Project Overview

Summary:

In January 2016, Dr. Sunshine Van Bael at Tulane University was awarded an RFP-V grant of $1,580,319 (later, modified to $1,597,491) to lead the GoMRI project entitled Chemical Evolution and Plant-Microbe Degradation of Petroleum in Saline Marsh Plants and Soils consisted of 2 collaborative institution and approximately 19 research team members (including students).

     The importance of bacteria for biodegradation of petroleum is well described for contaminated seawater and coastal soils, but very little is known about the role of symbiotic plant bacteria in degrading petroleum. Endophytes are bacteria and fungi that live as symbionts within plant roots, stems and leaves. These symbionts are closely associated with the plant and some endophyte species serve the dual purpose of promoting plant growth and degrading petroleum inside of plant tissues. In an extreme environment such as a salt marsh, where oxygen is limited in soils, plants may be especially dependent on endophytic bacteria for resilience to stress and to respond to petroleum contamination.

     Preliminary research since the Deepwater Horizon (DWH) oil spill has shown that when coastal grasses are contaminated with petroleum, the bacterial communities in their tissues incorporate more taxa with known roles in biodegradation. This preliminary work has led to the hypotheses driving this proposal: that endophyte communities inside of coastal plants will shift to incorporate and amplify endophytic bacteria that are tolerant to petroleum and can biodegrade it inside of plant tissues, and that plant delivery of oxygen and endophytic bacteria to polluted soils will hasten the chemical evolution of petroleum. Addressing these hypotheses at the mechanistic level involves describing the transport mechanisms and the catabolic activities of bacteria with respect to petroleum inside of plant tissues and at the interface of grass roots and the rhizosphere. These processes are not well characterized, particularly in saline marshes. Such a knowledge gap is a problem because it prohibits a mechanistic understanding of how grasses, symbiotic bacteria and polluted soil interact, and thus slows the development of remediation tools that use plant-delivered, naturally occurring bacteria to clean up polluted soils.

     The overall goal of the funded research was to develop a mechanistic understanding of plant bacterial symbioses in relation to petroleum/dispersant pollution in saline marshes. The funded work characterized the transport, fate and catabolic activities of bacterial communities in petroleum-polluted soils and within plant tissues. The project focused on Spartina alterniflora (smooth cordgrass), the foundational grass species within salt marshes along Atlantic and Gulf coasts. The specific goals were (1) to use next-generation genomic technology for characterizing the taxonomy and function of microbial communities inside of S. alterniflora tissues and in the rhizosphere, while relating these communities to the chemical evolution of crude oil constituents in plant tissues and in soil; and (2) to use new visualization and computational modeling approaches for investigating the biomechanical and chemical influences on bacteria movement at the interface of roots and soil to mechanistically relate bacterial chemotaxis to the presence of petroleum, dispersant, oxygen and root exudates. The proposed research goals directly addressed GoMRI research theme two, as each ultimately relates plant-symbiont interactions to the chemical evolution and biodegradation of petroleum and dispersants in coastal ecosystems. Pursuing these goals advanced understanding of key processes that occurred in the DWH spill and may occur in future spills.

     The outcomes of the proposed research included (1) a deeper knowledge of the functional genomics of petroleum degradation and uptake of petroleum into plants, (2) the first descriptions and computational models for the biomechanical and chemical aspects of bacterial movement at the root: rhizosphere interface in response to petroleum and dispersant, and (3) the first determination of how plant-endophyte symbioses influence the fate of petroleum in marsh ecosystems. Developing a mechanistic understanding of plant-symbiont-petroleum interactions provides a foundation for the development of remediation tools using naturally occurring plant-bacteria combinations. Such strategies are being developed in other ecosystems but have not yet been extended to include coastal plants in the Gulf of Mexico (GoM), where there is a persistently high threat of petroleum contamination.

Research Highlights

As of December 31, 2019, this project’s research resulted in,   6 peer-reviewed publications and 28 scientific presentations and  16 datasets being submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are/will be made available to the public. The project also engaged 4 Master’s level and 2 PhD level students over its award period. Significant outcomes of this project’s research according to GoMRI Research Themes are highlighted below.

Theme Two:

Goal 1. We characterized the taxonomy and genomic function of bacterial communities inside of S. alterniflora tissues and in the rhizosphere, relating these communities to the transport phenomena of petroleum into plant tissues and to the chemical evolution of crude oil constituents within plant tissues and in soil.

Accomplishments

• Field sampling was completed and a paper about fungi was published in Science of the Total Environment. Work on the bacterial samples is still in progress. A related manuscript was just accepted at the American Journal of Botany.
• All samples were received at Duke University and work began on extracting DNA, library preparation and sequencing. Some work went into optimizing extraction of root DNA for analysis on the GeoChip, a microarray for studying functional genes.  
• Analyses of PAHs from the field were completed. Sampling in the field was finished. This year, most time was spent developing and refining techniques to use confocal microscopy. The goal for this is to see how and where PAHs are sequestered in leaf tissue. Analyses of greenhouse samples for PAHs was also completed.

Major findings

• In our paper about fungi, we showed that even six years after the oil spill, fungal communities associated with Spartina alterniflora had decreased diversity compared to sites without oil. This is significant because most microbial studies after the oil spill focused on bacteria and not fungi. 
• To summarize our preliminary analyses with microbial communities in our greenhouse experiment, microbial communities were shaped by the presence of a plant, oil, and soil inoculum.  However the effect was more pronounced in bacteria than in fungi, particularly with regards to oil.
• In addition, we found that plant growth is slower when oil is present, though the effect may be dependent on soil microbes or repeated oiling. Finally, oil decay was faster when a plant was present or when the soil microbial community composition changed, but there was no interaction of the two.
• Confocal imaging of phenanthrene vapor penetrations into abaxial leaf tissues were performed to better understand the mechanism of PAH interactions of Spartina and Avicennia (black mangrove) plant tissues. Imaging confirms that PAHs move into the interlamellar tissues between the leaf tissue cells after passing through the cuticle. Tissues around salt glands in Avicennia and leaf ridges on Spartina are areas of significant accumulation of PAHs.

Goal 2. We investigated and modelled the biomechanical and chemical influences on

bacterial movement at the interface of roots and soil, relating bacterial chemotaxis to the presence of petroleum, oxygen and root exudates.

Accomplishments 

• One experiment with a strain of endophytic bacteria was fully completed and the manuscript was published in the Journal of Environmental Chemistry.
• Computational work has been completed on bacterial transport in confined spaces (like soil pores or plant roots). The model of bacterial flagella developed for this project has also been adapted to study the buckling of actin filaments in compressive flows. A manuscript entitled “Elastohydrodynamics of swimming helices: effects of flexibility and confinement” by J. Lagrone, R. Cortez, L. Fauci has published in Phys. Rev. Fluids. The math modeling group continued to develop models of bacterial chemotaxis using a Kirchhoff rod model and explored different stochastic models of reorientation of swimming direction based upon chemical cues.

 Major findings

• The endophyte, Pseudomonas putida, isolated from Spartina alterniflora, can degrade oil droplets.
• The swimming performance of an actuated, helical filament (i.e., a bacterial helical bundle) is enhanced when confined to a cylindrical tube.
•  A helical filament whose axis is not aligned with the tube axis can exhibit centering behavior in the narrowest tubes.
• Fast multipole methods can be used to resolve the complex buckling of flexible fibers in flow. 
•  Long, flexible passive fibers in shear can develop multiple buckling sites, depending upon the elasto-viscous number.

  Proposal Abstract - RFP-V PI Sunshine Van Bael