Core metabolism plasticity in phytoplankton: Response of Dunaliella tertiolecta to oil exposure.

Human alterations to the marine environment such as an oil spill can induce oxidative stress in phytoplankton. Exposure to oil has been shown to be lethal to most phytoplankton species, but some are able to survive and grow at unaffected or reduced growth rates, which appears to be independent of the class and phylum of the phytoplankton and their ability to consume components of oil heterotrophically. The goal of this article is to test the role of core metabolism plasticity in the oil-resisting ability of phytoplankton. Experiments were performed on the oil- resistant chlorophyte, Dunaliella tertiolecta, in control and water accommodated fractions of oil, with and without metabolic inhibitors targeting the core metabolic pathways. We observed that inhibiting pathways such as photosynthetic electron transport (PET) and pentose-phosphate pathway were lethal; however, inhibition of pathways such as mitochondrial electron transport and cyclic electron transport caused growth to be arrested. Pathways such as photorespiration and Kreb's cycle appear to play a critical role in the oil-tolerating ability of D. tertiolecta. Analysis of photo-physiology revealed reduced PET under inhibition of photorespiration but not Kreb's cycle. Further studies showed enhanced flux through Kreb's cycle suggesting increased energy production and photorespiration counteract oxidative stress. Lastly, reduced extracellular carbohydrate secretion under oil exposure indicated carbon and energy conservation, which together with enhanced flux through Kreb's cycle played a major role in the survival of D. tertiolecta under oil exposure by meeting the additional energy demands. Overall, we present data that suggest the role of phenotypic plasticity of multiple core metabolic pathways in accounting for the oxidative stress tolerating ability of certain phytoplankton species.

Physiological response of 10 phytoplankton species exposed to macondo oil and the dispersant, Corexit.

Culture experiments were conducted on ten phytoplankton species to examine their biological and physiological responses during exposure to oil and a combination of oil and dispersant. The species tested included a range of taxa typically found in the Gulf of Mexico such as cyanobacteria, chlorophytes, and diatoms. Cultures were exposed to Macondo surrogate oil using the water accommodated fraction (WAF), and dispersed oil using a chemically enhanced WAF (CEWAF) and diluted CEWAF, to replicate conditions following the Deepwater Horizon spill in the Gulf of Mexico. A range of responses were observed, that could broadly class the algae as either "robust" or "sensitive" to oil and/or dispersant exposure. Robust algae were identified as Synechococcus elongatus, Dunaliella tertiolecta, two pennate diatoms Phaeodactylum tricornutum and Navicula sp., and Skeletonema grethae CCMP775, and were largely unaffected by any of the treatments (no changes to growth rate or time spent in lag phase relative to controls). The rest of the phytoplankton, all centric diatoms, exhibited at least some combination of reduced growth rates or increased lag time in response to oil and/or dispersant exposure. Photophysiology did not have a strong treatment effect, with significant inhibition of photosynthetic efficiency (Fv /Fm ) only observed in the CEWAF, if at all. We found that the effects of oil and dispersants on phytoplankton physiology were species-dependent, and not always detrimental. This has significant implications on how oil spills might impact phytoplankton community structure and bloom dynamics in the Gulf of Mexico, which in turn impacts higher trophic levels.

Molecular mechanism of oil induced growth inhibition in diatoms using Thalassiosira pseudonana as the model species.

The 2010 Deepwater Horizon oil-spill exposed the microbes of Gulf of Mexico to unprecedented amount of oil. Conclusive evidence of the underlying molecular mechanism(s) on the negative effects of oil exposure on certain phytoplankton species such as Thalassiosira pseudonana is still lacking, curtailing our understanding of how oil spills alter community composition. We performed experiments on model diatom T. pseudonana to understand the mechanisms underpinning observed reduced growth and photosynthesis rates during oil exposure. Results show severe impairment to processes upstream of photosynthesis, such as light absorption, with proteins associated with the light harvesting complex damaged while the pigments were unaffected. Proteins associated with photosynthetic electron transport were also damaged, severely affecting photosynthetic apparatus and depriving cells of energy and carbon for growth. Negative growth effects were alleviated when an organic carbon source was provided. Further investigation through proteomics combined with pathway enrichment analysis confirmed the above findings, while highlighting other negatively affected processes such as those associated with ferroxidase complex, high-affinity iron-permease complex, and multiple transmembrane transport. We also show that oxidative stress is not the primary route of negative effects, rather secondary. Overall, this study provides a mechanistic understanding of the cellular damage that occurs during oil exposure to T. pseudonana.

Influence of nutrient status on the response of the diatom Phaeodactylum tricornutum to oil and dispersant.

Phytoplankton play a central role in our ecosystems, they are responsible for nearly 50 percent of the global primary productivity and major drivers of macro-elemental cycles in the ocean. Phytoplankton are constantly subjected to stressors, some natural such as nutrient limitation and some manmade such as oil spills. With increasing oil exploration activities in coastal zones in the Gulf of Mexico and elsewhere, an oil spill during nutrient-limited conditions for phytoplankton growth is highly likely. We performed a multifactorial study exposing the diatom Phaeodactylum tricornutum (UTEX 646) to oil and/or dispersants under nitrogen and silica limitation as well as co-limitation of both nutrients. Our study found that treatments with nitrogen limitation (-N and-N-Si) showed overall lower growth and chlorophyll a, lower photosynthetic antennae size, lower maximum photosynthetic efficiency, lower protein in exopolymeric substance (EPS), but higher connectivity between photosystems compared to non-nitrogen limited treatments (-Si and +N+Si) in almost all the conditions with oil and/or dispersants. However, certain combinations of nutrient limitation and oil and/or dispersant differed from this trend indicating strong interactive effects. When analyzed for significant interactive effects, the-N treatment impact on cellular growth in oil and oil plus dispersant conditions; and oil and oil plus dispersant conditions on cellular growth in-N-Si and-N treatments were found to be significant. Overall, we demonstrate that nitrogen limitation can affect the oil resistant trait of P. tricornutum, and oil with and without dispersants can have interactive effects with nutrient limitation on this diatom.

Trait-dependent variability of the response of marine phytoplankton to oil and dispersant exposure.

The Deepwater Horizon oil spill released millions of barrels of crude oil into the Gulf of Mexico, and saw widespread use of the chemical dispersant Corexit. We assessed the role of traits, such as cell size, cell wall, motility, and mixotrophy on the growth and photosynthetic response of 15 phytoplankton taxa to oil and Corexit. We collected growth and photosynthetic data on five algal cultures. These responses could be separated into resistant (Tetraselmis astigmatica, Ochromonas sp., Heterocapsa pygmaea) and sensitive (Micromonas pusilla, Prorocentrum minimum). We combined this data with 10 species previously studied and found that cell size is most important in determining the biomass response to oil, whereas motility/mixotrophy is more important in the dispersed oil. Our analysis accounted for a third of the variance observed, so further work is needed to identify other factors that contribute to oil resistance.

Diatom aggregation when exposed to crude oil and chemical dispersant: Potential impacts of ocean acidification.

Diatoms play a key role in the marine carbon cycle with their high primary productivity and release of exudates such as extracellular polymeric substances (EPS) and transparent exopolymeric particles (TEP). These exudates contribute to aggregates (marine snow) that rapidly transport organic material to the seafloor, potentially capturing contaminants like petroleum components. Ocean acidification (OA) impacts marine organisms, especially those that utilize inorganic carbon for photosynthesis and EPS production. Here we investigated the response of the diatom Thalassiosira pseudonana grown to present day and future ocean conditions in the presence of a water accommodated fraction (WAF and OAWAF) of oil and a diluted chemically enhanced WAF (DCEWAF and OADCEWAF). T. pseudonana responded to WAF/DCEWAF but not OA and no multiplicative effect of the two factors (i.e., OA and oil/dispersant) was observed. T. pseudonana released more colloidal EPS (< 0.7 µm to > 3 kDa) in the presence of WAF/DCEWAF/OAWAF/OADCEWAF than in the corresponding Controls. Colloidal EPS and particulate EPS in the oil/dispersant treatments have higher protein-to-carbohydrate ratios than those in the control treatments, and thus are likely stickier and have a greater potential to form aggregates of marine oil snow. More TEP was produced in response to WAF than in Controls; OA did not influence its production. Polyaromatic hydrocarbon (PAH) concentrations and distributions were significantly impacted by the presence of dispersants but not OA. PAHs especially Phenanthrenes, Anthracenes, Chrysenes, Fluorenes, Fluoranthenes, Pyrenes, Dibenzothiophenes and 1-Methylphenanthrene show major variations in the aggregate and surrounding seawater fraction of oil and oil plus dispersant treatments. Studies like this add to the current knowledge of the combined effects of aggregation, marine snow formation, and the potential impacts of oil spills under ocean acidification scenarios.

Growth dynamics and domoic acid production of Pseudo-nitzschia sp. in response to oil and dispersant exposure.

The diatom genus Pseudo-nitzschia is a common component of phytoplankton communities in the Gulf of Mexico and is potentially toxic as some species produce the potent neurotoxin domoic acid. The impact of oil and chemical dispersants on Pseudo-nitzschia spp. and domoic acid production have not yet been studied; preliminary findings from a mesocosm experiment suggest this genus may be particularly resilient. A toxicological study was conducted using a colony of Pseudo-nitzschia sp. isolated from a station off the coast of Louisiana in the Gulf of Mexico. The cultures were exposed to a water accommodated fraction (WAF) of oil and a diluted chemically enhanced WAF (DCEWAF) which was a mix of oil and dispersant (20:1). Exposure to WAF induced a lag phase but did not inhibit growth rates once in exponential growth. Cultures grown in DCEWAF did not experience a lag phase but had significantly lower growth rates than the Control and WAF cultures. The cellular quota of domoic acid was higher in cultures treated with DCEWAF and WAF relative to their control values, and half of the domoic acid had leaked out of the cells into the surrounding seawater in the DCEWAF cultures while all the domoic acid remained inside the cells in WAF-treated cultures. These results suggest that the presence of oil could lead to toxic blooms, but that the application of dispersant could decrease bioaccumulation of domoic acid through the food web.

Response of natural phytoplankton communities exposed to crude oil and chemical dispersants during a mesocosm experiment.

During the 2010 Deepwater Horizon oil spill, the chemical dispersant Corexit was applied over vast areas of the Gulf of Mexico. Marine phytoplankton play a key role in aggregate formation through the production of extracellular polymeric materials (EPS), an important step in the biological carbon pump. This study examined the impacts of oil and dispersants on the composition and physiology of natural marine phytoplankton communities from the Gulf of Mexico during a 72-hour mesocosm experiment and consequences to carbon export. The communities were treated using the water accommodated fraction (WAF) of oil, which was produced by adding Macondo surrogate oil to natural seawater and mixed for 24 h in the dark. A chemically enhanced WAF (CEWAF) was made in a similar manner, but using a mixture of oil and the dispersant Corexit in a 20:1 ratio as well as a diluted CEWAF (DCEWAF). Phytoplankton communities exposed to WAF showed no significant changes in PSII quantum yield (Fv/Fm) or electron transfer rates (ETRmax) compared to Control communities. In contrast, both Fv/Fm and ETRmax declined rapidly in communities treated with either CEWAF or DCEWAF. Analysis of other photophysiological parameters showed that photosystem II (PSII) antenna size and PSII connectivity factor were not altered by exposure to DCEWAF, suggesting that processes downstream of PSII were affected. The eukaryote community composition in each experimental tank was characterized at the end of the 72 h exposure time using 18S rRNA sequencing. Diatoms dominated the communities in both the control and WAF treatments (52 and 56% relative abundance respectively), while in CEWAF and DCEWAF treatments were dominated by heterotrophic Euglenozoa (51 and 84% respectively). Diatoms made up the largest relative contribution to the autotrophic eukaryote community in all treatments. EPS concentration was four times higher in CEWAF tanks compared to other treatments. Changes in particle size distributions (a proxy for aggregates) over time indicated that a higher degree of particle aggregation occurred in both the CEWAF and DCEWAF treatments than the WAF or Controls. Our results demonstrate that chemically dispersed oil has more negative impacts on photophysiology, phytoplankton community structure and aggregation dynamics than oil alone, with potential implications for export processes that affect the distribution and turnover of carbon and oil in the water column.

Diagnostic tool to ascertain marine phytoplankton exposure to chemically enhanced water accommodated fraction of oil using Fourier Transform Infrared spectroscopy.

Phytoplankton alter their macromolecule composition in response to changing environmental conditions. Often these changes are consistent and can be used as indicators to predict their exposure to a given condition. FTIR-spectroscopy is a powerful tool that provides rapid snapshot of microbial samples. We used FTIR to develop signature macromolecular composition profiles of three cultures: Skeletonema costatum, Emiliania huxleyi, and Navicula sp., exposed to chemically enhanced water accommodated oil fraction (CEWAF) in artificial seawater and control. Using a multivariate model created with a Partial Least Square Discriminant Analysis of the FTIR-spectra, classification of CEWAF exposed versus control samples was possible. This model was validated using aggregate samples from a mesocosm study. Analysis of spectra and PCA-loadings plot showed changes to carbohydrates and proteins in response to CEWAF. Overall we developed a robust multivariate model that can be used to identify if a phytoplankton sample has been exposed to oil with dispersant.

Extracellular Enzyme Activity Profile in a Chemically Enhanced Water Accommodated Fraction of Surrogate Oil: Toward Understanding Microbial Activities After the Deepwater Horizon Oil Spill.

Extracellular enzymes and extracellular polymeric substances (EPS) play a key role in overall microbial activity, growth and survival in the ocean. EPS, being amphiphilic in nature, can act as biological surfactant in an oil spill situation. Extracellular enzymes help microbes to digest and utilize fractions of organic matter, including EPS, which can stimulate growth and enhance microbial activity. These natural processes might have been altered during the 2010 Deepwater Horizon oil spill due to the presence of hydrocarbon and dispersant. This study aims to investigate the role of bacterial extracellular enzymes during exposure to hydrocarbons and dispersant. Mesocosm studies were conducted using a water accommodated fraction of oil mixed with the chemical dispersant, Corexit (CEWAF) in seawater collected from two different locations in the Gulf of Mexico and corresponding controls (no additions). Activities of five extracellular enzymes typically found in the EPS secreted by the microbial community - α- and ß-glucosidase, lipase, alkaline phosphatase, leucine amino-peptidase - were measured using fluorogenic substrates in three different layers of the mesocosm tanks (surface, water column and bottom). Enhanced EPS production and extracellular enzyme activities were observed in the CEWAF treatment compared to the Control. Higher bacterial and micro-aggregate counts were also observed in the CEWAF treatment compared to Controls. Bacterial genera in the order Alteromonadaceae were the most abundant bacterial 16S rRNA amplicons recovered. Genomes of Alteromonadaceae commonly have alkaline phosphatase and leucine aminopeptidase, therefore they may contribute significantly to the measured enzyme activities. Only Alteromonadaceae and Pseudomonadaceae among bacteria detected here have higher percentage of genes for lipase. Piscirickettsiaceae was abundant; genomes from this order commonly have genes for leucine aminopeptidase. Overall, this study provides insights into the alteration to the microbial processes such as EPS and extracellular enzyme production, and to the microbial community, when exposed to the mixture of oil and dispersant.