higher resolution time points immediately following the start of the experiment, as compared to previous ADDOMEx experiments where the first collection time point was 4 days, This allowed us to identify and assess the rate of rapid oil incorporation into MOS within the first two days, and the rapid removal of aliphatic and aromatic oil-associated organic compounds within MOS by Day 4 mainly via microbial processing. MOS is further demonstrated to represent a critical microenvironment for oil incorporation and degradation along its transit below the euphotic zone to sediments. Analysis of exoenzymes for which assays to measure their rates was combined with metatranscriptomic analysis of all known exoenzymes to show how microbes use enzymes to degrade oil both alone and in the presence of dispersant.

In addition to looking at how oil degraded in marine oil snow, mesocosm experiments conducted during ADDOMEx and ADDOMEx2 revealed that hydrocarbon-oxidizing microbes selectively colonize oil droplets in the water column, that the number of resultant oil-microbe micro-aggregates is directly proportional to the amount of dissolved oil in the water, which is enhanced by application of dispersant, and that the number of micro-aggregates increases and decreases with the oxidation rate of alkanes and PAHs in the overall community. It was further shown that the bacteria found on the micro-aggregates have hydrocarbon oxidation genes that allow for the degradation of a diverse suite of hydrocarbon compounds.

In the last mesocosm experiment (M7), we examined hydrocarbon concentrations in dissolved vs. particle-associated phases, and targeted metabolites were measured in the dissolved phase. We observed metabolic intermediate compounds from naphthalenes during microbial oxidation. This observation highlights the partial degradation of hydrocarbons may occur during a marine oil spill as opposed to complete mineralization to carbon dioxide. Additionally, we were able to integrate metagenomic, metatranscriptomic and metabolomic analysis for the first time in the ADDOMEx project. This combination of analyses allows us to tie specific gene activity from specific microbes to the chemical products made by those activities.

In the past decades, modeling studies have suggested that fragmentation of marine snow during its transit to depth, is of central importance. Fragmentation would potentially shift the balance between the export of aggregated material to the seafloor and its biodegradation at depths. As small fragments are not believed to sink, but likely would be very bioavailable once removed from the nutrient poor surface ocean, fragmentation could greatly impact the fate of oil in the water. Our group conducted laboratory experiments to investigate the fragmentation potential of marine oil snow to help predicting the ultimate fate of sinking oil in the ocean. MOS formed in roller tanks filled with cultured diatoms and crude oil was physically stronger under our laboratory conditions compared with marine snow formed from the same diatom culture w/o the oil amendment. We assume that oil droplets may lead to a tighter packaging of cells within MOS, possibly decreasing aggregate porosity, which could explain a greater physical strength of MOS compared to non-oil containing aggregates. As a consequence, flux attenuation of sinking MOS may be lowered relative to marine snow. A relatively high connectivity between MOS formation at the surface and deposition at the seafloor may thus explain the large extent of the MOS sedimentation event in the aftermath of the 2020 DwH oil spill.

 Proposal Abstract - RFP-VI PI Antonietta Quigg