Marine microbial intact polar diacylglycerolipids and their application in the study of nutrient stress and bacterial production

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2013Intact polar diacylglycerolipids (IP-DAGs) were used to study microbial dynamics in the surface ocean. IP-DAGs from surface ocean seawater were quantified using high performance liquid chromatography-mass spectrometry (HPLC-MS), after first developing a sensitive, high throughput molecular ion independent triple quadrupole MS method for quantification. Using this analytical technique I examined the distribution of the nine most abundant classes of IPDAGs across the Mediterranean, and found that phospholipids as a percent of total IP-DAGs correlated with phosphate concentration. Furthermore, phospholipids were a higher percent of total particulate phosphorus where phosphate was higher, ranging from 1-14%. Thus IP-DAGs can play not only a significant but also a dynamic role in defining planktonic nutrient needs and cellular C:N:P ratios in the environment. Additionally, microcosm incubations were amended with phosphate and ammonium, and in the course of several days this elicited a shift in the ratios of IP-DAGs. This study was the first to demonstrate the dynamic response of membrane lipid composition to changes in nutrients in a natural, mixed planktonic community, and indicated that the change in IP-DAG ratios in response to changing nutrients may be a useful indicator of microbial nutrient stress. In the surface waters of the western North Atlantic I used three experimental approaches to identify the microbial sources of the nine most abundant classes of IP-DAGs. Phytoplankton are the primary source of one class of sulfolipid, sulfoquinovosyldiacylglycerol, and one class of betaine lipid, diacylglyceryl-trimethyl-homoserine, while heterotrophic bacteria are the dominant source of the phospholipids phosphatidylglycerol and phosphatidylethanolamine. In regrowth experiments in the Sargasso Sea and the North Pacific I demonstrated that phospholipid specific production rate is representative of heterotrophic bacterial cell specific growth rate. I measured phospholipid specific production rate and bacterial production rate using uptake of 3H-leucine (3H-Leu) and 3H-thymidine (3H-TdR) across the North Atlantic, across the Mediterranean, and in the North Pacific subtropical gyre. I found that phospholipid specific production rates estimate heterotrophic bacterial cell specific growth rates that are on the order of 1 per day, an order of magnitude faster than cell specific growth rates suggested by uptake of 3H-Leu and 3H-TdR.This research was supported by a National Science Foundation Graduate Research Fellowship, a National Defense Science and Engineering Graduate Fellowship, and grants from the U.S. Office of Naval Research (N00014-09-1-0091 and N00014-06-1-0134) and the U.S. National Science Foundation (OCE-1031143, OCE-1029687, and OCE-0646944)

Variable phosphorus uptake rates and allocation across microbial groups in the oligotrophic Gulf of Mexico

Microbial uptake of dissolved phosphorus (P) is an important lever in controlling both microbial production and the fate and cycling of marine P. We investigated the relative role of heterotrophic bacteria and phytoplankton in P cycling by measuring the P uptake rates of individual microbial groups (heterotrophic bacteria and the phytoplankton groups Synechococcus, Prochlorococcus, and picoeukaryotic phytoplankton) in the P-depleted Gulf of Mexico. Phosphorus uptake rates were measured using incubations with radiolabeled phosphate and adenosine triphosphate coupled with cell sorting flow cytometry. We found that heterotrophic bacteria were the dominant consumers of P on both a biomass basis and a population basis. Biovolume normalized heterotrophic bacteria P uptake rate per cell (amol P μm-3 hr-1) was roughly an order of magnitude greater than phytoplankton uptake rates, and heterotrophic bacteria were responsible for generally greater than 50% of total picoplankton P uptake. We hypothesized that this variation in uptake rates reflects variation in cellular P allocation strategies, and found that, indeed, the fraction of cellular P uptake utilized for phospholipid production was significantly higher in heterotrophic bacteria compared to cyanobacterial phytoplankton. These findings indicate that heterotrophic bacteria have a uniquely P-oriented physiology and play a dominant role in cycling dissolved P

Variability of Microbial Particulate ATP Concentrations in Subeuphotic Microbes Due to Underlying Metabolic Strategies in the South Pacific Ocean

Adenosine triphosphate (ATP) is the primary energy storage molecule in metabolic pathways. It is common in marine studies to use particulate ATP (PATP) concentrations as representative of microbial biomass. However, there is growing evidence from culture studies, models, and transcriptional data that PATP concentration varies across microbes and conditions, thus compromising interpretations in environmental settings. Importantly, there is a lack of open ocean studies assessing variations in PATP concentrations and thus a deficiency of information on the key biogeochemical drivers for variability in microbial PATP independent of biomass. In sampling the U.S. GO-SHIP P06E zonal transect (32.5°S) across the eastern South Pacific, from the subtropical gyre to the upwelling waters off Chile, we conducted the first comprehensive transect survey quantifying PATP. PATP concentrations increased toward the upwelling region of the transect, but varied vertically when normalized against three measures of biomass: particulate phosphorus, microbial abundance, and microbial biovolume. Generally, greater biomass-normalized PATP concentrations were observed below the deep chlorophyll maximum. Subdividing the P06E transect into four biogeochemical regimes highlighted distinct metabolic strategies used by microbes. Between these regimes, we found PATP concentrations were representative of biomass in upper surface waters. However, below the deep chlorophyll maximum we observed higher biomass normalized PATP concentrations that we hypothesize were due to less availability of energy sources in those subeuphotic zone waters and abundances of chemoautotrophs in the microbial community. This finding suggests that stored energy was more important for these deeper microbes

Controls on the Fate of Dissolved Organic Carbon Under Contrasting Upwelling Conditions

To understand controls on the production and remineralization of recalcitrant dissolved organic carbon (DOC) produced in association with positive net community production (NCP), we simulated upwelling systems of different intensities by combining and incubating whole seawater collected from different depths in the Florida Strait (27°N, ∼79°W). The natural microbial communities in the treatments grew under controlled light and temperature for 15 days (i.e., the autotrophic phase); they were subsequently incubated for 35 days in the dark heterotrophic phase. We analyzed the phytoplankton composition and pigment fluorescence intensity during the light phase, and dissolved organic and inorganic variables during both phases. Initial high or low availability of inorganic nutrients controlled phytoplankton growth and the magnitude of NCP. In the strong upwelling treatment with higher initial inorganic nutrients, 25% of NCP accumulated as DOC after 15 days, however, this material was in turn fully remineralized during the dark phase. In contrast, low nutrients in the weak upwelling treatment limited the magnitude of NCP and accumulated DOC, which represented 11% of NCP. Surprisingly, most of this fraction resisted microbial remineralization in the dark phase, suggesting that upwellings of different intensities affect the quality of dissolved organic matter produced, thereby affecting the timing and location of its remineralization and, hence, its prospects for export to the deep ocean

Filtration efficiency of air conditioner filters and face masks to limit exposure to aerosolized algal toxins

Harmful algal blooms (HABs) can generate toxins that can be aerosolized and negatively impact human health through inhalation. HABs are often found in waterways near residences, therefore, aerosolized HAB toxins can potentially affect both indoor and outdoor air quality. Given that HABs are predicted to increase worldwide, effective mitigation strategies are needed to prevent the inhalation of aerosolized HAB toxins. In this work, we characterized both the particle filtration efficiency using particle sizing instruments as well as the mass concentration of different congeners of aerosolized microcystin (MC) toxins that penetrate through commercially available face masks and air conditioner (AC) filters. Particles were generated from cultures of the toxin-producing cyanobacteria Microcystis aeruginosa. Hydrophobic congeners of microcystin including MC-LF and MC-LW were enriched in aerosols compared to water, with MC-LR being the most abundant, which has implications for the toxicity of inhalable particles generated from HAB-contaminated waters. Particle transmission efficiencies and toxin filtration efficiencies scaled with the manufacturer-provided filter performance ratings. Up to 80% of small, microcystin-containing aerosols were transmitted through AC filters with low filter performance ratings. In contrast, both face masks as well as AC filters with high filter performance ratings efficiently removed toxin-containing particles to below limits of quantification. Our findings suggest that face masks and commercially available AC filters with high filtration efficiency ratings are suitable mitigation strategies to avoid indoor and outdoor air exposure to aerosolized HAB toxins. This work also has relevance for reducing airborne exposure to other HAB toxins, non-HAB toxins, pathogens, and viruses, including SARS-CoV-2, the virus responsible for the COVID-19 pandemic

African biomass burning is a substantial source of phosphorus deposition to the Amazon, Tropical Atlantic Ocean, and Southern Ocean

The deposition of phosphorus (P) from African dust is believed to play an important role in bolstering primary productivity in the Amazon Basin and Tropical Atlantic Ocean (TAO), leading to sequestration of carbon dioxide. However, there are few measurements of African dust in South America that can robustly test this hypothesis and even fewer measurements of soluble P, which is readily available for stimulating primary production in the ocean. To test this hypothesis, we measured total and soluble P in long-range transported aerosols collected in Cayenne, French Guiana, a TAO coastal site located at the northeastern edge of the Amazon. Our measurements confirm that in boreal spring when African dust transport is greatest, dust supplies the majority of P, of which 5% is soluble. In boreal fall, when dust transport is at an annual minimum, we measured unexpectedly high concentrations of soluble P, which we show is associated with the transport of biomass burning (BB) from southern Africa. Integrating our results into a chemical transport model, we show that African BB supplies up to half of the P deposited annually to the Amazon from transported African aerosol. This observational study links P-rich BB aerosols from Africa to enhanced P deposition in the Amazon. Contrary to current thought, we also show that African BB is a more important source of soluble P than dust to the TAO and oceans in the Southern Hemisphere and may be more important for marine productivity, particularly in boreal summer and fall

Variable phosphorus uptake rates and allocation across microbial groups in the oligotrophic Gulf of Mexico

Microbial uptake of dissolved phosphorus (P) is an important lever in controlling both microbial production and the fate and cycling of marine P. We investigated the relative role of heterotrophic bacteria and phytoplankton in P cycling by measuring the P uptake rates of individual microbial groups (heterotrophic bacteria and the phytoplankton groups Synechococcus, Prochlorococcus, and picoeukaryotic phytoplankton) in the P-depleted Gulf of Mexico. Phosphorus uptake rates were measured using incubations with radiolabeled phosphate and adenosine triphosphate coupled with cell sorting flow cytometry. We found that heterotrophic bacteria were the dominant consumers of P on both a biomass basis and a population basis. Biovolume normalized heterotrophic bacteria P uptake rate per cell (amol P μm-3 hr-1) was roughly an order of magnitude greater than phytoplankton uptake rates, and heterotrophic bacteria were responsible for generally greater than 50% of total picoplankton P uptake. We hypothesized that this variation in uptake rates reflects variation in cellular P allocation strategies, and found that, indeed, the fraction of cellular P uptake utilized for phospholipid production was significantly higher in heterotrophic bacteria compared to cyanobacterial phytoplankton. These findings indicate that heterotrophic bacteria have a uniquely P-oriented physiology and play a dominant role in cycling dissolved P

HPLC and 32P-radiolabeling method for quantification of microbial adenylate concentrations and turnover rates in seawater

We optimized a high throughput method to quantify turnover rates of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in marine microbes from simultaneous measures of the respective stocks and phosphorylation rates. We combined a microbial adenylate extraction method using boiling 20 mM Tris buffer with purification and analysis by high pressure liquid chromatography optimized to quantify these intracellular adenylate concentrations in marine microbes. Additionally, we incorporated radiolabeled phosphate (P-32(i)) incubations to quantify phosphorus (P) uptake rates and the phosphorylation rates for these adenylate compounds in microbial cells. With this method, we can directly assess the variations in microbial growth rates, metabolic turnover rates, energy charge, and adenylate storage. We applied and validated this method application with environmental samples from Biscayne Bay, Florida, and quantified adenylate turnover times of 12, 15, and 73 min, for ATP, ADP, and AMP, respectively. Future incorporation of this method into experiments and geographic surveys across marine environments will allow for direct assessments of changes in microbial metabolic activity in relation to other ecological variables

Microbial sources of intact polar diacylglycerolipids in the Western North Atlantic Ocean

► Microibal sources of intact polar diacylglycerolipids were identified. ► Photoautotrophs are sole source of sulfoquinovosyldiacylglycerol. ► Eukaryotic phytoplankton are likely source of a betaine lipid. ► Heterotrophic bacteria are sole source of phosphatidylglycerol.Intact polar membrane lipids are essential components of microbial membranes and recent work has uncovered a diversity of them occurring in the ocean. While it has long been understood that lipid composition varies across microbial groups, the microbial origins of the intact polar lipids in the surface ocean remain to be fully explained. This study focused on identifying the microbial sources of intact polar diacylglycerolipids (IP-DAGs) in the surface waters of the western North Atlantic Ocean. We used three approaches to define these microbial sources: (i) 13C tracing to identify photoautotrophic and heterotrophic production of the major classes of IP-DAGs, (ii) cell sorting flow cytometry of Prochlorococcus, Synechococcus and heterotrophic bacteria to determine IP-DAG composition and (iii) regrowth incubations targeting IP-DAG production by heterotrophic bacteria. Stable isotope tracing indicated that sulfoquinovosyldiacylglycerol (SQDG) and diacylglyceryl-trimethyl-homoserine (DGTS) were produced predominantly by photoautotrophs, while phosphatidylglycerol (PG) production was dominated by heterotrophic bacteria. Of the cells sorted with flow cytometry, Prochlorococcus and Synechococcus were found to have abundant glycolipids, while heterotrophic bacteria were dominated by phospholipids. The regrowth incubations showed that the growth of heterotrophic bacteria correlated with an increase in the concentration of PG, phosphatidylethanolamine (PE) and monoglycosyldiacylglycerol (MGDG). The finding of MGDG in heterotrophic bacteria differs from previous work, which had asserted that the membranes of heterotrophic bacteria in this environment were composed entirely of phospholipids. Overall, our findings indicate that phytoplankton are the primary source of SQDG and DGTS, while heterotrophic bacteria are the dominant source of PG, making these three compounds promising biomarkers for the study of microbes in the surface ocean

Molecular Ion‐Independent Quantification of Polar Glycerolipid Classes in Marine Plankton Using Triple Quadrupole MS

Polar glycerolipids are a diverse family of lipid molecules that form the bulk of bacterial and eukaryotic microbial membranes. The earth and ocean sciences has a long history of using fatty acids as biomarkers for microbes, but have only recently begun to examine the intact polar lipids from which they are derived. Current analytical approaches rely on laboriously quantifying the molecular ions of each of these species independently. Thus, we saw a need for a method for quantifying polar glycerolipid classes that was: (i) selective for individual classes, (ii) inclusive of all species within a class, (iii) independent of foreknowledge of the molecular ions of the polar glycerolipid, and (iv) amenable to automated, high‐throughput data analysis methods. Our new HPLC‐electrospray‐ionization triple‐quadrupole MS (HPLC‐ESI‐TQMS) method can be applied to quantify the nine major classes of polar glycerolipid in planktonic communities: the phospholipids phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the glycolipids monoglycosyldiacylglycerol, diglycosyldiacylglycerol and sulfoquinovosyldiacylglycerol; and the betaine lipids diacylglyceryl trimethyl homoserine, diacylglyceryl hydroxymethyl trimethyl‐β‐alanine, and diacylglyceryl carboxyhydroxymethylcholine. The analyses rely on neutral loss and parent ion scan events that yield one chromatogram for each class of polar glycerolipid, simplifying downstream analysis and increasing sample throughput. The efficacy of the method was demonstrated by analyzing plankton community samples from a variety of marine environments